Introduction

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(±)-Epibatidine is a potent nAChR ligand (Qian et al., 1993; Badio and Daly, 1994; Sullivan et al., 1994) isolated from the frog skin of Epipedobates tricolor (Spande et al., 1992) which has analgesic activity 200 times more potent than morphine. These analgesic actions are insensitive to opioid receptor antagonists and are blocked by selective nAChR antagonists, e.g., mecamylamine, which indicates a role for nAChR mechanisms in nociceptive signaling (Jinno et al., 1994; Damaj et al., 1993; Puttfarcken et al., 1997). (±)-Epibatidine, although a potent ligand for the major nAChR subtype in the brain, the 42 (Flores et al., 1992), also has potent agonist activity at sympathetic ganglionic-like (3 containing) and neuromuscular (11) nAChR subtypes (Sullivan et al., 1994; Briggs et al., 1995). Activity at these peripheral nAChRs apparently is responsible for the marked hypertension and muscular paralysis observed with the in vivo use of (±)-epibatidine, which results in a very limited therapeutic index that has precluded its clinical usefulness as an analgesic (Sullivan et al., 1994). The identification and development of novel nAChR ligands with antinociceptive efficacy similar to (±)-epibatidine and a reduced side-effect profile based on selective interactions with mammalian nAChR subtypes offers the potential for developing a novel class of analgesic agents distinct from the opioids.

In situ hybridization studies and binding studies with [³H]()-nicotine and [³H]()-cytisine have identified an abundance of nAChRs along processing centers of the pain pathway (Khan et al., 1994; Zoli et al., 1995). Tissues that contain nAChRs include the DRG, the dorsal horn of the spinal cord, brainstem nuclei (e.g., raphe nucleus and locus coeruleus), thalamic relay stations, limbic association areas (e.g., amygdala) and the cerebral cortex. Moreover, the diversity of neuronal nAChR subunits expressed within these critical processing regions suggests that multiple nAChRs may exist to modulate nociceptive transmission, not unlike the diversity that exists for the opioid receptor system (Cherney, 1996). Some initial work examining this issue suggests that a sensory ganglion-like (3 containing) nAChR may be involved at some levels of nociceptive transmission (Flores et al., 1996; Puttfarcken et al., 1997), whereas the -Btx-sensitive nAChRs (e.g., 7 in the central nervous system and 11 at the neuromuscular junction) are less likely to be involved (Khan et al., 1994; Rao et al., 1996). Thus, specific milestones to advancing compounds for clinical evaluation requires the identification of ligands that retain activity at nAChRs involved with nociceptive processing and have diminished interactions with sympathetic ganglion and neuromuscular nAChRs.

This article describes the in vitro pharmacological characterization of ABT-594 (fig. 1), a novel 3-pyridyl ether nAChR agonist (Holladay et al., 1998) that was identified in classical animal models of acute, persistent and neuropathic pain (Bannon et al., 1998a, b; Decker et al., in press, 1998) and, as now characterized pharmacologically at the neurochemical level, demonstrates superior selectivity for neuronal nAChRs.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structures of ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine], A-98593 [(S)-5-(2-azetidinylmethoxy)-2-chloropyridine], (±)-epibatidine and ()-nicotine.

	Materials and Methods

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ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine; fig. 1], and its S-enantiomer, A-98593, were synthesized at Abbott Laboratories as described by Holladay et al. (1998). (±)-Epibatidine was purchased from Research Biochemicals International (Natick, MA). ()-Nicotine (hydrogen tartrate salt), morphine sulfate, atropine, mecamylamine hydrochloride, acetylcholine chloride, collagenase Type 1A and gentamicin were obtained from Sigma Chemical Co. (St. Louis, MO). All radioligands were obtained from NEN Life Sciences (Boston, MA). -Btx was obtained from Molecular Probes (Eugene, OR). Tricaine (3-aminobenzoic acid ethyl ester methanesulfonate; Finquel) was obtained from Argent Chemical Laboratories (Fisheries Chemical Division, Redmond, WA).

The human neuroblastoma cell line, IMR-32, was obtained from ATCC (Rockville, MD) and maintained in a log phase of growth as described by Lukas (1993). The DRG hybrid cell line F11 was a generous gift from Dr. Richard J. Miller (University of Chicago) and was maintained as described (Puttfarcken et al., 1997). Cell lines stably expressing the human 42 and 7 nAChR subtypes (designated K177 and K28, respectively) were maintained as described previously (Gopalakrishnan et al., 1995, 1996).

