Introduction

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A substantial body of literature has demonstrated that muscarinic cholinergic agonists as well as cholinesterase inhibitors produce antinociception (e.g., see review by Hartvig et al., 1989). Among early studies, the nonselective muscarinic agonists tremorine (Chen, 1958), arecoline (Herz, 1962), oxotremorine (George et al., 1962) and RS86 (Loew and Taeschler, 1964) were demonstrated to have antinociceptive effects in a variety of species and in a variety of antinociceptive tests. Subsequently, muscarinic agonists were demonstrated to be as efficacious as opioids in the mouse tail-flick procedure (e.g., Harris et al., 1969; Howes et al., 1969), a test in which opioid mixed agonist/antagonists such as nalorphine, pentazocine and cyclorphan were ineffective. In addition, it has been reported that physostigmine and tacrine produced analgesia in humans and potentiated the analgesic effects of morphine (Flodmark and Wramner, 1945; Stone et al., 1961; Peterson et al., 1986). It seems that clinical studies have not been conducted with direct-acting muscarinic agonists, perhaps because the currently available muscarinic agonists produce prominent parasympathomimetic effects. Although the currently available clinical data are limited, they are consistent with the idea that modulation of muscarinic receptor neurotransmission might produce clinically useful analgesia.

The parasympathomimetic effects, including bradycardia, hypotension, diarrhea, urination, salivation and lacrimation, produced by nonselective muscarinic agonists are mediated by multiple subtypes of muscarinic receptors. Subtypes of muscarinic receptors have been distinguished pharmacologically based on the affinity of antagonists and termed M₁, M₂, M₃ and M₄ (Hammer et al., 1980; Michel and Whiting, 1987; Lambrecht et al., 1989; Waelbroeck et al., 1990). Further, five gene products, termed m₁ to m₅, have been cloned (Peralta et al., 1987; Bonner et al., 1987; Buckley et al., 1988). The genetic m₁ to m₄ correspond to the pharmacologically defined M₁ to M₄ receptors; a pharmacological M₅ receptor has yet to be defined. The five genetic receptor subtypes are differentially distributed in brain (e.g., Bonner et al., 1987; Brann et al., 1988). In the periphery, pharmacological and genetic M₁ receptors are located in sympathetic ganglia, gastrointestinal tissue, rabbit vas deferens and salivary glands (Brown et al., 1980; Dörje et al., 1991; Eltze, 1988; North et al., 1985). M₂ receptors constitute approximately 90% of the muscarinic receptors in heart, and also are abundant in the ileum and peripheral lung (Dörje et al., 1991; Barnes, 1989). M₃ receptors occur predominantly in glands of secretion and on smooth muscle such as bladder (Dörje et al., 1991; Levey, 1993; Noronha-Blob et al., 1989). Peripheral M₄ receptors have been identified in rabbit peripheral lung, uterus and ileum (Dörje et al., 1991; Levey, 1993). However, M₅ receptors have yet to be observed in peripheral tissues. Muscarinic receptors are abundant in the substantia gelatinosa of the dorsal spinal cord (Gillberg and Aquilonius, 1985; Gillberg et al., 1988) as well as in the thalamus (Mesulam, 1990; Levey, 1993), areas of the central nervous system involved in pain transmission. However, the specific muscarinic receptor subtypes located in pain pathways have not been determined. Nevertheless, the delineation of multiple muscarinic receptor subtypes suggests that muscarinic agonists selective for one or a few receptor subtypes may be effective analgesics, but with a pharmacological profile devoid of the undesirable side effects of nonselective agonists such as oxotremorine, as well as of the opioids.

