Discovery of P2X7 receptor-selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states.

ATP-sensitive P2X(7) receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X(7) receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1beta and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Both the localization and functional consequences of P2X(7) receptor activation indicate a role in inflammatory processes. The phenotype of P2X(7) receptor gene-disrupted mice also indicates that P2X(7) receptor activation contributes to ongoing inflammation. More recently, P2X(7) receptor knockout data has also suggested a specific role in inflammatory and neuropathic pain states. The recent discovery of potent and highly selective antagonists for P2X(7) receptors has helped to further clarify P2X receptor pharmacology, expanded understanding of P2X(7) receptor signaling, and offers new evidence that P2X(7) receptors play a specific role in nociceptive signaling in chronic pain states. In this review, we incorporate the recent discoveries of novel P2X(7) receptor-selective antagonists with a brief update on P2X(7) receptor pharmacology and its therapeutic potential.

P2X7-related modulation of pathological nociception in rats.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

Pharmacological characterization of P2X7 receptors in rat peritoneal cells.

OBJECTIVE AND DESIGN: P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). MATERIALS AND METHODS: RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RESULTS: RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. CONCLUSIONS: RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

The role of neuronal nicotinic acetylcholine receptors in antinociception: effects of ABT-594.

ABT-594, a nicotinic acetylcholine receptor agonist, has antinociceptive effects in rat models of acute thermal, persistent chemical, and neuropathic pain. Direct injection of ABT-594 into the nucleus raphe magnus (NRM) is antinociceptive in a thermal threshold test and destruction of serotonergic neurons in the NRM attenuates the effect of systemic ABT-594. However, lidocaine-inactivation of the NRM prevents the antinociceptive effect of systemic (-)-nicotine but not that of systemic ABT-594.

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective analgesic acting via neuronal nicotinic acetylcholine receptors: I. In vitro characterization.

The discovery of (+/-)-epibatidine, a naturally occurring neuronal nicotinic acetylcholine receptor (nAChR) agonist with antinociceptive activity 200-fold more potent than that of morphine, has renewed interest in the potential role of nAChRs in pain processing. However, (+/-)-epibatidine has significant side-effect liabilities associated with potent activity at the ganglionic and neuromuscular junction nAChR subtypes which limit its potential as a clinical entity. ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine] is a novel, potent cholinergic nAChR ligand with analgesic properties (see accompanying paper by Bannon et al., 1998b) that shows preferential selectivity for neuronal nAChRs and a consequently improved in vivo side-effect profile compared with (+/-)-epibatidine. ABT-594 is a potent inhibitor of the binding of [3H](-)-cytisine to alpha 4 beta 2 neuronal nAChRs (Ki = 37 pM, rat brain; Ki = 55 pM, transfected human receptor). At the alpha 1 beta 1 delta gamma neuromuscular nAChR labeled by [125I] alpha-bungarotoxin (alpha-Btx), ABT-594 has a Ki value of 10,000 nM resulting in a greater than 180,000-fold selectivity of the compound for the neuronal alpha 4 beta 2 nAChR. In contrast, (+/-)-epibatidine has Ki values of 70 pM and 2.7 nM at the alpha 4 beta 2 and alpha 1 beta 1 delta gamma nAChRs, respectively, giving a selectivity of only 38-fold. The S-enantiomer of ABT-594, A-98593 has activity at the neuronal alpha 4 beta 2 nAChR identical with ABT-594 (Ki = 34-39 pM), which demonstrates a lack of stereospecific binding similar to that reported previously for (+/-)-epibatidine. A similar lack of stereoselectivity is seen at the human alpha 7 receptor. However, A-98593 is 3-fold more potent at the neuromuscular nAChR (Ki = 3420 nM) and the brain alpha-Btx-sensitive nAChR (Ki = 4620 nM) than ABT-594. ABT-594 has weak affinity in binding assays for adrenoreceptor subtypes alpha-1B (Ki = 890 nM), alpha-2B (Ki = 597 nM) and alpha-2C (Ki = 342 nM), and it has negligible affinity (Ki > 1000 nM) for approximately 70 other receptors, enzyme and transporter binding sites. Functionally, ABT-594 is an agonist. At the transfected human alpha 4 beta 2 neuronal nAChR (K177 cells), with increased 86Rb+ efflux as a measure of cation efflux, ABT-594 had an EC50 value of 140 nM with an intrinsic activity (IA) compared with (-)-nicotine of 130%; at the nAChR subtype expressed in IMR-32 cells (sympathetic ganglion-like), an EC50 of 340 nM (IA = 126%); at the F11 dorsal root ganglion cell line (sensory ganglion-like), an EC50 of 1220 nM (IA = 71%); and via direct measurement of ion currents, an EC50 value of 56,000 nM (IA = 83%) at the human alpha 7 homooligimeric nAChR produced in oocytes. A-98593 is 2- to 3-fold more potent and displays approximately 50% greater intrinsic activity than ABT-594 in all four functional assays. In terms of potency, ABT-594 is 8- to 64-fold less active than (+/-)-epibatidine and also has less IA in these functional assays. ABT-594 (30 microM) inhibits the release of calcitonin gene-related peptide from C-fibers terminating in the dorsal horn of the spinal cord, an effect mediated via nAChRs. Pharmacologically, ABT-594 has an in vitro profile distinct from that of the prototypic nicotinic analgesic (+/-)-epibatidine, with the potential for substantially reduced side-effect liability and, as such, represents a potentially novel therapeutic approach to pain management.