All experiments involving animals were conducted according to protocols approved by the Abbott Institutional Animal Care and Use Committee.

Receptor Binding Assays

Membrane preparations. Frozen rat cerebral cortical membrane pellets (ABS, Wilmington, DE) were thawed, washed and resuspended in 30 volumes of assay buffer (composition, mM: NaCl, 120; KCl, 5; CaCl₂, 2; MgCl₂, 2 and Tris-HCl, 50; pH 7.2, at 4°C). Confluent K177 cells stably expressing the human 42 subunit were rinsed with ice-cold Tris binding buffer (composition, mM: Tris-HCl, 50; NaCl, 120; KCl, 5; MgCl₂, 1 and CaCl₂, 2.5; pH 7.4 at 4°C), mechanically disaggregated and homogenized with a polytron for 10 s. The homogenate was centrifuged at 45,000 × g for 20 min at 4°C and the pellet resuspended in ice-cold buffer at a concentration of 40 to 50 µg protein. Confluent K28 cells stably expressing the human 7 subunit were rinsed with ice-cold HEPES binding buffer (composition, mM: NaCl, 118: KCl, 4.8: CaCl₂, 2.5; MgSO₄, 1.2; HEPES, 20; pH 7.5), mechanically disaggregated and homogenized with a polytron for 10 s. The homogenate was centrifuged at 45,000 × g for 20 min at 4°C and the pellet resuspended in ice-cold buffer at a concentration of 40 to 50 µg protein.

[³H] ()-cytisine binding. Binding conditions were as described previously (Anderson et al., 1995; Gopalakrishnan et al., 1996). Samples containing 20 to 200 µg of protein, 0.7 nM [³H]()-cytisine (30 Ci/mmol) and the indicated concentrations of test compound were incubated in a final volume of 500 µl for 75 min at 4°C in triplicate. Nonspecific binding was determined in the presence of 10 µM ()-nicotine.

[¹²⁵I]-Btx binding. [¹²⁵I]-Btx binding was determined with membranes prepared from rat brain and K28 cells as described by Gopalakrishnan et al. (1995) and from Torpedo californica electroplax. A solid-phase binding assay with a 96-well microtiter plate was used to measure the binding of [¹²⁵I]-Btx (106 Ci/mmol) to T. californica electroplax membranes (Wilson et al., 1988).

Additional receptor binding studies. The selectivity of ABT-594 as an nAChR ligand was evaluated in a receptor binding selectivity screen (see table 2) by use of standard receptor binding protocols (Cerep, Celle l'Evescault, France). Additional assays to establish K_i values at adrenoreceptors of the alpha-1B, alpha-2B and alpha-2C subtypes were conducted as described by Hancock et al. (1995).

Functional Assays

⁸⁶Rb⁺ efflux. Experimental cells were seeded at a density of 500,000 cells/ml into a 24-well tissue culture dish. Plated cells were allowed to proliferate for at least 48 h before loading with 8 µCi/ml of ⁸⁶Rb⁺ (35 Ci/mmol) overnight at 37°C. The ⁸⁶Rb⁺ efflux assays were performed as described by Sullivan et al. (1994), except serum-free Dulbecco's Modified Eagle's Medium (Gibco BRL, Gaithersburg, MD) was used during the cell rinsing and agonist-induced efflux steps. The following cell lines were used with the expressed nAChR given in parentheses: K177 (42); IMR 32 (34); F11 (34).

Channel currents. The preparation of Xenopus laevis oocytes, injection with receptor RNA and measurement of 7 nAChR responses using two-electrode voltage-clamp was carried out as described previously (Briggs et al., 1995). Sections of one ovary (generally three to four lobes) were removed surgically under tricaine anesthesia (0.28% in deionized water) and oocytes were prepared after incubation for 1 to 2 h at room temperature in collagenase (Sigma type 1A, 2 mg/ml) in low-Ca⁺⁺ Barth's solution (pH 7.55) containing , mM: NaCl, 87.5; KCl, 2.5; MgCl₂, 1; Na-HEPES buffer, 10; and 100 µg/ml gentamicin. Oocytes were maintained at 17-18°C in normal Barth's solution (containing, mM; NaCl, 90; KCl, 1; NaNO₃, 0.66; CaCl₂, 0.74; MgCl₂, 0.82; NaHCO₃, 2.4; sodium pyruvate, 2.5 Na-HEPES buffer, 10; pH 7.55) containing 100 µg/ml gentamicin. Oocytes were injected with 40 to 50 nl of human 7 nAChR RNA within 24 h of their preparation and were used 2 to 7 days after injection. Responses were measured with two-electrode voltage-clamp (60 mV) in Barth's solution containing 10 mM BaCl₂ and lacking CaCl₂ and MgCl₂ (Ba⁺⁺-Barth's) to prevent secondary activation of Ca⁺⁺-dependent Cl current. Atropine (2 µM) was included to block activation of endogenous muscarinic ACh receptors.

