Carolyn J. Spencer

Characterization of muscarinic agonists in recombinant cell lines

Using recombinant CHO cells that express Hm1-Hm5 receptors, reference muscarinic agonists have been characterized with respect to their activity in receptor binding and second messenger assays. In whole cell [3H]-N-methyl scopolamine binding, no agonist was found to be truly subtype selective, although some showed marked differences between several of the subtypes (e.g. m1 vs. m2). As a functional index of receptor activation, phosphatidyl-inositol (PI) turnover was measured for m1, m3, and m5 receptors while inhibition of forskolin-stimulated cAMP accumulation was measured for m2 and m4 receptors. Both full and partial agonists were delineated in PI turnover, but all agonists showed similar responses on cAMP. Alkylation studies with propylbenzylcholine mustard showed that both efficacy and potency were markedly affected in the functional assays by the number of free receptors. Thus, receptor reserve appears to play a major role in the determination of subtype selectivity for agonists using functional measures. Even with these limitations, however, the use of transformed cell lines is playing a pivotal role in the discovery of selective agonists.

Loss of muscarinic M1 receptors with aging in the cerebral cortex of fisher 344 rats

Age-related changes in central cholinergic muscarinic receptors were measured in young (3-6 month), middle-aged (15-17 month), and aged (22-26 month) male Fisher 344 rats by receptor binding techniques. Using [3H]-quinuclidinyl benzilate as the ligand, a significant decrease (14-19%) in the number of muscarinic cortical receptors was measured in aged rats compared to both young and middle-aged rats. With the selective M1 antagonist, [3H]-pirenzepine, a 17% decrease in receptor density was observed in the cortex of aged animals compared to young rats. For both ligands no differences were observed in the striatum or hippocampus between any age group and there was no change in affinity (Kd) in any of the three brain regions for the three age groups. Additionally, there was no difference in choline acetyltransferase activity measured in cortex, hippocampus, or striatum of young and aged rats. Thus, there is a loss of M1 muscarinic receptors in the cerebral cortex of aged male Fisher 344 rats.

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.

CI-1017, a functionally M1-selective muscarinic agonist: design, synthesis, and preclinical pharmacology.

The five muscarinic receptor subtypes (M1-M5) are characterized by seven helices that define a transmembrane cavity which serves as the binding pocket for agonists and antagonists. The five cavities appear to be topographically different enough to permit subtype selectivity among antagonists but not among classical agonists which tend to be smaller in size than antagonists. It was reasoned that synthesis of muscarinic agonists longer/larger than their classical counterparts might result in subtype selectivity. M1 subtype selectivity was found in a class of 1-azabicyclo[2.2.1]heptan-3-one, O-(3-aryl-2-propynyl) oximes. One of these, CI-1017, improved spatial memory of hippocampally deficient mice and nbM-lesioned rats at doses of 1.0-3.2 and 0.1-0.3 mg/kg, respectively, while producing parasympathetic side effects only at very high doses (100-178 mg/kg). Additionally, CI-1017 inhibited production of amyloidogenic A beta and increased secretion of soluble APP. Thus, CI-1017, besides treating AD symptomatically, may also retard its progression. CI-1017 has recently completed phase I clinical trials.

Selective Muscarinic Agonists for Alzheimer Disease Treatment

Replacement therapy in Alzheimer Disease (AD) with muscarinic agonists may be of therapeutic benefit in alleviating certain cognitive deficits associated with the disorder. Based upon this “cholinergic hypothesis” (Bartus et al., 1982), a number of clinical trials were conducted over the last decade with a variety of agonists (Table I). The overall results with these compounds were equivocal with some patients reporting positive responses, some showing no responses, and others unable to complete the trials due to troublesome cholinergic side effects. Two conclusions from these studies were that either the design (e.g. outcome measures) of the trials were insufficient to determine efficacy or that the compounds themselves had major deficiencies which prevented the cholinergic hypothesis from being adequately tested.

Acetylcholine releasing agents as cognition activators. Chemistry and pharmacology of a series of ureas

Abstract We have sought to improve upon the activity of the well known AcCh-releasing agents, 4-aminopyridine and 3,4-diaminopyridine. This work has led to the discovery of a series of ureas with AcCh-releasing properties. From this series, compound 5 has emerged as a potent AcCh-releasing agent with promising in vivo activity.

Pharmacological Characterization of PD151832, an M1 Muscarinic Receptor Agonist

Alzheimer’s disease (AD) is an age-related disorder characterized by progressive neurological impairment. Among neurotransmitters affected, the loss of forebrain cholinergic neurons has been well documented with the results contributing to the idea that therapies based on cholinergic pharmacology would be useful treatments in AD (Bartus et al., 1982). Among those suggested have been acetylcholinesterase (AChE) inhibitors and muscarinic agonists. Clinical studies have shown that the AC inhibitor tacrine is therapeutically active (Knapp et al., 1994). However, robust and reproducible clinical data with classical muscarinic agonists have been variable and no agonists have been approved to date for AD treatment.

Control of the Release of [3H]-Acetylcholine from Rat Hippocampal Slices by Aminopyridines and Phencyclidine

Release of acetylcholine (ACh) resulting from the depolarization of cholinergic nerve terminals appears to involve the movement of specific ions across the nerve membrane. Experimentally, electrical stimulation and veratridine release ACh by increasing the flux of Na+ as shown by their sensitivity to the Na+ channel blocker tetrodotoxin (TTX), while elevated K+ releases ACh by a TTX insensitive mechanism. In addition, all three methods appear to require the presence of extracellular Ca++ in order to release ACh from vesicular stores (3, 9, 11). Electrophysiological data have suggested that blockade of K+ channels will also enhance neurotransmitter release by increasing Ca++ entry or utilization, due to the prolongation of the repolarization period (14).