Butylthio[2.2.2] (LY297802/NNC11-1053) is a mixed muscarinic cholinergic receptor agonist/antagonist that crosses the blood-brain barrier and produces antinociception in mice and rats (Olesen et al., 1996; Sauerberg et al., 1995; Swedberg et al., 1995, 1997). As such, butylthio[2.2.2] may have therapeutic utility in the treatment of pain. The present report summarizes the preclinical pharmacology of butylthio[2.2.2]. In vitro, the affinity of butylthio[2.2.2] for several neurotransmitter receptors and uptake sites was evaluated, as well as its efficacy and potency at muscarinic receptors in isolated tissue segments of rabbit vas deferens and guinea pig atrium, urinary bladder and ileum. In vivo, the effects of butylthio[2.2.2] on neurotransmitter levels, ex vivo receptor binding, gastrointestinal function, body temperature, tremor, salivation, ventilation and heart rate were determined. The present results demonstrate that butylthio[2.2.2] is a novel muscarinic receptor mixed agonist/antagonist. Preliminary portions of these data were presented at the Sixth International Symposium on Subtypes of Muscarinic Receptors (Sauerberg et al., 1995; Swedberg et al., 1995; Shannon et al., 1995).

	Methods

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Materials. Butylthio[2.2.2] (fig. 1) was synthesized at Novo Nordisk (Måløv, Denmark) and Lilly Research Laboratories (Indianapolis, IN). Radioactive isotopes were obtained from New England Nuclear Corporation (Boston, MA) and Amersham, Inc. (Arlington Heights, IL). All chemicals used were of reagent grade.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structure of butylthio[2.2.2].

Radioligand displacement assays. The methodologies used for examination of binding to neurotransmitter receptors and uptake sites are given in table 1.

Rabbit vas deferens. Vasa deferentia were isolated from male New Zealand White rabbits weighing 2.5 to 4.0 kg (Hazelton Research Products, Denver, PA) which had been sacrificed by i.v. overdose with sodium pentobarbital. As described previously in detail (Shannon et al., 1993), each vas deferens was dissected free from surrounding tissue, divided into a prostatic and an epididymal segment and placed in modified Krebs' solution of the following composition (mM): NaCl, 134; KCl, 3.4; CaCl₂, 2.8; KH₂PO₄, 1.3; NaHCO₃, 16; MgSO₄, 0.6; and glucose, 7.7. The pH of the Krebs' solution was maintained at 7.4 during all experiments by constant bubbling with 95% O₂/5% CO₂. Changes in isometric tension were recorded and analyzed with an M5000 Signal Processing Center with XYZ Realtime software (Modular Instruments, Inc., Malvern, PA) and a Compaq Deskpro 386 computer (Compaq Computer Corporation, Houston, TX).

Guinea pig atria. Male Hartley guinea pigs (Møllegård, Ry, Denmark) were sacrificed by cervical dislocation. The hearts were quickly removed and the atria dissected free from the surrounding tissue. The atria were suspended in 10-ml organ baths in modified Krebs-Henseleit solution of the following composition (mM): NaCl, 118; NaHCO₃, 25; KCl, 4.7; CaCl₂, 2.5; MgCl₂, 2.1; NaH₂PO₄, 1.03; and glucose, 11. The solution was continuously bubbled with 95% O₂ and 5% CO₂ and maintained at 37°C. The mechanical activity of the tissue was measured by a Hugo Sachs Electronics (March-Hugstetten, Germany) type 351 isometric force transducer connected via a HSE type 301 bridge amplifier to a Kontron type 340 potentiometric pen recorder.

Guinea pig urinary bladder. Whole urinary bladders were isolated from male Hartley guinea pigs weighing 250 to 350 g (Charles River Laboratories, Inc., Portage, MI), washed in modified Krebs' solution of the following composition (mM): NaCl, 134; KCl, 3.4; CaCl₂, 2.8; KH₂PO₄, 1.3; NaHCO₃, 16; MgSO₄, 0.6; and glucose, 7.7, then transferred to fresh Krebs' solution maintained at 7.4. Three equatorial rings approximately 1.0 mm thick were cut from each bladder body above the level of the ureters. Each ring was suspended from a platinum/iridium hook at a passive force of 0.5 g in a 10-ml organ bath maintained at 31°C and attached to a Grass FT.03 force transducer. Changes in isometric tension were recorded and analyzed with an M5000 Signal Processing Center with XYZ Realtime software (Modular Instruments, Inc.) and a Compaq Deskpro 386 computer (Compaq Computer Corp.).