Fluorescence imaging. Agonist-induced Ca⁺⁺ dynamics were assessed in K177 (42) cells. The cell permeant acetoxymethyl (AM) ester form of the intracellular Ca⁺⁺ probe, Fluo-3 (Molecular Probes, Eugene, OR; Minta et al., 1989) was dissolved in anhydrous dimethyl sulfoxide with 10% pluronic acid and diluted in growth media to a final concentration of 4 mM. The dye was placed on the cells for 1 h at 37°C and unincorporated dye was removed by washing with the assay buffer [HEPES-Salts buffer (pH 7.5) composition, mM; HEPES, 20; NaCl, 120; KCl, 5; MgCl_2,1; glucose, 5; and CaCl_2, 5]. After agonist addition, Ca⁺⁺ dynamics were recorded in a FLIPR (Molecular Devices, Sunnyvale, CA) equipped with an Argon laser (wavelength, 480 nm) and a CCD camera on a second time scale. Independent measurements of 0.1 mM nicotine (100%) and unloaded cells (0%) were performed on each plate of cells with an average fluorescence range of 20,000 units.

Neurotransmitter release. Release of the nociceptive transmitter, CGRP, in response to capsaicin was measured as CGRP-LI from dorsal spinal cord slices by a modification of the methods of Garry et al. (1994) and Chen et al., (1996). Capsaicin was used at a concentration of 1 µM to restrict responses to the C-type sensory cells of the DRG (Gamse et al., 1979). Male rats (180-200 g, Harlan Sprague Dawley, Indianapolis, IN) were decapitated, and the spinal cords were removed by hydraulic extrusion with sterile saline. The lumbar enlargement was isolated, and the dorsal half of the lumbar portion of the spinal cord was placed on a McIlwain tissue chopper (Brinkmann, Westbury, NY). Before superfusion, tissue slices (250 × 250 µm) were placed in oxygenated buffer of the following composition, mM: HEPES-NaOH, 15; NaCl, 137; KCl, 4.7; MgS0₄,1; ascorbic acid, 0.1; CaCl₂, 2.5; NaH₂PO₄, 0.125; glucose, 10; and 0.1% bovine serum albumin; 20 µM bacitracin; 1 µM phosphoramidin and 1 µM thiorphan, (pH 7.4) alone or containing ABT-594, and incubated for 30 min at 37°C. After pretreatment, the tissue was placed in release chambers (Brandel Superfusion System, Gaithersburg, MD) and perfused with buffer alone for 30 min to stabilize release. Basal release (fractions 1 and 2) was established by perfusing the tissue with buffer alone for 6 min. To evoke peptide release, spinal cord tissue was perfused with 1 µM capsaicin for 6 min. Subsequently, the tissue was perfused with oxygenated buffer for 12 min to demonstrate a return to basal release. Fractions (perfusate) were collected every 3 min into tubes containing 100 µM 2-[N-morpholino] ethanesulfonic acid to maintain the pH of the samples. At the end of each experiment, the tissue was removed from each chamber and placed in a test tube containing 2 ml of 0.1 N HCl. Lysates were sonicated, boiled for 20 min, centrifuged at 38,000 × g for 20 min and adjusted to pH 7 with 1 M Tris.

Data Analysis

In competition experiments, the compound concentration producing 50% inhibition (IC₅₀) of radioligand binding and the Hill coefficient (n_H) were determined from plots of log (B₀  B)/B versus log (concentration of drug), where B₀ and B are specific binding in the absence and presence of competitor, respectively, by a four-parameter logistics program in RS/1 (Bolt, Beranek and Newman Inc., Cambridge, MA). Inhibition constant (K_i) values were determined with the Cheng-Prusoff equation (Cheng and Prusoff, 1972). EC₅₀ values for the ⁸⁶Rb⁺ efflux assays were determined by nonlinear least-squares regression analysis and statistical significance by one-site analysis of variance or paired t test with Graphpad Prism Software (San Diego, CA).

