Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.     

Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors

1. The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric effects on radioligand-binding characteristics at other G-protein-coupled receptors, such as cerebral A1 adenosine receptors (Gi-coupled), cardiac left ventricular alpha1-adrenoceptors (Gq), and beta-adrenoceptors (Gs). 2. The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [3H]-N-methylscopolamine (NMS) from muscarinic M2-receptors: 30 micromol l(-1) W84, 30 micromol l(-1) alcuronium, 1000 micromol l(-1) gallamine. As radioligands, we used the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthine (CPX), the alpha1-adrenoceptor ligand [3H]-prazosin (PRAZ), and the beta-adrenoceptor ligand (-)-[125I]-iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively. 3. CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88+/-0.15 and 12.27+/-0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36+/-2% of the control value with alcuronium and to 42+/-2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half-life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve. 4. In summary, the allosteric action of the applied agents is not a common feature of G-protein-coupled receptors and appears to be specific for muscarinic receptors.     

Lack of intrinsic activity and significant subtype selectivity of SR 95639A at muscarinic receptors.

We assessed the intrinsic activity of the purported selective muscarinic M1 receptor agonist SR 95639A (morpholinoethylamino-3-benzocyclohepta-(5,6-c)-pyridazine) in inducing several receptor-mediated signals. Our results indicate that SR 95639A lacks the ability to activate phosphoinositide hydrolysis in rat cerebral cortex or in Chinese hamster ovary cells transfected with the genes of the muscarinic m1 and m3 receptors. Similarly, this compound did not exhibit intrinsic activity in stimulating muscarinic receptors which inhibit cyclic AMP synthesis and did not suppress acetylcholine release in rat striatum. In addition, SR 95639A did not show a marked selectivity at the level of the ligand recognition site at the muscarinic M1, M2 and M3 receptors, since it bound to these receptor subtypes with equilibrium dissociation constants of 4, 6 and 11 microM, respectively.     

In vitro characterization of tripitramine, a polymethylene tetraamine displaying high selectivity and affinity for muscarinic M2 receptors.

1. The antimuscarinic effects of tripitramine were investigated in vitro in isolated driven left (force) and spontaneously beating right (force and rate) atria as well as in the ileum of guinea-pig and rat and in the trachea and lung strip of guinea-pig and compared with the effects of methoctramine. 2. Tripitramine was a potent competitive antagonist of muscarinic M2 receptors in right and left atria. The pA2 values ranged from 9.14 to 9.85. However, in the guinea-pig and rat left atria but not in guinea-pig right atria, tripitramine at lower concentrations (3-10 nM) produced a less than proportional displacement to the right of agonist-induced responses owing to the presence of a possible saturable removal process. 3. Tripitramine was about three orders of magnitude less potent in ileal and tracheal than in atrial preparations (pA2 values ranging from 6.34 to 6.81) which makes it more potent and more selective than methoctramine. 4. Another intriguing finding was the observation that the pA2 value of 7.91 observed for tripitramine in guinea-pig lung does not correlate with that found at both muscarinic M2 and M3 receptor subtypes, which clearly indicates that the contraction of guinea-pig lung strip is not mediated by these muscarinic receptor subtypes. 5. A combination of tripitramine with atropine resulted in addition of the dose-ratios for left atria as required for two antagonists interacting competitively with the same receptor site, whereas the same combination gave a supra-additive antagonism on guinea-pig ileum which suggests that tripitramine interacts with a second interdependent site. 6. Tripitramine was more specific than methoctramine since, in addition to muscarinic receptors, it inhibited only frog rectus abdominis muscular (pIC50 value of 6.14) and rat duodenum neuronal (pIC50 value of 4.87) nicotinic receptors among receptor systems investigated, namely alpha 1-, alpha 2-, and beta 1-adrenoceptors, H1- and H2-histamine receptors, and muscular and neuronal nicotinic receptors.     

p-Fluoro-hexahydro-sila-difenidol: affinity for vascular muscarinic receptors

The M3-selective antagonist, p-fluorohexahydro-sila-difenidol was used to characterize muscarinic receptors in two vascular preparations, the rabbit ear artery with an endothelium-dependent relaxation and the bovine coronary artery with an endothelium-independent contractile response. pKB values were consistent with the presence of M3 receptors, 7.9 and 7.5 in coronary and ear arteries, respectively. These findings confirm that muscarinic receptors of the rabbit ear artery and bovine coronary artery have similar characteristics and belong to the M3 subtype.     

