Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer's disease.

It could be argued that clinical experience with cholinergic drugs in the therapy of AD has not yet shown relevant symptomatic improvements. The main reasons for this might be attributed to peripheral cholinergic effects and the liver toxicity of some of these drugs, which limit their use and prevent confirmation of the cholinergic hypothesis (Gray et al., 1989). The main disadvantages of the cholinesterase inhibitors used in clinical trials are the short duration of action in the case of physostigmine and the potential for liver toxicity seen with the aminoacridine derivatives. The results presented with SDZ ENA 713 indicate that the disadvantages of AChE inhibitors might be overcome by improving CNS selectivity and thereby decreasing the peripheral cholinergic effects and toxicity. Clinico-pharmacological studies with SDZ ENA 713 have been performed in healthy volunteers; while central activity was clearly demonstrated in an EEG-sleep study (Holsboer et al., 1992), no prohibitive peripheral side effects were seen, confirming in humans the results obtained in experimental animals (Enz et al., 1991). A multicentre clinical investigation in AD patients has been performed in Europe and is currently being evaluated.

Muscarinic activity of the thiolactone, lactam, lactol, and thiolactol analogues of pilocarpine and a hypothetical model for the binding of agonists to the m1 receptor.

Pilocarpine isosteres have been synthesized and characterized with regard to their in vitro muscarinic properties. The results indicate that the carbonyl oxygen of the lactone function of pilocarpine is of primary importance for agonist activity with the ether oxygen being of lesser or secondary importance. An X-ray structure determination for the hydrogen O,O'-ditoluoyltartrate salt of thiolactone pilocarpine isostere 2a has been performed. This compound has an unusual pharmacological profile exhibiting M1-agonist selectivity as well ass presynaptic antagonism. As a result this compound is also viewed as having therapeutic potential for Alzheimer's disease. A model for the binding of pilocarpine and other muscarinic agonists to the third transmembrane helix of the human m1 muscarinic receptor has been developed.

Presynaptic muscarinic receptors mediating inhibition of neurogenic contractions in rabbit vas deferens are of the ganglionic M1-type.

The present study was designed to further characterize the presynaptic muscarinic M1-receptor responsible for the inhibition of neurogenic contractions in the isolated rabbit vas deferens. Electrically induced twitch contractions of this preparation were inhibited by the M1-agonist, McN-A-343, and by some of its analogs: 4-chloro-phenyl derivative greater than McN-A-343 greater than trans-olefinic analog greater than cis-olefinic analog. The same rank order of potency was observed for these agonists to raise the blood pressure of pithed rats by stimulation of M1-receptors in sympathetic ganglia. A highly significant correlation was found between the antimuscarinic potencies of atropine, pirenzepine and a series of 9 antagonists structurally related to the ganglionic M1 beta-receptor selective compounds, hexocyclium and hexahydro-difenidol, to antagonize the McN-A-343-induced inhibition of twitch contractions in rabbit vas deferens or the muscarine-induced depolarization in rat isolated superior cervical ganglia. It is suggested that the presynaptic muscarinic receptor that mediates inhibition of neurogenic contractions in rabbit vas deferens is of the ganglionic M1 beta-type.

The pharmacological assessment of RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion hydrobromide). A potent, specific muscarinic acetylcholine receptor agonist.

The effects of the compound RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion hydrobromide) in a number of in vitro and in vivo test systems for muscarinic cholinergic activity were analyzed and compared to those of classical muscarinic receptor agonists. In radioligand binding assays RS 86 presented high nanomolar apparent affinity only for sites labeled by 3H-muscarinic receptor agonists while its apparent affinity for sites labeled by 3H-muscarinic receptor antagonists including [3H]QNB, [3H]NMS and [3H]pirenzepine was in the micromolar range. RS 86 had no or only low affinity (IC50 greater than 10 microM) for other neurotransmitter or drug receptor sites. The compound induced scopolamine-sensitive contractions of the isolated guinea-pig ileum showing a pD2 of 6 in this model. In the isolated rat superior cervical ganglion RS 86 was also an agonist with a pD2 of 6.7. When given to mice or rats by different routes RS 86 induced central and peripheral effects typical of a muscarinic receptor agonist, such as hypothermia, tremor, mydriasis, salivation, lacrimation, diarrhoea and modification of behavior as observed in an open field. In several of these tests RS 86 was about 10 times less potent than oxotremorine but more potent than arecoline, pilocarpine, aceclidine or the compound (cis) AF-30. The ED50 values for some central effects, including the induction of hypothermia and alert non-mobile behavior were lower than those for tremor and peripheral effects. Some of the effects lasted for up to 6 h, depending on the dose. Finally, RS 86 administration resulted in modifications of brain acetylcholine turnover and high affinity choline uptake typical of a central muscarinic receptor agonist. Taken together these results demonstrate clearly that RS 86 is a potent, centrally acting, selective muscarinic receptor agonist. RS 86 appears to be an adequate tool for the clinical examination of the cholinergic hypothesis of Alzheimer's disease.

Effects of gamma-aminobutyrate and bicuculline on primary afferent depolarization of cutaneous fibres in the cat spinal cord.

The excitability of single cutaneous primary afferent fibres (sural nerve) was tested by focal stimulation in the dorsal horn of the cat spinal cord, and recording the antidromically conducted action potential in the peripheral nerve. To induce primary afferent depolarization, which is an expression of presynaptic inhibition, the superficial peroneal nerve was stimulated. The primary afferent depolarization was measured as the concomitant excitability change in the antidromically excited sural fibre. This primary afferent depolarization was reduced by 32% during microelectrophoretic release of bicuculline methochloride near the microstimulation electrode in the dorsal horn. Microelectrophoresis of gamma-aminobutyrate increased excitability in sural nerve fibres which correlated with the primary afferent depolarization induced by stimulation of the superficial peroneal nerve. The results suggest a possible role for gamma-aminobutyrate in presynaptic inhibition of cutaneous afferent fibres in the cat.

Muscarinic receptors on intact, cultured neurons. Characterization by [3H]quinuclidinylbenzilate binding.

[3H]Quinuclidinylbenzilate (QNB) was used to identify muscarinic cholinergic receptors on intact, cultured neurons from fetal rat brains. Scatchard analysis revealed a single binding site with a dissociation constant Kd = approximately or equal to 170 pM. The rank order of potency of cholinergic drugs to displace [3H]QNB from intact neurons was similar to that observed using isolated membranes of brain homogenates. No difference in the rank order was observed with cultures of neurons from different brain regions which vary in their neuronal composition.