Electrophysiological responses were quantified by measuring the peak current amplitude relative to the base-line holding current determined immediately preceding agonist application. Agonist dose-response curve parameters were determined by nonlinear curve fitting of the Hill equation (Sigmaplot software, Jandel Scientific, San Rafael, CA).

Levels of CGRP-LI were determined by comparing the percent radioactivity bound in unknown samples to a standard curve with an 11-point nonlinear least-squares regression program (Graphpad Prism Software, San Diego, CA). To determine the effects of ABT-594 on capsaicin-evoked release, Student's t test was used to compare the quantity of CGRP-LI release evoked by capsaicin alone to that measured in the presence of ABT-594. Statistical significance was defined at P < .01 level. Mean values are shown with error bars indicating ± S.E.M.

	Results

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Receptor Binding

ABT-594 displaced specific [³H]()-cytisine binding to the human 42 nAChR stably expressed in the K177 cell line (Gopalakrishnan et al., 1996) with a K_i value of 55 ± 5 pM and a Hill coefficient of 0.99 ± 0.04 (table 1). The S-enantiomer of ABT-594, A-98593, had a K_i value of 34 ± 5 pM at the human 42 nAChR. Corresponding K_i values for ABT-594 and A-98593 at the 42 nAChR in rat brain membranes were 37 ± 3 pM and 39 ± 3 pM, respectively. In K177 cells, ()-nicotine had a K_i value of 1.05 ± 0.09 nM and in rat brain, a K_i value of 1.05 ± 0.02 nM. The analgesic opioid, morphine, had negligible activity at the 42 nAChR (K_i > 10,000 nM; data not shown).

View this table: [in this window] [in a new window]   TABLE 1 nAChR binding properties of ABT-594, A-98593, (±)-epibatidine and ()-nicotine Receptor binding studies were performed as described under "Materials and Methods." Results are the means ± S.E.M. of three to five separate determinations. The Hill coefficients were not significantly different from unity in all cases (data not shown).

At the rat brain -Btx-sensitive nAChR (7), ABT-594 had a K_i value of 13,800 ± 390 nM, an approximately 250,000-fold lower affinity than that observed at the human 42 nAChR. At the human 7 nAChR subtype expressed in K28 cells, ABT-594 had a K_i value of 1560 ± 170 nM, 28,000-fold less than its activity at the human 42 nAChR (table 1). At the 11 nAChR, ABT-594 had a K_i value of 10,000 ± 500 nM, which shows a selectivity of greater than 180,000-fold. A-98593, the S-enantiomer of ABT-594, also displayed weak affinity for the rat brain -Btx-sensitive (7) and 11 nAChRs (K_i values = 4620 and 3420 nM, respectively) but unlike 42 binding, showed stereoselectivity being approximately 3-fold more potent than ABT-594. At the human 7, A-98593 had activity almost identical with ABT-594 (K_i = 1780 nM), which again demonstrates a lack of stereoselectivity. ()-Nicotine displaced [¹²⁵I]-Btx binding from rat brain and human 7 with K_i values of 4000 ± 890 nM and 7130 ± 780 nM, respectively. ()-Nicotine was also a weak inhibitor (K_i > 1000 nM) of the binding of [¹²⁵I]-Btx to the 11 nAChR (table 1). In all the binding assays above, morphine was essentially inactive with K_i values of greater than 10,000 nM (data not shown). In contrast, (±)-epibatidine displayed high affinity (K_i = 2.7 nM) for the 11 nAChR subtype (Sullivan et al., 1994).

ABT-594 was examined in more than 70 other receptor, enzyme and uptake binding assays (table 2) and demonstrated weak relative affinities (K_i > 1000 nM) for opioid, muscarinic, purinergic P1 and P2, glycine, 5-HT₃ and benzodiazepine receptors as well as other members of the ligand-gated ion channel and G-protein-coupled superfamilies including GABA_A, GABA_B, N-methyl-D-aspartate, quisqualate, kainate, L-, N- and T-calcium, chloride and potassium channels, serotonin, dopamine, adrenergic, atrial naturetic peptide, angiotensin, vasopressin, cholecystokinin, somatostatin, glucagon, endothelin, neurokinins 1-3, phencyclidine, neuropeptide Y, vasoactive intestinal peptide; choline, norepinephrine, serotonin, adenosine, GABA and dopamine uptake sites; and did not inhibit the activity of monoamine oxidase (A and B), phosphodiesterases I-V, or cyclooxygenases (COX-1 and COX-2). Comparatively weak affinities were detected at the adrenergic subtypes tested and these were reexamined to derive K_i values as noted: alpha-2C (human cloned), 342 nM; alpha-2B (rat neonatal lung), 597 nM; and alpha-1B (hamster cloned), 890 nM.