Studies on the affinity and selectivity of tiquizium bromide (HSR-902), a novel spasmolytic agent, for muscarinic receptors

The affinity and selectivity of a novel spasmolytic agent, tiquizium bromide (HSR-902), for muscarinic receptors were studied by the radioligand binding technique using 3H-quinuclidinyl benzilate. The parameters (Ki, nH) of HSR-902 obtained from competition experiments in cerebral cortex and heart muscarinic receptors showed that HSR-902 was an atropine-type, nonselective muscarinic antagonist. The affinity of HSR-902 toward the stomach and ileal muscarinic receptors was about 3-4 times more potent than atropine.     

Hexocyclium derivatives with a high selectivity for smooth muscle muscarinic receptors.

1. The affinity of a number of derivatives of the muscarinic antagonist, hexocyclium, containing an amidine cationic head, for guinea-pig cardiac and ileal receptors was investigated. 2. All the compounds studied displayed a greater affinity for muscular than for cardiac muscarinic receptors. 3. The 5 fold ileal selectivity of hexocyclium was increased by a number of chemical substitutions. The largest discrimination between receptors (about 200 fold) was found for the formamidine derivative. 4. The selectivity displayed by the hexocyclium derivatives stemmed from a greater decrease in affinity towards cardiac as compared to ileal receptors.     

Quest for agonist and antagonist selectivity at muscarinic receptors in guinea-pig smooth muscles and cardiac atria

Summary Potencies of 11 muscarinic agonists in eliciting contraction of smooth muscle in guinea-pig ileum, trachea, urinary bladder and uterus and in inhibiting the rate of contractions of cardiac atria were compared. While acetylcholine (ACh) was the most potent agonist on the ileum, uterus and cardiac atria, cis-l(+)-dioxolane was equally as potent as ACh on the ileum and more potent on the urinary bladder and trachea. Compared to ACh, methylfurmethide, oxotremorine, acetoxybut-2-inyl-trimethylammonium and cis-l(+)-dioxolane acted weakly on the atria. Aceclidine, arecoline and acetyl--methylcholine displayed selectivity for the urinary bladder and pilocarpine for the tracheal and urinary bladder smooth muscles. Oxotremorine had very low activity on the uterus. The stereoselectivity of muscarinic ACh receptors (mAChRs) for cis-l(+)- and cis-d(–)-dioxolane was low in the urinary bladder and uterus and high in the ileum and trachea.Most antagonists showed little selectivity between different organs, but S(–)-phenylcyclohexylglycoloyl choline was 6 times more active on the urinary bladder than on the ileum and AF-DX 116 was 12–30 times more active on the atria than on the smooth muscles. Among the N-alkyl derivatives of benzilylcholine, the octyl derivative was 400 times more active on the ileum than on the atria, while among the N-alkyl derivatives of QNB, the N-decyl derivative was 41 times more active on the ileum.The observed differences in the potency of various agonists and their stereoisomers on different smooth muscles cannot be explained by differences in the accessibility of receptors or in receptor reserve. It is suggested that they reflect the heterogeneity of muscarinic receptors in different smooth muscles and differences between smooth muscles regarding the preferential coupling of their mAChRs to different G proteins. The observed selectivity of S(–)-phenylcyclohexylglycoloyl choline suggests a difference between the mAChRs responsible for the contraction of ileal and urinary bladder smooth muscles.     

Calcitonin: antagonism at intestinal muscarinic receptors.