Guinea pig ileum. Ilea were isolated from male Hartley guinea pigs (Charles River Laboratories, Inc.) weighing 250 to 450 g. Myenteric plexus-longitudinal muscle strips were prepared by the method of Paton and Zar (1968), as modified by Shannon and Sawyer (1989), and placed in Krebs' solution of the following composition (mM): NaCl, 134; KCl, 3.4; CaCl₂, 2.8; KH₂PO₄, 1.3; NaHCO₃, 16; and glucose, 7.7. Changes in isometric tension were recorded and analyzed with an M5000 Signal Processing Center with XYZ Realtime software (Modular Instruments, Inc.) and a Compaq Deskpro 386 computer (Compaq Computer Corp.).

Neurotransmitter levels and ex vivo receptor binding. Male Sprague-Dawley rats (Charles River Laboratories) weighing approximately 100 to 150 g were injected orally with saline or a dose of drug and sacrificed 40 min later by decapitation. As suggested by Sethy and Francis (1988), rats were sacrificed by decapitation rather than microwave irradiation to permit ex vivo binding determinations. Striatum and cortex were rapidly dissected, weighed and then homogenized by sonification in 0.4 ml of 0.1 N trichloroacetic acid containing 10 µM acetylthiocholine and 1 µM 5-hydroxyindolcarboxylic acid as internal standards. ACh, choline and monoamines and their metabolites in striatum were determined by electrochemical detection by high-performance liquid chromatography as described previously (Fuller and Perry, 1989; Bymaster et al., 1993). For determination of ex vivo binding to brain receptors, the cerebral cortex was homogenized in 10 volumes of 20 mM Tris-Cl buffer, pH 7.4, containing 1 mM MnCl₂. The butylthio[2.2.2]-induced inhibition of ex vivo binding of 1 nM [³H]pirenzepine (87.0 Ci/mmol, New England Nuclear) to muscarinic receptors was determined by adding 0.1 ml of tissue whole homogenate and radioisotope in 1.1 ml total volume of the buffer above. After incubation for 1 h at 25°C, the homogenates were filtered through glass filters (Whatman, GF/c) with vacuum. The filters were washed three times with 2 ml cold buffer, and placed in scintillation vials containing 10 ml of scintillation fluid (Ready Protein+, Beckman, Fullerton, CA). Filters were presoaked in 0.1% polyethylenimine for several hours before use. Radioactivity trapped on the filters was determined by liquid scintillation spectrometry. Nonspecific binding was determined with 1 µM atropine.

Effects on charcoal meal transit. Male CD-1 mice (Charles River Laboratories) weighing 20 to 25 g were used. Mice were fasted for 2 h and administered varying doses of butylthio[2.2.2] or morphine orally (in a total volume of 0.25 ml). Fifteen minutes later, the mice were given a standard charcoal meal (0.3 ml) by gavage. The mice were sacrificed 30 min after administration of the charcoal meal, and the distance the charcoal meal had traveled was measured. The data were expressed as the percent of the gut the charcoal meal traveled. A group of five mice was tested at each dose level, and the experiment was repeated at least three times. Data from different experiments were pooled. Statistical significance was determined by a paired t-test, and P < .05 was considered statistically significant.