Functional Studies

Ion flux. Human 42. ABT-594 activated ⁸⁶Rb⁺ efflux through the human 42 nAChR in K177 cells with an EC₅₀ of 140 ± 16 nM and an intrinsic activity (IA) compared with ()-nicotine of 130% (fig. 2A). Comparable data for ()-nicotine were: EC₅₀ = 4.2 ± 1.1 µM; IA = 100%; ABT-594 was thus nearly 30-fold more potent and had 30% more IA than ()-nicotine (table 3). ABT-594 was eight times less potent than (±)-epibatidine (EC₅₀ = 17 ± 2 nM; IA = 156%). A-98593, the S-enantiomer of ABT-594, was also a functional agonist at the human 42 nAChR being greater than 2-fold more potent than ABT-594 (EC₅₀ = 60 ± 15 nM; IA = 157%) with an IA similar to (±)-epibatidine. The peak responses for ABT-594, A-98593, (±)-epibatidine and ()-nicotine were attenuated by the noncompetitive nAChR antagonist, mecamylamine (100 µM) (fig. 2B).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effect of ABT-594, (±)-epibatidine, A-98593 and ()-nicotine on the activation of cation (86Rb+) efflux from K177 cells expressing the human 42 subunit combination (A) and attenuation of peak responses by 100 µM mecamylamine (B). Cells that had been loaded with 86Rb+ were exposed for 5 min to the concentrations of the compounds indicated. Values are the mean ± S.E.M., n = 3-5.

View this table: [in this window] [in a new window]   TABLE 3 Summary of functional attributes for ABT-594, A-98593, (±)-epibatidine and ()-nicotine Functional assays were performed as described under "Materials and Methods." Data from representative figures as indicated in text. Results are means ± S.E.M. of three to five separate determinations.

sympathetic ganglion-3-containing

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of ABT-594, (±)-epibatidine, A-98593 and ()-nicotine to activate cation (86Rb+) efflux from ganglionic cell lines expressing the human 3-containing combination in the IMR-32 cells (A) or the rat 3-containing subunit combination in the F11 cells (B). Cells that had been loaded with 86Rb+ were exposed for 5 min to the concentrations of compound indicated. Values are the mean ± S.E.M., n = 3-5.

sensory ganglion-3-containing

Channel currents. Human 7 (oocytes). ABT-594 had an EC₅₀ of 56 ± 20 µM with a Hill coefficient (n_H) relative to ACh of 0.77 ± 0.18 and an IA of 83 ± 7% at the human 7 nAChR (fig. 4; table 3). A-98593 was 2.7-fold more potent than ABT-594 (EC₅₀ = 21 ± 7 µM). Comparable literature data on (±)-epibatidine and ()-nicotine (Briggs et al., 1995), respectively, were 1.30 ± 0.11 µM and 83 ± 10 µM. ABT-594 was approximately 1.5-fold more potent than ()-nicotine and 43-fold less potent than (±)-epibatidine at this nAChR subtype.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Activation of human 7 nAChR in oocytes by ABT-594 and A-98593 compared with both (±)-epibatidine and ()-nicotine. Example responses from one oocyte are shown for ABT-594 in the inset. The smaller, slower response is to 10 µM ABT-594 and the faster spike-like response is to 100 µM ABT-594. Calibration lines indicate 100 nA and 200 ms. Concentration-response data points are shown as mean ± S.E.M. with the curves representing the Hill equations fit to these data. For each compound, the 7 nAChR response amplitudes were normalized to the response to 10 mM ACh (a maximal response) in the same oocyte to account for variability in receptor expression. Data are from 4 oocytes for ABT-594 (solid circles), 6 oocytes for A-98593 (open circles), 18 oocytes for (±)-epibatidine (inverse triangle) and 4 oocytes for ()-nicotine (open triangles).

Ca⁺⁺ dynamics. Human 42. Plasma levels of ABT-594 that elicit maximal antinociceptive behavior correspond to plasma concentrations of approximately 25 nM (Bannon et al., 1998b). Free intracellular Ca⁺⁺ levels in K177 cells rose after exposure to both ABT-594 and (±)-epibatidine (final concentration = 25 nM; fig. 5) by 69 and 92%, respectively, relative to that induced by 100 µM nicotine, the concentration that elicits a peak response in the K177 cell line (fig. 2).