The action of calcitonin was studied on the motility of isolated innervated segments of rabbit and guinea-pig intestines as well as longitudinal muscle with adherent myenteric plexus dissected from the guinea-pig ileum. Calcitonin (0.25 muM) antagonized contractile responses to acetylcholine and the cholinergic response to electrical field stimulation. This hormonal effect was relatively specific since it was not observed at nicotinic receptors or adrenoceptors, nor did calcitonin act as a local anaesthetic or directly on the contractile machinery of smooth muscle. Perivascular adrenergic and intrinsic non-adrenergic inhibitory responses also were unaffected by calcitonin. However, calcitonin did have antihistaminic properties directed against H1-receptors. The concentration of calcitonin required to achieve muscarinic antagonism in our experiments is not reached at the resting level of circulating hormone.     

Probing the selectivity of allosteric modulators of muscarinic receptors at other G‐protein‐coupled receptors

1 The aim of the present investigation was to analyse whether three prototype allosteric modulators of ligand binding to muscarinic receptors, i.e. alcuronium, gallamine, and the alkane‐bis‐ammonium compound W84 (hexane‐1,6‐bis[dimethyl‐3′‐phthalimidopropyl‐ammonium bromide]), may have allosteric effects on radioligand‐binding characteristics at other G‐protein‐coupled receptors, such as cerebral A1 adenosine receptors (Gi‐coupled), cardiac left ventricular α1‐adrenoceptors (Gq), and β‐adrenoceptors (Gs).
2 The modulators were applied at concentrations known to be high with regard to the allosteric delay of the dissociation of the antagonist [³H]‐N‐methylscopolamine (NMS) from muscarinic M2‐receptors: 30 μmol l?1 W84, 30 μmol l?1 alcuronium, 1000 μmol l?1 gallamine. As radioligands, we used the adenosine A1‐receptor ligand [³H]‐cyclopentyl‐dipropylxanthine (CPX), the α1‐adrenoceptor ligand [³H]‐prazosin (PRAZ), and the β‐adrenoceptor ligand (?)‐[¹²⁵I]‐iodocyanopindolol (ICYP). Allosteric actions on ligand dissociation and the equilibrium binding were measured in the membrane fractions of rat whole forebrain (CPX) and of rat cardiac left ventricle (PRAZ, ICYP, NMS), respectively.
3 CPX and PRAZ showed a monophasic dissociation with half‐lives of 5.88±0.15 and 12.27±0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influenced by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36±2% of the control value with alcuronium and to 42±2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately accelerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was still bound, so that a reliable half‐life could not be calculated. ICYP dissociation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the control curve.
4 In summary, the allosteric action of the applied agents is not a common feature of G‐protein‐coupled receptors and appears to be specific for muscarinic receptors.     

Effects of cyproheptadine and pizotifen on central muscarinic receptors

The affinities of cyproheptadine, pizotifen and (+/-)-quinuclidinyl xanthane-9-carboxylate hemioxylate (QNX) were determined at muscarinic autoreceptors and postsynaptic (IP1 formation) receptors in rat hippocampal slices. The affinity values for QNX were 8.2 and 8.5 respectively. Cyproheptadine and pizotifen were less potent than QNX. Pizotifen was slightly (2-fold) less active at antagonizing IP1 formation than blocking the autoreceptors whereas cyproheptadine was equally active at antagonizing the two hippocampal muscarinic receptors.     

Effects of alkanols and halothane on rat brain muscarinic and alpha-adrenergic receptors.

Effects of the primary alcohols ethanol, butanol, pentanol and of halothane were measured on the binding functions of muscarinic and alpha-adrenergic receptor preparations in rat brain homogenates, with the use of the antagonists 3H-quinuclidinyl benzilate and 3H-WB-4101. IC50 concentrations of the alkanols for the muscarinic and alpha-receptors respectively were: ethanol, 2.0 M and 1.4 M; butanol, 0.24 M and 0.16 M; heptanol, 3.7 X 10(-3) M and 2.6 X 10(-3) M. The plot of IC50 values versus number of carbon atoms in the alkanol was linear and of the same slope as the plot of membrane fluidity changes, thus indicating the importance of the membrane/water partition coefficient of the alkanol. Halothane at clinical concentrations had no effect on the receptors, although significant inhibition of radioligand binding was produced by 2.5 mM halothane, and inhibition was complete in presence of 17.5 mM anesthetic. From the correlation of receptor binding inhibitions with membrane fluidity changes reported by other workers, it is suggested that the activity of membrane receptors may be modulated by the fluidity of their membranes.     