Body temperature, tremor, salivation. Male CF1 mice (Harlan Sprague-Dawley, Indianapolis, IN) weighing 18 to 25 g were used. Rectal temperature was measured (model BAT 8, Bailey Instruments, Saddle Brook, NJ) immediately before and 30 min after a s.c. injection of vehicle, a dose of butylthio[2.2.2] or oxotremorine. In these same mice, tremor and salivation were scored on a scale of 0 = no effect, 1 = moderate tremor or salivation and 2 = marked tremor or salivation. In antagonism experiments, varying doses of butylthio[2.2.2] were administered a few seconds before oxotremorine (1 mg/kg), and the degree of salivation or tremor scored 30 min later. Five mice were used per treatment group. Ambient room temperature was maintained at 22 ± 1°C.

Heart rate in conscious rats. Sprague-Dawley rats (Harlan Sprague-Dawley) weighing 150 to 250 g were used. During experimental sessions, the rats were loosely restrained in a cylindrical tube made of 1/8-inch stainless steel rods spaced approximately 3/8 inch apart. The animals were adapted to restraint before the initiation of the studies. Heart rate was monitored by subcutaneous electrocardiograph electrodes, and all data were recorded on-line by an IBM/AT computer with LabLinc interface modules (Coulbourn Instruments, LeHigh Valley, PA). The average heart rate during a 1-min period was recorded every 5 min.

Ventilation in guinea pigs. Male Hartley guinea pigs were obtained from Charles River Laboratories, Inc. Under fluothane anesthesia, each guinea pig was prepared with a saphenous vein catheter. Animals were allowed to regain consciousness in a pressure plethysmograph for approximately 1 h before drug administration. The signal from the plethysmograph was sensed by a differential pressure transducer (Validyne model DP45-14, Northridge, CA) coupled to a pulmonary mechanics analyzer (model 6, Buxco Electronics, Sharon, CT). V_T and f were recorded on a multichannel CRT Visicorder (Honeywell model 1858, Denver, CO) as described previously (Silbaugh et al., 1989).

	Results

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Receptor binding profile. A summary of the radioligand binding studies is given in table 2. Butylthio[2.2.2] had highest affinity for [³H]oxotremorine-M and [³H]pirenzepine-labeled muscarinic receptors (IC₅₀ values = 0.32 and 1.4 nM, respectively). Butylthio[2.2.2] had an 11 nM affinity for [³H]QNB-labeled muscarinic receptors. Butylthio[2.2.2] did not inhibit mu or kappa opioid receptor binding at concentrations up to 10 µM. Butylthio[2.2.2] had only weak (>0.3 µM) or negligible affinity (>10 µM) for the remaining neurotransmitter receptor, ion channel and uptake sites tested.

Rabbit vas deferens. Butylthio[2.2.2] produced a concentration-dependent inhibition of the electrically stimulated twitch response in isolated rabbit vas deferens over the concentration range of 0.01 to 100 nM (fig. 2). Maximum inhibition was 75 ± 7% of control twitch height and occurred at a concentration of 30 nM. The IC₅₀ for butylthio[2.2.2] was 0.33 ± 0.04 nM. In comparison, the IC₅₀ for carbachol was 208 ± 15 nM. In addition, butylthio[2.2.2] did not increase the electrically stimulated twitch response in the isolated rabbit vas deferens at concentrations ranging from 0.01 to 100 nM (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-related decreases in twitch height in the electrically stimulated rabbit vas deferens by butylthio[2.2.2] in comparison with the nonselective muscarinic agonist carbachol. Each point represents the mean of six tissues. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as the percentage of control twitch height in each tissue.

Guinea pig atria. Alone, butylthio[2.2.2] was without significant effect in the guinea pig atria (data not shown). However, butylthio[2.2.2] produced a concentration-dependent antagonism of the negative inotropic effects of the nonselective muscarinic agonist carbachol in the guinea pig atria (fig. 3). A Schild plot of the data in figure 3 yielded a pA₂ value of 6.9 with a slope of 1.5, which was significantly different from unity.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Concentration-related antagonism by butylthio[2.2.2] of the negative inotropic effects of the nonselective muscarinic agonist carbachol in guinea pig atria. Each point represents the mean of 6 to 12 tissues. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as the percentage reduction of the force of contraction.