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of ABT-594 (25 nM) and (±)-epibatidine (25 nM) in modulating intracellular Ca++ dynamics. K177 cells were loaded with Fluo-3 for 30 to 60 min before exposure to the behaviorally relevant concentrations of the compound indicated. Values are the mean ± S.E.M., n = 3 and normalized to the peak response of ()-nicotine (100 µM) in this cell line (maximum response of ()-nicotine represented by the dashed line).

CGRP release studies. In initial studies, the effects of 1 µM capsaicin on CGRP-LI release from rat dorsal horn spinal cord slices in 15 fractions collected every 3 min gave low base-line levels in fractions 1 to 2 and 9 to 15 (data not shown). Thus only fractions 3 to 8 were used to assess evoked CGRP-LI release. Capsaicin (1 µM) produced a 1.8-fold increase in CGRP-LI (0.35 ± 0.03%) over that observed under basal conditions (0.21 ± 0.04%) (fig. 6A). Pretreatment with ABT-594 (1-30 µM) attenuated capsaicin-evoked CGRP-LI release (fig. 6B). ABT-594 (30 µM) inhibited the CGRP-LI release evoked by 1 µM capsaicin by approximately 40%. The effects of 30 µM ABT-594 were attenuated by pretreatment with 100 µM mecamylamine (fig. 6B).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   ABT-594 inhibition of capsaicin-evoked CGRP-LI release from C-fibers in rat spinal cord slices. (A) Before superfusion, slices of the dorsal half of the lumbar enlargement of the spinal cord were pretreated with buffer alone, ABT-594 (1-30 µM) or 100 µM mecamylamine at 37°C for 30 min. CGRP-LI activity was assayed in both perfusates and lysates from fraction 3 through 8, according to the procedures stated under "Materials and Methods." The tissue was then superfused with buffer alone (open square), 1 µM capsaicin in the absence (closed square) and presence of 30 µM ABT-594 (asterisk) or 30 µM ABT-594 + 100 µM mecamylamine (open circle) (n = 5). Fractions were collected every 3 min into tubes containing 1 M 2-[N-morpholino] ethanesulfonic acid. (B) Peptide release expressed as the sum of three fractions (two during and one immediately following treatment). Pretreatment with ABT-594 (30 µM) significantly attenuated 1 µM capsaicin-evoked CGRP-LI release (** P < .01). CGRP-LI levels were significantly higher in the presence of capsaicin, 100 µM mecamylamine and 30 µM ABT-594 than those measured in the presence of capsaicin and 30 µM ABT-594 ( P < .05)

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

In the present paper, the in vitro pharmacological properties of ABT-594, a novel, nAChR agonist with broad spectrum antinociceptive activity (Bannon et al., 1998a, b; Holladay et al., 1998) are described and compared with those of the S-enantiomer, A-98953, (±)-epibatidine and ()-nicotine in standard receptor binding assays, functional assays (involving either ion flux, channel currents or Ca⁺⁺ dynamics) and CGRP-LI release from rat dorsal spinal cord.

ABT-594 is a potent and selective ligand for neuronal nAChRs that exhibits the potent analgesic activity seen with (±)-epibatidine (Bannon et al., 1998b) whereas substantially reducing the potent sympathetic ganglion and neuromuscular side effects that contributed to the limited therapeutic index of (±)-epibatidine. Thus, in contrast to (±)-epibatidine, ABT-594 is 28,000 to 250,000 times less active at the 7 and 11 nAChRs than the human 42 nAChR (table 1). (±)-Epibatidine shows only a 38-fold selectivity for the human 42 nAChR. This poor selectivity profile for (±)-epibatidine is thought to account for its side-effect liabilities (Sullivan and Bannon, 1996). In addition, ABT-594 has negligible activity in binding assays for more than 70 other receptors, enzymes and transmitter uptake sites (table 2). Like (±)-epibatidine (Badio and Daly, 1994), ABT-594 (R-isomer) and the corresponding S-isomer, A-98593 showed no stereoselectivity for binding at either the 7 or 42 nAChR. However, A-98593 was approximately three times more potent than ABT-594 at the 11 nAChRs. When comparing the radioligand binding data obtained in this study with those recently reported for A-85380 (K_i = 0.04 nM; Sullivan et al., 1996), ABT-594 exhibits affinity similar to the related azetadine analog, A-85380, at the 42 subtype. However, at either the -Btx-sensitive binding site present on the human 7 subtype in K28 cells or 1 in Torpedo electroplax, ABT-594 is significantly less potent (K_i = >10,000 nM) than A-85380 (K_i = 148 nM and 314 nM, respectively, Sullivan et al., 1996).