Identification and initial structure-activity relationships of (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), a potent, orally active, non-opiate analgesic agent acting via neuronal nicotinic acetylcholine receptors.

New members of a previously reported series of 3-pyridyl ether compounds are disclosed as novel, potent analgesic agents acting through neuronal nicotinic acetylcholine receptors. Both (R)-2-chloro-5-(2-azetidinylmethoxy)pyridine (ABT-594, 5) and its S-enantiomer (4) show potent analgesic activity in the mouse hot-plate assay following either intraperitoneal (i.p.) or oral (p.o.) administration, as well as activity in the mouse abdominal constriction (writhing) assay, a model of persistent pain. Compared to the S-enantiomer and to the prototypical potent nicotinic analgesic agent (+/-)-epibatidine, 5 shows diminished activity in models of peripheral side effects. Structure-activity studies of analogues related to 4 and 5 suggest that the N-unsubstituted azetidine moiety and the 2-chloro substituent on the pyridine ring are important contributors to potent analgesic activity.

Broad-spectrum, non-opioid analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors.

Development of analgesic agents for the treatment of severe pain requires the identification of compounds that are devoid of opioid receptor liabilities. A potent (inhibition constant = 37 picomolar) neuronal nicotinic acetylcholine receptor (nAChR) ligand called ABT-594 was developed that has antinociceptive properties equal in efficacy to those of morphine across a series of diverse animal models of acute thermal, persistent chemical, and neuropathic pain states. These effects were blocked by the nAChR antagonist mecamylamine. In contrast to morphine, repeated treatment with ABT-594 did not appear to elicit opioid-like withdrawal or physical dependence. Thus, ABT-594 may be an analgesic that lacks the problems associated with opioid analgesia.

Synthesis and structure-activity relationships of pyridine-modified analogs of 3-[2-((S)-pyrrolidinyl)methoxy]pyridine, A-84543, a potent nicotinic acetylcholine receptor agonist.

Analogs of 3-[2-((S)-pyrrolidinyl)methoxy]pyridine, (A-84543, 1) with 2-, 4-, 5-, and 6-substituents on the pyridine ring were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 9,000 nM when tested in vitro for neuronal nicotinic acetylcholine receptor binding activity. Assessment of functional activity at subtypes of neuronal nicotinic acetylcholine receptors indicates that pyridine substitution can have a profound effect on efficacy at these subtypes, and several subtype-selective agonists and antagonists have been identified.