Introduction of unsaturation into theN-n-alkyl chain of the nicotinic receptor antagonists, NONI and NDNI: Effect on affinity and selectivity

N-n-octylnicotinium iodide (NONI) and N-n-decylnicotinium iodide (NDNI) are selective nicotinic receptor (nAChR) antagonists mediating nicotine-evoked striatal dopamine (DA) release, and inhibiting [3H]nicotine binding, respectively. This study evaluated effects of introducing unsaturation into the N-n-alkyl chains of NONI and NDNI on inhibition of [3H]nicotine and [3H]methyllycaconitine binding (alpha4beta2* and alpha7* nAChRs, respectively), (86)Rb+ efflux and [3H]DA release (agonist or antagonist effects at alpha4beta2* and alpha6beta2*-containing nAChRs, respectively). In the NONI series, introduction of a C3-cis- (NONB3c), C3-trans- (NONB3t), C7-double-bond (NONB7e), or C3-triple-bond (NONB3y) afforded a 4-fold to 250-fold increased affinity for [3H]nicotine binding sites compared with NONI. NONB7e and NONB3y inhibited nicotine-evoked 86Rb+ efflux, indicating alpha4beta2* antagonism. NONI analogs exhibited a 3-fold to 8-fold greater potency inhibiting nicotine-evoked [3H]DA overflow compared with NONI (IC50 = 0.62 microM; Imax = 89%), with no change in Imax, except for NONB3y (Imax = 50%). In the NDNI series, introduction of a C4-cis- (NDNB4c), C4-trans-double-bond (NDNB4t), or C3-triple-bond (NDNB3y) afforded a 4-fold to 80-fold decreased affinity for [3H]nicotine binding sites compared with NDNI, whereas introduction of a C9 double-bond (NDNB9e) did not alter affinity. NDNB3y and NDNB4t inhibited nicotine-evoked 86Rb+ efflux, indicating antagonism at alpha4beta2* nAChRs. Although NDNI had no effect, NDNB4t and NDNB9e potently inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.02-0.14 microM, Imax = 90%), as did NDNB4c (IC50 = 0.08 microM; Imax = 50%), whereas NDNB3y showed no inhibition. None of the analogs had significant affinity for alpha7* nAChRs. Thus, unsaturated NONI analogs had enhanced affinity at alpha4beta2*- and alpha6beta2*-containing nAChRs, however a general reduction of affinity at alpha4beta2* and an uncovering of antagonist effects at alpha6beta2*-containing nAChRs were observed with unsaturated NDNI analogs.     

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine.

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.     

Effects of halothane on high affinity agonist binding and guanine nucleotide sensitivity of muscarinic acetylcholine receptors from brainstem of rat

The influence of halothane on muscarinic receptors with a high affinity for agonists was studied using [3H]oxotremorine-M. [3H]Oxotremorine-M bound with high affinity (KD = 2.8 nM) to a subpopulation of muscarinic receptors in the brainstem of rat, representing 32% of the total receptor pool. Agonist affinity for binding sites for [3H]oxotremorine-M was not affected by a guanine nucleotide (5'-guanylylimidodidiphosphate; Gpp(NH)p), although the level of binding was decreased, presumably due to the conversion of receptors to lower affinity conformations. However, only 58% of 3 nM binding of [3H]oxotremorine-M was sensitive to Gpp(NH)p. Halothane had two effects on the binding of [3H]oxotremorine-M: halothane (1) decreased the level of binding of [3H]oxotremorine-M without affecting agonist affinity for the surviving sites, and (2) lowered the sensitivity of the binding of [3H]oxotremorine-M to Gpp(NH)p by a factor of 120. The decrease in binding of [3H]oxotremorine-M binding was nonselective with regard to the sensitivity of the receptors to the guanine nucleotide, insofar as Gpp(NH)p inhibited the binding of [3H]oxotremorine-M to the same extent in the presence and absence of halothane. These results suggest that halothane (1) converts both G protein-coupled and -uncoupled muscarinic receptors to states of lower agonist affinity and (2) lowers the affinity of receptor-G protein complexes for guanine nucleotides.     