Functionally, A-98593 was approximately 2- to 4-fold more potent than ABT-594 in the functional ion flux assays examined that involved the activation of 7, 3- containing and 42 nAChRs. In addition, A-98593 had 20 to 40% greater IA than ABT-594. A-98593 had an IA profile similar to (±)-epibatidine across all four receptors but was 3.5- to 28-fold less active (table 3). These in vitro findings thus may account for the increased toxicities seen with A-98593 as compared with ABT-594 (Holladay et al., 1998) and also suggest that increased IA at sympathetic 3-containing ganglionic nAChRs contribute to the side effects seen with (±)-epibatidine, e.g., greater pressor responses in anesthetized dogs (Sullivan and Bannon, 1996; Holladay et al., 1998), albeit to a much greater degree than for A-98593. Previous studies with the selective 42 nAChR ligands, ABT-418 (Arneric et al., 1995), ABT-089 (Sullivan et al., 1997), ()-nicotine and (±)-epibatidine suggested a potential correlation between potency and/or efficacy at the putative "34" nAChR in the IMR-32 cell line with cardiovascular side-effect potential. Compounds more potent than ()-nicotine on IMR-32 cation flux, like (±)-epibatidine (Sullivan and Bannon, 1996), displayed large cardiovascular pressor effects in dogs, whereas compounds less potent than ()-nicotine, like ABT-418 or ABT-089 (Arneric et al., 1997), exhibited reduced or negligible pressor changes, respectively. Together with the in vitro findings, these data suggest that ABT-594 should exhibit diminished adverse cardiovascular effects via activation of the autonomic nervous system relative to the more potent (±)-epibatidine. Initial experiments in anesthetized dogs, where ABT-594 was given i.v. (bolus), showed significantly reduced (>80%) effects on both diastolic blood pressure and heart rate compared with either A-98593 or (±)-epibatidine (Holladay et al., 1998).

Compared with ()-nicotine, ABT-594, A-98593 and (±)-epibatidine all exhibit greater IA at the 42 and the IMR 32, sympathetic ganglionic-like (3-containing) nAChRs. At the F11, sensory ganglion (3-containing) nAChRs, and the 7 nAChRs expressed in oocytes, A-98593 and (±)-epibatidine have efficacy identical with ()-nicotine, whereas ABT-594 has lower IA. This suggests that the nAChRs in F11 cells are less sensitive to the agonist properties of nAChR ligands than those present in the IMR 32 cell line, which, as already discussed, apparently mediates the sympathetic ganglion/cardiovascular effects of (±)-epibatidine.

Opioids affect calcium dynamics (Smart and Lambert, 1996), and the antinociceptive activity of ()-nicotine can be modulated further by compounds that affect intracellular calcium levels (Damaj et al., 1993). Previous studies (Bannon et al., 1995) also have shown that the analgesic effects of (±)-epibatidine could be potentiated by Bay K 8644, a Ca⁺⁺ channel agonist, which suggests that raising intracellular Ca⁺⁺ can further enhance the analgesic actions of nAChR ligands. Because nAChR interactions also can modulate calcium dynamics (Role and Berg, 1996), experiments were performed to establish whether behaviorally relevant concentrations of ABT-594 would alter intracellular levels of Ca⁺⁺. Both ABT-594 and (±)-epibatidine, at 25 nM, a concentration of ABT-594 corresponding to plasma levels producing antinociception (Bannon et al., 1998b), induced an elevation of intracellular free Ca⁺⁺ in K177 cells that was attenuated by the nAChR antagonist, mecamylamine (data not shown). These data suggest that enhancement of intracellular Ca⁺⁺ dynamics may be an important link in mediating the antinociceptive effects of compounds like ABT-594 and (±)-epibatidine.