Structure-activity studies related to ABT-594, a potent nonopioid analgesic agent: effect of pyridine and azetidine ring substitutions on nicotinic acetylcholine receptor binding affinity and analgesic activity in mice.

Analogs of A-98593 (1) and its enantiomer ABT-594 (2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties.

Accumulating preclinical and clinical evidence data suggests that compounds that selectively activate neuronal nicotinic acetylcholine receptor (nAChR) subtypes may have therapeutic utility for the treatment of several neurological disorders. In the present study, the in vitro pharmacological properties of the novel cholinergic channel modulator ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine], are described. In radioligand binding studies, ABT-089 was shown to display selectivity toward the high-affinity (-)-cytisine binding site present on the alpha4beta2 nAChR subtype (Ki = 16 nM) relative to the [125I]alpha-bungarotoxin binding site present on the alpha7 (Ki > or = 10,000 nM) and alpha1beta1deltagamma (Ki > 1000 nM) nAChR subtypes. In cation flux and channel current studies, ABT-089 displayed a more complex profile than (-)-nicotine having agonist, partial agonist and inhibitory activities depending on the nAChR subtype with which it interacts. ABT-089 differentially stimulated neurotransmitter release. The compound displayed a similar potency and efficacy to (-)-nicotine to facilitate ACh release (ABT-089, EC50 = 3 microM; (-)-nicotine, EC50 = 1 microM), but was markedly less potent and less efficacious than (-)-nicotine to stimulate dopamine release (ABT-089, EC50 = 1.1 microM; (-)-nicotine, EC50 = 0.04 microM). Additionally, ABT-089 was neuroprotective against the excitotoxic insults elicited by exposure to glutamate in both rat cortical cell cultures (EC50 = 10 +/- 3 microM) and differentiated human IMR32 cells (EC50 = 3 +/- 2 microM). The differential full agonist/partial agonist profile of ABT-089, as compared with (-)-nicotine and ABT-418, illustrates the complexity of nAChR activation and the potential to target responses at subclasses of the neuronal and peripheral receptors.

Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron: studies with F11 cells.

F11 cells are a dorsal root ganglion (DRG) cell line used to model the function of authentic type C, peptidergic, nociceptive neurons. The cellular events underlying the antinociceptive effects of (+/-)-epibatidine, a nicotinic acetylcholine receptor (nAChR) ligand that is 200-fold more potent than morphine, is unknown. The present study investigated the ability of cholinergic channel activators (ChCAs) to effect nAChR-gated ion flux and modulate the release of substance P (SP), a neuropeptide identified to play a critical role in nociception. The prototypical agonists (-)-nicotine and (-)-cytisine, the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium, the novel ChCA ABT-418 [(S)-3-methyl-5-(-1-methyl-2-pyrrolidinyl)isoxazole], and (+/-)-epibatidine evoked a concentration-dependent stimulation of rubidium (86Rb+) efflux with EC50 values of 14.2 +/- 1.6, 63.4 +/- 24, 3.8 +/- 2.0, 29.8 +/- 2.6, and 0.019 +/- 0.001 microM as well as maximal intrinsic activities of 100, 97, 69, 75, and 102%, respectively. The noncompetitive nAChR antagonist mecamylamine potently antagonized (-)-nicotine-evoked ion flux, whereas the competitive antagonist dihydro-beta-erythroidine was a weak antagonist, giving support to an alpha3beta4 nAChR subtype. In addition, concentrations of (+/-)-epibatidine, similar to those necessary to induce maximal 86Rb+ efflux, evoked spontaneous release of SP from these cells, which was blocked by mecamylamine. Furthermore, prolonged exposure to (+/-)-epibatidine desensitized the functional response of the nAChR in this cell line (IC50 = 12 +/- 9 nM). These findings in F11 cells provide a model to investigate the role nAChRs play in modulating DRG cell function, and may lead to insights into the role these receptors have in modulating nociceptive transmission.

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo.

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

Structure-activity studies on 2-methyl-3-(2(S)-pyrrolidinylmethoxy) pyridine (ABT-089): an orally bioavailable 3-pyridyl ether nicotinic acetylcholine receptor ligand with cognition-enhancing properties.