Guinea pig bladder. Alone, butylthio[2.2.2] was without significant effect in isolated guinea pig bladder rings (data not shown). However, butylthio[2.2.2] produced a concentration-dependent antagonism of the contractions produced by carbachol (fig. 4). A Schild plot of the data presented in figure 4 yielded a pA₂ value of 7.4 for butylthio[2.2.2] with a slope of 2.1 ± 0.3, which was significantly different from unity.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Concentration-related antagonism by butylthio[2.2.2] of contractions produced by the nonselective muscarinic agonist carbachol in the guinea pig urinary bladder. Each point represents the mean of six tissues. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 3 µM carbachol.

Guinea pig ileum. Butylthio[2.2.2] produced an inverted-U concentration-response curve for contractions of the guinea pig ileum myenteric plexus-longitudinal muscle with a maximal effect of approximately 25% produced by 100 nM (fig. 5, closed triangles). Carbachol (10-300 nM; fig. 5, closed squares) produced concentration-dependent contractions of the guinea pig ileum myenteric plexus-longitudinal muscle with an EC₅₀ of 57 nM. Butylthio[2.2.2] (50, 100 and 200 nM) shifted the carbachol concentration-response curve to the right in a concentration-dependent manner. A Schild plot of the data presented in figure 5 yielded a pA₂ value of 8.3 for butylthio[2.2.2] with a slope of 1.3 ± 0.4, which was not significantly different from unity.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Partial agonist effects, and antagonism of carbachol, by butylthio[2.2.2] in the guinea pig ileum myenteric plexus-longitudinal muscle preparation. Each point represents the mean of six tissues. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 1 µM carbachol.

Neurotransmitter levels. After oral administration, doses of 3 and 10 mg/kg p.o. of butylthio[2.2.2] produced small but significant increases in tissue levels of the dopamine metabolite DOPAC (table 3), which suggested a small increase in dopamine turnover. However, butylthio[2.2.2] had no substantial effect on brain levels of dopamine or its metabolite HVA, on acetylcholine or on 5HT and its metabolite 5HIAA (table 3).

Ex vivo receptor binding. An estimation of levels of butylthio[2.2.2] (or butylthio[2.2.2] and metabolites that inhibit muscarinic binding) may be determined in brain by ex vivo binding. Treatment of rats with butylthio[2.2.2] p.o. for 40 min produced a dose-related decrease in ex vivo binding of [³H]pirenzepine to cortex whole homogenates (table 4). The dose of butylthio[2.2.2] required to inhibit ex vivo binding 50% (ED₅₀) was calculated to be 4 mg/kg p.o.

Effects on charcoal meal transit. Morphine (3-30 mg/kg p.o.) produced a dose-related inhibition of gastrointestinal transit in mice after oral administration (fig. 6). In contrast, butylthio[2.2.2] p.o. had no substantial effect on motility.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Lack of effect of butylthio[2.2.2], and dose-related decrease by morphine, on charcoal meal transit after oral administration in mice. Each point represents the mean of four to six mice. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. Shaded area represents the mean ± 1 S.E.M. of transit after administration of vehicle. Data are expressed as the percent distance of the gastrointestinal tract that the charcoal meal traveled.

