Roy D. Schwarz

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.

Preclinical and phase 1 clinical characterization of CI-979/RU35926, a novel muscarinic agonist for the treatment of Alzheimer's disease.

In vitro and in vivo characterization in rodents and monkeys shows that CI-979/RU35926 is a partial muscarinic agonist with equal affinity for the five subtypes of muscarinic receptors. It activates central cholinergic receptors as shown by its ability to decrease body temperature, enhance local cortical blood flow and increase cortical arousal measured by QEEG. Further, it reverses spatial memory deficits in rats with ibotenic acid-induced lesions of forebrain cholinergic neurons. Signs of peripheral cholinergic stimulation appear at doses higher or equal to those necessary to produce central activity. In a single-dose tolerance study in young, healthy human volunteers, CI-979/RU35926 was well tolerated at doses of 0.002-1.0 mg with cholinergic symptoms such as hypersalivation and sweating, observed at 2-4 mg. It demonstrated linear pharmacokinetic behavior over a dose range of 0.1 to 4 mg and elimination half-life varied from 2-5 hours. Measurement of unchanged drug in urine suggests that the drug was extensively metabolized. Thus, the safety profile supported further clinical evaluation and CI-979/RU35926 is currently in Phase II clinical trials.

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.

Inhibitors of V-Type ATPases, Bafilomycin A1 and Concanamycin A, Protect Against β-Amyloid-Mediated Effects on 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction

Abstract: The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide‐induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysbsomal acidification in amyloid peptide‐treated differentiated PC12 cell cultures using selective vacuolar‐type (N‐type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V‐type ATPase. Treating nerve growth factor‐differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of β‐amyloid peptides Aβ(1‐42), Aβ(1‐40), and Aβ(25‐35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Aβ uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Aβ‐mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Aβ effects on MTT dye conversion.

Chapter 54: Subtype selective muscarinic agonists: potential therapeutic agents for Alzheimer's disease

Publisher Summary Senile dementia of Alzheimer type (AD) is a progressive neurodegenerative disease of unknown etiology. AD and related dementias are characterized by significant neuronal pathology in discrete cortical and subcortical brain regions. The basal forebrain cholinergic system, among other neurotransmitter systems, is severely affected in Alzheimers disease. Significant loss of forebrain cholinergic neurons accompanied by decreased neocortical choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, the major anabolic and catabolic enzymes for acetylcholine, is consistently seen in the brains of demented subjects. Moreover, reduction in ChAT and AChE activity are correlated with the degree of dementia and severity of neuropathological hallmarks of AD. A close association, therefore, exists between cholinergic biochemical abnormalities and this disease. Replacement of lost cholinergic function should provide palliative relief from the cognitive symptoms accompanying AD. Several approaches to cholinergic replacement have been tried in AD. Clinical studies with cholinesterase inhibitors and acetylcholine releasing agents have shown significant, albeit weak activity. The activity of AChE inhibitors and ACh releasing agents is dependent on the state of forebrain cholinergic neurons. In contrast, postsynaptic muscarinic receptors on cholinoceptive neurons in the neocortex and hippocampus are relatively spared in AD. Agents acting directly at these sites and mimicking the actions of acetylcholine should restore lost cholinergic function and retain efficacy throughout AD. Unfortunately, reducing theory to practice in this area has been difficult and the clinical efficacy of muscarinic agonists in AD is equivocal. The limited clinical utility of muscarinic agonists can be attributed to their propensity to induce unwanted cholinergic-mediated parasympathetic effects— that is, (nausea, hypersalivation, sweating), poor oral bio-availability, short durations of action and perhaps subtype selectivity for receptors mediating unwanted effects. This limited clinical utility should not be surprising since these are old agents that were not specifically designed for the use in treating the cognitive decline associated with aging and dementia. Newer muscarinic agonists with improved efficacy to side effect ratios and optimized durations of action are needed.

A silica gel plate-based qualitative assay for acetylcholinesterase activity : a mass method to screen for potential inhibitors

A procedure for the qualitative assessment of inhibitory activity towards acetylcholinesterase for a given compound is described. Solutions of the compounds of interest are spotted on silica gel TLC plates in a matrix pattern. The silica gel plate is sprayed with a solution of acetylthiocholine iodide and 5,5-dithiobis(2-nitrobenzoic acid) followed by a solution of acetylcholinesterase. The enzyme reaction produces a yellow background color with inhibitor compounds exposed as white zones where color has failed to develop. The results for a test set of compounds were compared to those obtained using the standard Ellman assay procedure and found to agree for virtually all of these compounds. The conditions of silica gel plate thickness, reagent concentration, and enzyme source under which this procedure is suitable were investigated. This represents an extremely rapid method to screen large numbers of compounds to uncover new inhibitors of acetylcholinesterase and potentially other enzymes as well.

Differential expression of group I metabotropic glutamate receptors (mGluRs) in the rat pheochromocytoma cell line PC12: role of nerve growth factor and ras

Glutamate treatment of PC12 cells has been shown to result in the accumulation of intracellular inositol phosphates suggesting the presence of glutamate metabotropic receptors (mGluRs) positively coupled to phospholipase C. The present study examined the expression of group I mGluRs (mGluR1 and mGluR5) in PC12 cells. Undifferentiated PC12 cells were found to express both mGluR5 mRNA and receptor protein by reverse transcription polymerase chain reaction (RT-PCR) and western blot techniques. However, mGluR1 mRNA was not detected in these cells and western blot analysis showed only faint mGluR1alpha immunoreactivity suggesting a very low level of mGluR1 expression. Nerve growth factor-induced differentiation of PC12 cells resulted in the induction of mGluR1alpha and mGluR1beta mRNA and mGluR1alpha protein. PC12 cells overexpressing dominant negative ras revealed that NGF-induced mGluR1 induction, but not mGluR5 expression, is dependent on ras pathway activation in these cells. These results suggest PC12 cells may be a useful model for investigating the regulation and expression of group I mGluR isoforms and their role in neuronal processes in vitro.

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.

Synthesis and Sar of bulky 1-azabicyclo[2.2.1]-3-one oximes as muscarinic receptor subtype selective agonists

The synthesis of a series of potent and efficacious 1-azabicyclo[2.2.1]heptan-3-one oxime muscarinic agonists is described. The oximes have extended appendages designed to span the cavity defined by the seven transmembrane helices of the muscarinic receptor. Some members of the series are selective for receptors of the m1 subtype. One such oxime, 31, shows affinity and functional selectivity for m1 over m2, m3, and m4 muscarinic receptor types.