Temperature coefficients of affinity and entropies of adsorption from enantiomeric pairs of compounds acting at muscarinic receptors in the guinea-pig ileum.

1 The affinities of a series of enantiomeric pairs of esters of phenylcyclohexylglycollic acid, of a pair of esters of alpha-methyltropic acid and of hyoscyamine methiodide have been measured for the muscarinic receptors of the guinea-pig ileum at 30 degrees and 37 degrees C and estimates have been made of their free energies, enthalpies and entropies of adsorption. 2 With (-)-S-hyoscyamine methiodide the enthalpy of adsorption is negative whereas with the (+)-R-enantiomer it is positive. 3 With the esters of phenylcyclohexylglycollic acid the size of the onium group appears to be as important as the stereochemical configuration in determining entropy. Large onium groups appear to be associated with an increase in entropy though this can only be measured approximately because of the narrow range of temperature which can be used.     

Allosterism at muscarinic receptors: ligands and mechanisms

The evaluation of allosteric ligands at muscarinic receptors is discussed in terms of the ability of the experimental data to be interpreted by the allosteric ternary complex model. The compilation of useful SAR information of allosteric ligands is not simple, especially for muscarinic receptors, where there are multiple allosteric sites and complex interactions.     

Tetrahydropyrimidine derivatives display functional selectivity for M1 muscarinic receptors in brain

Selective muscarinic agonists might be useful in the treatment of Alzheimer's disease. Previous studies identified several amidine derivatives as selective and efficacious m1 agonists, using muscarinic receptor subtypes expressed in cell lines. In the present studies, the functional selectivities, side effect profiles and memory-enhancing properties of these ligands were examined through a series of in vitro and in vivo experiments. CDD-0078, CDD-0097, and CDD-0102 behaved as partial agonists by stimulating PI turnover in rat cortical slices to roughly 100% above basal levels. Pirenzepine was more potent than either AF-DX 116 or p-F-hexahydrosiladifenidol (p-F-HHSiD) in blocking the PI responses of each ligand, suggesting that the responses were due to activation of M₁ receptors. The time course of pharmacological responses was examined following i.p. injections of muscarinic agonists. Low does (0.1 and 1.0 mg/kg) of CDD-0078, CDD-0097, and CDD-0102 did not elicit signs of cholinergic activity during the 2-hr testing period. The highest dose tested (10 mg/kg) produced a modest degree of salivation and lacrimation during the first 30-min period. Core body temperature remained unaffected. Central nervous system (CNS) penetration was evaluated through ex vivo binding studies. CDD-0097 inhibited 1.0 nM [³H]pirenzepine binding in a dose-dependent manner 30 min following i.p. injections. In behavioral studies, CDD-0097 (1.0 mg/kg) completely reversed the memory deficits induced by hemicholinium-3 or by IgG-192 saporin in two types of memory tasks. It did not impair the performance of control animals in either task. In summary, CDD-0097 displayed a limited side-effect profile and the ability to penetrate into the central nervous system and stimulate M₁ receptors. The amidine derivatives should be useful in further exploring the functional consequences of activating M₁ muscarinic receptors in the CNS. The beneficial effects of CDD-0097 on memory function warrant further examination of the compound as a selective M₁ agonist for the treatment of Alzheimer's disease. Drug Dev. Res. 40:171–184, 1997. © 1997 Wiley-Liss, Inc.