Body temperature, tremor and salivation. The nonselective muscarinic receptor agonist oxotremorine produced a dose-related decrease in body temperature in mice over the dose range of 0.01 to 1.0 mg/kg s.c. (fig. 7, upper panel); doses of 0.03 to 1.0 mg/kg significantly decreased body temperature. Butylthio[2.2.2] produced a dose-related decrease in body temperature over the dose range of 1.0 to 10 mg/kg s.c. (fig. 7, upper panel), however the magnitude of the change in body temperature was not as great as that observed with oxotremorine. In addition, oxotremorine produced dose-related salivation and tremor, with marked, whole-body tremors and copious salivation at a dose of 1.0 mg/kg (data not shown). Butylthio[2.2.2] did not produce salivation or tremors at doses up to 10 mg/kg s.c. (fig. 7, lower panel, open symbols). Rather, butylthio[2.2.2] produced a dose-related antagonism of oxotremorine (1.0 mg/kg)-induced salivation and tremor (fig. 7, lower panel, closed symbols).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effects of butylthio[2.2.2] on body temperature (upper panel) and dose-related antagonism of oxotremorine (1.0 mg/kg)-induced salivation and tremor (lower panel) in mice after s.c. administration. Abscissa, dose of butylthio[2.2.2] in milligrams per kilogram; ordinate, upper panel, mean change in rectal body temperature in °C. Vertical lines represent ±S.E.M. and are absent when less than the size of the point. Ordinate, lower panel, mean salivation or tremor score. Each point represents the mean of one observation in each of five mice. Points above Veh represent the effects of vehicle alone or vehicle plus 1.0 mg/kg oxotremorine. * P < .05 vs. vehicle alone.

Heart rate in conscious rats. Dose-response curves for the nonselective muscarinic agonist oxotremorine, the nonselective muscarinic antagonist scopolamine and butylthio[2.2.2] were determined by administering sequentially increasing doses s.c. at 15-min intervals. Oxotremorine produced a dose-related decrease in heart rate which reached a magnitude of approximately 25% 15 min after 1.0 mg/kg s.c. of oxotremorine (fig. 8, upper panel). In contrast, scopolamine produced a dose-related increase in heart rate which reached a magnitude of approximately 30% (fig. 8, upper panel). Butylthio[2.2.2] produced only modest decreases in heart rate which were not different from those obtained after the repeated administration of vehicle (fig. 8, upper panel).

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Upper panel, dose-response curves for the decrease in heart rate produced by oxotremorine (closed square), the increase in heart rate produced by scopolamine (closed diamond) and the lack of effect of butylthio[2.2.2] (open circle) after s.c. administration in conscious rats. Lower panel, sequential dose-response curve for the nonselective muscarinic agonist carbachol after the administration of vehicle (closed circle) or 1.0 mg/kg butylthio[2.2.2] (open circle) on heart rate at 5-min intervals after s.c. administration in conscious rats. Abscissa, dose of drug in milligrams per kilogram; ordinate, percent change in heart rate from base line. Points above baseline represent means before drug administration. Points above Pre represent the effects of vehicle or butylthio[2.2.2] administration alone. Each point represents the mean of one observation in each of eight (upper panel) or four (lower panel) rats. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. * P < .05 vs. control.

Ventilation in guinea pigs. Before the start of i.v. dosing with butylthio[2.2.2], morphine or saline, V_T was 2.34 ± 0.15, 2.33 ± 0.20 and 2.23 ± 0.15 ml, respectively, and f was 93.0 ± 3.2, 81.8 ± 6.9 and 93.0 ± 3.0 breaths/min, respectively. There were no significant differences in these base-line ventilatory parameters among the three groups. Administration of 3 volumes of saline had no effect on V_T and f (fig. 9). Morphine (0.03-3.0 mg/kg i.v.) produced a dose-related decrease in V_T (fig. 9, upper panel), but had no effect on f (fig. 9, lower panel). Over the dose range of 0.001 to 0.1 mg/kg i.v., butylthio[2.2.2] decreased V_T but had no effect on f. A dose of 0.3 mg/kg i.v., however, stimulated respiration and increased V_T and f to approximately 175% of base-line values.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Dose-response curves for butylthio[2.2.2] and morphine on tidal volume (upper panel) and rate of breathing (lower panel) after i.v. administration in guinea pigs. Doses were administered sequentially at 5-min intervals. The effects of three sequential administrations of saline are presented for comparison. Abscissa, dose of drug in milligrams per kilogram; ordinate, upper panel, tidal volume (VT) expressed as percent of base line; ordinate, lower panel, breathing frequency (f) expressed as percent of base line. Each point represents the mean of four to eight guinea pigs. Vertical lines represent ±1 S.E.M. and are absent when less than the size of the point. * P < .05 vs. control.