The antinociceptive actions of ABT-594 were explored further at the neurochemical level by examining the effects of the compound on capsaicin-induced release of the putative nociceptive transmitter CGRP from dorsal horn nerve terminals in vitro. Capsaicin, an agent known to cause heterologous desensitization of nociceptive fibers to noxious stimuli as well as the depletion of releasable pools of substance P and CGRP, was used at 1 µM to restrict responses to the C-type sensory cells (Gamse et al., 1979). ABT-594 inhibited the capsaicin-evoked CGRP-LI release, an effect mediated via nAChRs, because it was blocked by mecamylamine. These findings with ABT-594 are similar to findings with (±)-epibatidine, where pretreatment with 1 µM (±)-epibatidine inhibited capsaicin-evoked CGRP release from rat spinal cord slices (P. Puttfarcken, unpublished observation). ABT-594 (30 µM) also has been found to attenuate capsaicin-evoked substance P release (Bannon et al., 1998a), another nociceptive transmitter involved in the cellular phenotype of inflammatory pain (Neumann et al., 1996). These data are complementary to those showing that capsaicin pretreatment can significantly inhibit [³H]epibatidine binding to laminae I and II, areas of input from primary afferents, of the dorsal horn (Khan et al., 1997). Taken together, these data suggest that the presence of nAChRs on C-fiber afferents may modulate nociceptive input and that modulation of nociceptive neurotransmitter release may be, in part, one of the mechanisms underlying the antinociceptive properties of ABT-594. However the cellular mechanism responsible for these inhibitory actions remains unclear. Although both ABT-594 and (±)-epibatidine exhibited an affinity similar to the 42 subtype, these similarities were not observed in release assays. This discrepancy may be caused by the state of the nAChR measured in each assay. Whereas binding assays reflect the affinity of a ligand for the desensitized state of the nAChR, functional studies measure the open state of the receptor. Alternatively, the activation of the 42 subtype may not be responsible for the inhibition of capsaicin-evoked CGRP release. Indeed, both ABT-594 and (±)-epibatidine bind to other nAChRs, and the specific subtype responsible for modulating nociceptive transmission remains unknown.

Despite the extensive knowledge that has developed during the past decade regarding the various nAChR subtypes (Sargent, 1993; Arneric et al., 1995; Holladay et al., 1997), the nAChR subtype(s) responsible for analgesia remains unclear. The fact that nociceptive transmission can be modulated by both spinal and supraspinal mechanisms (Dray et al., 1994), and may thus involve the different nAChRs present at these levels, complicates the interpretation of data at the molecular level. The recent finding (Flores et al., 1996) that the major nAChR subtype in the trigeminal nerve appears to be the 34, suggests that 3-containing nAChRs may be important. However, based on the precedent of multiple opioid receptors regulating nociception (Cherney, 1996), it is conceivable that an analogously complex receptor pharmacology exists for neuronal nAChRs.

Based on the findings with ABT-594, it may be speculated that the 42 nAChR is involved in supraspinal analgesia. However, other compounds with similar preference for the 42 nAChR exhibit weak or no analgesic activity (M. Decker, unpublished observations). Furthermore, the reduced agonist activity of ABT-594 in the F11 functional assay may argue for discrete differences in the sensory ganglionic nAChR subtypes in this DRG cell line and the IMR 32 sympathetic ganglionic subtype. The 7 nAChR appears unlikely to mediate analgesia, because 7 selective ligands, like GTS-21, are weak analgesics (M. Decker, unpublished observations) and the 7 selective antagonist, methyllycaconitine, is ineffective in altering the antinociceptive effects of various nicotinic ligands (Rao et al., 1996). In addition, ABT-594 exhibited 400-fold greater potency in activating the neuronal 42 nAChR than the 7 subtype. It remains possible, given the recent identification of new functionally active nAChR subunit combinations with 3 (Forsayeth and Kobrin, 1997) and 6 (Gerzanich et al., 1997) that as yet unknown forms of the nAChR may subserve the effects of compounds like ABT-594 and (±)-epibatidine as potential analgesics.

In conclusion, the data presented provide evidence that ABT-594 has enhanced functional selectivity for neuronal, -Btx-insensitive nAChRs rather than the neuromuscular and sympathetic ganglionic subtypes. ABT-594 also can interact with nAChRs to modulate nociceptive neurotransmitter release from C-fibers at the level of the dorsal horn, a center highly involved in pain processing. The observed neuronal selectivity of ABT-594 may contribute to the substantial separation between the antinociceptive efficacy and the reduced cardiovascular side effects of the compound thereby supporting the increased safety index as compared to (±)-epibatidine (Decker et al., in press, 1998). ABT-594 offers the potential of being a novel and safe therapeutic alternative to the limited therapies presently existing for pain management.