Helmut Ensinger

Pramipexole binding and activation of cloned and expressed dopamine D2, D3 and D4 receptors

Pramipexole (SND 919; 2-amino-4,5,6,7-tetrahydro-6-propylamino-benzthiazole-dihydrochlor ide) is a potent dopamine autoreceptor agonist. We have carried out an analysis of the binding affinities of dopamine D2L, D2S, D3, and D4 receptors for pramipexole using both [3H]pramipexole and [3H]spiperone as radioligands at cloned and heterologously expressed receptors. Studies were carried out using rat and human D2L, D2S and D3 receptors with equivalent results. When the binding of pramipexole to the high affinity, guanine nucleotide-sensitive state of each receptor was analyzed, pramipexole is most selective for D3 compared to D2 and D4 receptors. These results indicate a 5-fold selectivity of pramipexole for D3 receptors, while quinpirole and bromocriptine are non-selective or more D2/D4 receptor selective. Two measurements of receptor activation for dopamine D2, D3, and D4 receptors also show that pramipexole is most potent for activation of D3 receptors. The dopamine D3 receptor selectivity of pramipexole may explain the previously described properties of this drug, including its potent autoreceptor preference.

Cloning and functional characterization of a human A1 adenosine receptor.

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.

Pharmacodynamic profile of the M1 agonist talsaclidine in animals and man

In functional pharmacological assays, talsaclidine has been described as a functionally preferential M1 agonist with full intrinsic activity, and less pronounced effects at M2- and M3 receptors. In accordance with this, cholinomimetic central activation measured in rabbits by EEG recordings occurred at a 10 fold lower dose than that inducing predominantly M3-mediated side effects. This pharmacological profile is also reflected in the clinical situation: Both in healthy volunteers and in Alzheimer patients--unlike after unspecific receptor stimulation through cholinesterase inhibitors--the mainly M3-mediated gastrointestinal effects (like nausea and vomiting) were not dose-limiting. Rather, sweating and hypersalivation, mediated through muscarinic receptors, occurred dose-dependently and were finally dose-limiting. In contrast to talsaclidine, sabcomeline had a less pronounced functional M1 selectivity in pharmacological assays. This was also shown in anaesthetized guinea pigs where sabcomeline alone induced bronchoconstriction, and in the rabbit EEG where central activation and cholinergic side effects occurred in the same dose range. Neither drug, however, showed convincing improvement of cognitive functions in patients with mild-to-moderate Alzheimers disease. This asks for a reassessment of the muscarinic hypothesis for the treatment of this disease.

WAL 2014 FU (Talsaclidine) : A preferentially neuron activating muscarinic agonist for the treatment of Alzheimer's disease

The functional selectivity of WAL 2014 FU with regard to stimulation of the neuronal muscarinic M1 receptor subtype in vitro and in vivo is shown in different receptor preparations, isolated organ models, whole animal testing, and finally humans. From receptor binding experiments in membrane preparations from rat tissues and from Chinese hamster ovary cells expressing human muscarinic receptor subtypes, it can be delineated that the ratio between M1 and M2 (hm1 and hm2) is shifted in favour of the M1 receptor affinity, when compared to several classic muscarinic agonists such as carbachol, arecoline, and oxotremorine. The intermediate GTP‐shift of 7.5 for WAL 2014 FU in a M2 muscarinic receptor preparation (rat heart) indicates only partial agonistic activity at this subtype, carbachol (e.g., shows a shift of 51). Moreover, the ratio from agonist to antagonist receptor binding comparing the affinities using [3H]cis‐methyldioxolane and [3H]N‐methylscopolamine as radioligands, suggests only a partial agonist behaviour at M2 receptors, too.

[3H]Mr 2034 labels a high affinity opioid κ-receptor not accessible to naloxazone

Abstract Saturation binding studies with [ 3 H]Mr 2034, an opioid κ-agonist, were carried out using brain homogenates from naloxazone-pretreated rats (200 mg/kg subcutaneously 16 hours before killing). [ 3H ]Mr 2034 showed unaffected binding characteristics, compared to membrane homogenates of untreated controls. However, in contrast to this finding, naloxazone blocked the high affinity binding of other tritiated opioid agonists and antagonists, among them the κ-agonist [ 3 H]ethylketocyclazocine. Displacement studies with [ 3 H]dihydromorphine, [ 3 H]ethylketocyclazocine and [ 3 H]Mr 2034 in brain membrane homogenates from untreated rats and using various opioid agonists and antagonists indicated that these substances are capable of differentiating between the two κ-ligands [ 3 H]ethylketocyclazocine and [ 3 H]Mr 2034, too.