	Discussion

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In rat brain membranes, butylthio[2.2.2] was relatively selective for muscarinic receptors. Butylthio[2.2.2] was most potent in inhibiting [³H]oxotremorine M and [³H]pirenzepine binding in rat brain homogenates with IC₅₀ values of 0.32 and 1.4 nM, respectively; butylthio[2.2.2] was less potent in inhibiting the nonselective muscarinic agonist [³H]QNB. In contrast, butylthio[2.2.2] had substantially less or no affinity for several other neurotransmitter receptor and uptake sites in brain, including nicotinic cholinergic and opioid receptors. Thus, the pharmacological effects of butylthio[2.2.2] in vivo are most likely caused by actions only at muscarinic cholinergic receptors.

In isolated tissues, butylthio[2.2.2] had high affinity for M₁ receptors in the rabbit vas deferens with an IC₅₀ of approximately 0.3 nM. At M₂ receptors in guinea pig atria, butylthio[2.2.2] was an antagonist with a pA₂ value of 6.9. At M₃ receptors in guinea pig urinary bladder (Noronha-Blob et al., 1989), butylthio[2.2.2] was an antagonist with a pA₂ value of 7.4. In the guinea pig longitudinal ileal preparation, butylthio[2.2.2] was a partial agonist with a maximal effect only approximately 25% that of carbachol. The muscarinic receptor population in the longitudinal ileal preparation is heterogeneous (e.g., Michel and Whiting, 1987), and the receptor subtypes involved in mediating contractions have not been definitively determined, but are most likely predominantly of the M₃ and M₂ subtype (Michel and Whiting, 1987), although M₁ receptors are present in the gut as well (Levey, 1993; Shannon et al., 1994). Taken together, the present data demonstrate that in functional studies in isolated tissues, butylthio[2.2.2] is an agonist at M₁ receptors and an antagonist or partial agonist at M₂ and M₃ muscarinic receptors.

Muscarinic agonists such as oxotremorine and RS86 produce antinociception in animals at doses that are accompanied by prominent parasympathomimetic side effects such as salivation and tremor (e.g., Sheardown et al., 1997; Swedberg et al., 1997). Butylthio[2.2.2] produces antinociception over the dose ranges of approximately 0.03 to 3.0 mg/kg s.c. and 0.3 to 30 mg/kg p.o. (Swedberg et al., 1997). In the present studies (see also Swedberg et al., 1997), butylthio[2.2.2] did not produce parasympathomimetic effects, including salivation and tremor, and produced only a modest hypothermia, up to a dose of 10 mg/kg s.c. and 30 mg/kg p.o. Rather, butylthio[2.2.2] antagonized the salivation and tremor produced by oxotremorine. The ED₅₀ values for butylthio[2.2.2] for antagonizing oxotremorine (present report) were as much as 10-fold higher than the ED₅₀ values for butylthio[2.2.2] for producing antinociception (Swedberg et al., 1997). Thus, at antinociceptive doses, butylthio[2.2.2] does not produce undesirable parasympathomimetic effects such as salivation and tremor, in part because it is an antagonist/partial agonist at M₂ and M₃ receptors.