2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, ABT-089 (S-4), a member of the 3-pyridyl ether class of nicotinic acetylcholine receptor (nAChR) ligands, shows positive effects in rodent and primate models of cognitive enhancement and a rodent model of anxiolytic activity and possesses a reduced propensity to activate peripheral ganglionic type receptors. The profiles of S-4, its N-methyl analogue, and the corresponding enantiomers across several measures of cholinergic channel function in vitro and in vivo are presented, together with in vitro metabolism and in vivo bioavailability data. On the basis of its biological activities and favorable oral bioavailability, S-4 is an attractive candidate for further evaluation as a treatment for cognitive disorders.

A-85380 [3-(2(S)-azetidinylmethoxy) pyridine]: in vitro pharmacological properties of a novel, high affinity alpha 4 beta 2 nicotinic acetylcholine receptor ligand.

The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2(S)-azetidinylmethoxy)pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human alpha 4 beta 2 nAChR subtype (Ki = 0.05 + 0.01 nM) relative to the human alpha 7 (Ki = 148 +/- 13 nM) and the muscle alpha 1 beta 1 dg subtype expressed in Torpedo electroplax (Ki = 314 +/- 12 nM). The R-enantiomer of A-85380, A-159470, displays little enantioselectivity towards the alpha 4 beta 2 and alpha 1 beta 1 delta gamma subtypes but does not display 12-fold enantioselectivity towards the alpha 7 subtype (Ki = 1275 +/- 199 nM). (+)- and(-)-Epibatidine display similar potencies at the human human alpha 4 beta 2 (Ki = 0.04 +/- 0.02 nM and 0.07 +/- 0.02 nM, respectively), human alpha 7 (Ki = 16 +/- 2 nM and 22 +/- 3 nM, respectively) and muscle alpha 1 beta 1 delta gamma g (Ki = 2.5 +/- 0.9 nM and 5.7 +/- 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human alpha 4 beta 2 (EC50 = 0.7 +/- 0.1 microM) and ganglionic (EC50 = 0.8 +/- 0.09 microM) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 microM). Further, A-85380 can activate (EC50 = 8.9 +/- 1.9 microM) currents through channels formed by injection of the human alpha 7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the alpha 7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (-)-nicotine but less potent than (+/-)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 +/- 0.001 microM which is equipotent to (+/-)-epibatidine, and 20-fold more potent than (-)-nicotine (EC50 = 0.04 +/- 0.009 microM). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [125I] alpha-BgT sites than [3H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the alpha 4 beta 2 subtype relative to the alpha 7 and alpha 1 beta 1 delta g nAChRs than (+/-)-epibatidine.

In vitro neuroprotective properties of the novel cholinergic channel activator (ChCA), ABT-418.

Recent literature has shown that compounds interacting with neuronal nicotinic acetylcholine receptors (nAChRs) have the potential to be neuroprotective both in vitro and in vivo. ABT-418 is a novel ChCA that selectively stimulates discrete subtypes of the nAChRs and exhibits cognitive enhancing activity. In the present study, the neuroprotective effects of ABT-418 and (-)-nicotine, as measured by the release of lactate dehydrogenase (LDH) into the media, were investigated in a glutamate (Glu)-induced cytotoxicity assay using either primary rat cortical neurons or a human differentiated cell line, IMR 32. The neuroprotection elicited by ABT-418 and (-)-nicotine is both time and concentration dependent with an optimal concentration of 10 microM and an optimal pretreatment time of 2 h. ABT-418 remained neuroprotective and not cytotoxic to rat cortical cells following subacute exposure for 7 days. Protection appears to be mediated via an interaction with nAChRs, possibly the alpha 7 subtype, since the neuroprotection was prevented by alpha-bungaratoxin (alpha-Bgt) and methyllycaconitine (MLA), both selective alpha 7 antagonists. Removal of extracellular Ca2+ prevented the neuroprotective effects of ABT-418 and (-)-nicotine, consistent with the known ability of alpha 7 nAChRs to modulate calcium dynamics. These data support the idea that ABT-418 not only enhances cognition, but may possibly slow the progression of the neurodegenerative process.

Novel 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved.

Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha 4 beta 2 receptor.

(-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (nAChR) has been shown to bind with high affinity to the rodent and avian alpha 4 beta 2 nAChR subtype. This subtype may represent a primary molecular target for some of the beneficial central nervous system effects i.e., cognitive enhancement, anxiolysis, analgesia, neuroprotection, of (-)-nicotine and related ligands. However, a detailed study of the human alpha 4 beta 2 subunit combination has not yet been reported. In this study, we stably coexpressed the human neuronal alpha 4 and beta 2 nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory properties. [3H]Cytisine bound to stably transfected cells with high affinity (KD value, 0.2 +/- 0.04 nM) and with a Bmax value of 1359 +/- 91 fmol/mg protein. A good correlation (r = 0.98) was observed between binding affinities in transfected cells and in native neuronal preparations for a series of nAChR ligands. 86Rb+ efflux studies showed that stably transfected cells express functional ion channels that are sensitive to blockade by dihydro-beta-erythroidine. (+/-)-Epibatidine, (-)-nicotine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine stimulated 86Rb+ efflux with EC50 values of 0.02, 3.9, 2.5, 10, 44 and 38 microM, respectively. Treatment of transfected cells with (-)-nicotine for 7 days led to a significant increase in the density of [3H](-)-cytisine binding sites (EC50 = 0.56 microM) and a significant enhancement in the sensitivity of ACh. Specific binding or (-)-nicotine-evoked cation efflux was not detected in untransfected cells. Analysis of total cellular RNA from transfected, but not untransfected cells, showed the expected fragment sizes corresponding to the human alpha 4 and beta 2 subunit mRNA. These results demonstrate that stable expression of the human alpha 4 beta 2 nAChR subunit combination can give rise to functional ion channels that bind [3H](-)-cytisine with high affinity, exhibit homologous regulation and evoke agonist-induced cation flux with pharmacological properties consistent with native neuronal alpha 4 beta 2 nAChR.

Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor.

The alpha 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca(2+)-permeable homooligomeric ion channel sensitive to alpha-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human alpha 7 cDNA in a mammalian cell line, HEK-293 and examined its pharmacologic properties. [125I] alpha-Bungarotoxin bound to transfected cells with a Kd value of 0.7 nM and a Bmax value of 973 pmoL/mg protein. No specific binding was detected in untransfected cells. Specific binding could be displaced by unlabeled alpha-bungarotoxin (Ki = 0.5 nM) and an excellent correlation was observed between binding affinities of a series of nicotinic cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expression of alpha 7 receptors was detected by fluorescein isothiocyanate-conjugated alpha-bungarotoxin in transfected cells. Whole cell currents sensitive to blockade by alpha-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upon rapid application of (-)-nicotine or acetylcholine with EC50 values of 49 microM and 155 microM respectively. We conclude that the human alpha 7 subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a unique system for studying human alpha 7 nicotinic receptor function and regulation, and for examining ligand interactions.

Functional modulation of human "ganglionic-like" neuronal nicotinic acetylcholine receptors (nAChRs) by L-type calcium channel antagonists.

Recent studies suggest that the neuronal nicotinic acetylcholine receptors present on chromaffin cells contain a 1,4-dihydropyridine-sensitive site whose occupation blocks membrane depolarization (1). In the present study, several L-type Ca2+ channel blockers inhibited the activation of the nAChRs present in the human neuroblastoma cell line, IMR 32, in a dose-dependent manner with IC50 values ranging from 0.8-3 microM. In contrast, omega-Conotoxin GVIA and omega-Agatoxin IVA had no effect up to 100 microM. Furthermore, the inorganic channel blocker, cadmium, had no effect either alone or on the modulatory role of the L-type antagonists, suggesting that the effects of these agents on nAChRs are not mediated via an interaction with calcium channels but possibly via a direct interaction with the nAChR ionophore.