In the isolated atria, butylthio[2.2.2] was an antagonist of the nonselective muscarinic agonist carbachol, which indicates that butylthio[2.2.2] is an M₂ receptor antagonist. In vivo, an antagonist at M₂ receptors would be expected to increase heart rate by blocking the endogenous parasympathomimetic tone and to decrease tissue levels of acetylcholine because of blockade of negative feedback inhibition at M₂ autoreceptors (e.g., Sethy and Francis, 1988; Hoss et al., 1990; Bymaster et al., 1993). Interestingly, butylthio[2.2.2] neither increased heart rate in rats, nor did it alter tissue levels of acetylcholine in rat brain. Butylthio[2.2.2] did, however, block the decrease in heart rate produced by carbachol (present report), which demonstrates that it could function as an M₂ antagonist in vivo as well as in isolated atria. Further studies are in progress to determine whether butylthio[2.2.2] can antagonize the effects of muscarinic agonists on acetylcholine release and tissue levels in brain. Thus, butylthio[2.2.2] is a unique muscarinic receptor antagonist. The reasons why butylthio[2.2.2] does not increase heart rate or alter brain tissue levels of acetylcholine, as do other antagonists at M₂ muscarinic receptors, are not readily apparent and require further research. In the isolated guinea pig atria, the slope of the Schild plot for butylthio[2.2.2] was significantly different from unity, which indicates that it antagonized carbachol in other than a competitive manner. Similarly, a Schild slope significantly different from unity was also obtained in the guinea pig bladder preparation for antagonism of carbachol by butylthio[2.2.2]. It may be that butylthio[2.2.2] interacts with a different site on the receptor than the muscarinic ligands such as carbachol and atropine.

Undesirable side effects produced by opioids include constipation and respiratory depression (Jaffe and Martin, 1990). An analgesic which does not produce these side effects might be expected to have an improved therapeutic profile relative to opioids. As demonstrated in the present studies, butylthio[2.2.2] had no effect on charcoal meal transit in mice at doses which produced antinociception (cf., Swedberg et al., 1997), whereas morphine produced the expected decrease in charcoal meal transit. Further, in guinea pigs, morphine produced dose-related decreases in V_T like those observed in previous studies with morphine by a similar methodology (Adcock et al., 1988). Butylthio[2.2.2] decreased V_T at lower doses, but at the highest dose tested, stimulated respiration and increased both V_T and f. Taken together, the present results suggest that butylthio[2.2.2] may have less liability to produce constipation and respiratory depression than morphine and thus may have an improved therapeutic profile over morphine.

The present studies characterized the pharmacology of the novel muscarinic ligand butylthio[2.2.2] which is equiefficacious to morphine in producing antinociception in animals (Swedberg et al., 1997). In vitro, butylthio[2.2.2] bound selectively to muscarinic receptors and was an agonist at M₁ receptors in the rabbit vas deferens, but an antagonist/partial agonist at M₂ and M₃ receptors in guinea pig atria, bladder and ileum. In vivo, butylthio[2.2.2] was devoid of parasympathomimetic effects, did not alter brain neurotransmitter levels, did not alter heart rate, did not decrease charcoal meal transit time and produced biphasic effects on V_T. The antinociception produced by butylthio[2.2.2] is antagonized in a competitive manner by the muscarinic antagonist scopolamine (Swedberg et al., 1997), which demonstrates that the antinociceptive effects of butylthio[2.2.2] are mediated by an agonist action at muscarinic receptors. In the present studies, butylthio[2.2.2] was an agonist at M₁ receptors and an antagonist at M₂ and M₃ receptors, which suggests that M₁ receptors may be involved in the analgesic effects of butylthio[2.2.2]. However, agonist activity at M₁ receptors is not required for producing muscarinic antinociception (Sheardown et al., 1997), and butylthio[2.2.2] is a partial agonist in cells transfected with the m₁ receptor (D. O. Calligaro and P. D. Suzdak, unpublished observations). It may be that M₄ and/or M₅ receptors are involved in mediating muscarinic receptor-mediated antinociception. In summary, butylthio[2.2.2] is a novel muscarinic ligand that produces antinociception equivalent to that of opioids at doses which do not produce parasympathomimetic effects. Butylthio[2.2.2] may thus have therapeutic utility in the clinical management of pain as an alternative to opioids.