Kathy M. O'Boyle

Drugs acting on brain dopamine receptors: a conceptual re-evaluation five years after the first selective D-1 antagonist.

Neurochimie, neuro-anatomie et neurobiologie des recepteurs dopaminergiques D1 et D2: selectivite des medicaments in vivo et in vitro, caracteristiques moleculaires, localisation, effets du developpement et du vieillissement sur ces recepteurs, regulation. Etudes fonctionnelles des recepteurs D1 et D2 en utilisant des medicaments selectifs

Effects of the putative D-1 antagonist SCH 23390 on stereotyped behaviour induced by the D-2 agonist RU24213

The effects of the putative selective dopamine D-1 antagonist benzazepine SCH 23390 and of the selective dopamine D-2 antagonist Ro22-2586 on stereotypy induced by the selective D-2 agonist RU24213 were compared. RU24213 (0.5–15 mg/kg) dose-dependently induced stereotyped behaviour characterised by continuous downward sniffing and locomotion. These responses were antagonised, as expected, by 40–200 μg/kg Ro22-2586, but surprisingly blocked by 40–200 μg/kg SCH 23390. The selectivities of these compounds for dopamine receptor subtypes were verified in terms of their relative abilities to displace the in vitro binding of 3H-piflutixol to striatal D-1 receptors and of 3H-spiperone to D-2 receptors. As SCH 23390 fails to influence D-2 mediated prolactin secretion or emesis in vivo, there appears to be no significant formation of an active metabolite of SCH 23390 with D-2 antagonist activity. Because SCH 23390 has some affinity for 5-hydroxytryptamine receptors, any effect on the serotonergic behavioural syndrome induced by 10 mg/kg 5-methoxy-N,N-dimethyltryptamine was also studied. The serotonergic responses of hind limb abduction, reciprocal forepaw treading and Straub tail were unaltered after 40–200 μg/kg SCH 23390, indicating no significant 5-HT blockade or non-specific depressant action at these doses which might influence the expression of stereotypy. Thus, these data are consistent with blockade of tonic D-1 dopaminergic activity that may influence the expression of behaviours initiated byD-2 dopaminergic stimulation.

Agonist and antagonist properties of benzazepine and thienopyridine derivatives at the D1 dopamine receptor

Nine structurally related 1-phenyl-1H-3-benzazepine derivatives and two thienopyridines were tested for agonist and antagonist properties at the adenylate cyclase-coupled D1 dopamine receptor in homogenates of the striatum of the rat. The benzazepines SK&F 77434 and SK&F 82958, both of which contain a catechol ring, were agonists; the intrinsic activity of SK&F 77434 was similar to that of SK&F 38393, whereas SK&F 82958 was a full agonist. The remaining benzazepines inhibited the stimulation of adenylate cyclase by dopamine. Antagonist potency depended on the nature of the substituent at position 7 of the benzazepine molecule, 7-halogen compounds being the most potent. The Ki values, obtained from analysis of the antagonism of dopamine-stimulated adenylate cyclase, were significantly correlated with the Ki values for displacement of D1 ligands in binding experiments. Furthermore, antagonist activity of the resolved racemic benzazepine SK&F 83566 resided almost exclusively in the R-enantiomer. The thienopyridine derivatives SK&F 89641 and SK&F 89145 were partial agonists with greater efficacies than SK&F 38393.

Loss of rat striatal dopamine receptors with ageing is selective for D-2 but not D-1 sites: association with increased non-specific binding of the D-1 ligand [3H]piflutixol.

The effects of age on the binding of dopaminergic ligands to rat striatal membranes were studied. When compared with four month old animals, at 22 months there was a significant decrease in the density of specific binding sites for the D-2 ligand [3H]spiperone while their affinity was unaltered. However, the specific binding of [3H]piflutixol to D-1 sites was unaltered between these age groups. The non-specific binding of [3H]piflutixol was significantly increased in aged preparations. Age-related loss of striatal D-2 dopamine receptors does not extend to the D-1 type. Alterations in non-specific [3H]piflutixol binding may reflect other pathophysiological changes associated with senescence.

Locomotor behaviors in response to new selective D-1 and D-2 dopamine receptor agonists, and the influence of selective antagonists

With the introduction of the selective D-1 dopamine receptor agonist and antagonist benzazepines, especially as enantiomeric pairs, there is now a range of D-1 compounds to complement the previously available selective D-2 agents. These have been used to investigate whether sub-types of dopamine receptors might be differentially involved in locomotor behavior. Stereotyped locomotion induced by the non-selective D-2 agonist apomorphine and by the selective D-2 agonist RU 24213 were blocked by the selective D-2 antagonists metoclopramide and Ro 22-2586 [-)-piquindone). Responses to either D-2 agonist were also blocked by the selective D-1 antagonists SCH 23390 and R-(but not S-) SK&F 83566. Non-stereotyped locomotion was induced by R- but not S-SK&F 38393, a stereoselective D-1 agonist, and was blocked by SCH 23390. Responses to the D-1 agonist were also antagonised by metoclopramide. Such results suggest concerted D-1:D-2 interplay in the regulation of at least some dopaminergic behaviors, such as locomotion.

Selective and stereospecific interactions of R-SK&F 38393 with [3H]piflutixol but not [3H]spiperone binding to striatal D1 and D2 dopamine receptors: Comparisons with SCH 23390

Four benzazepine derivatives, racemic SK&F 38393, its resolved R- and S-enantiomers, and SCH 23390 have been investigated for their interactions with striatal D1 and D2 dopamine receptors, as indexed by the binding of [3H] piflutixol and [3H]spiperone respectively. For the agonist SK&F 38393, its R-enantiomer was active in displacing [3H] piflutixol while its S- antipode was 100 fold less potent. In contrast, both enantiomers showed similar and negligible activity to displace [3H]spiperone. SCH 23390, an antagonist analogue with an R-configuration, potently displaced [3H] piflutixol but not [3H]spiperone. R-SK&F 38393 and SCH 23390 may help clarify the structural requirements for, and functional consequences of, selective actions at the D1 dopamine receptor.

Cholinergic activation of Cl- secretion in rat colonic epithelia

Acetylcholine receptor agonists and antagonists were used in a pharmacological analysis to identify which muscarinic receptor(s) may be involved in cholinergic regulation of Cl- secretion across rat colonic mucosa in vitro. A comparative ligand binding analysis for each of the antagonists was carried out in parallel. Both studies elicited identical rank order potencies (atropine > or = 4-diphenyl-acetoxy-N-piperidine methiodide (4-DAMP) > pirenzepine > 11-[[2[(diethylamino)methyl]-1-pipiridinyl]acetyl[5,11- dihydro-6H-pyrido[2,3-b]]1,4]benzodiazepine-6-one (AF-DX 116). Cholinomimetic-induced Cl- secretion was predominantly mediated by activation of muscarinic receptors in rat isolated colonic mucosa, with only a modest contribution from nicotinic receptors. Short circuit current responses evoked by the selective muscarinic M1 receptor agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-a minium chloride (McN-A-343) suggest that this receptor subtype, which is thought to be neuronally sited, also plays a minor role in regulation of intestinal ion transport. The principal epithelial cell receptors responsible for acetylcholine receptor-mediated Cl- secretion appear to belong to the M3 class.

Adenosine A1 receptor‐mediated inhibition of dopamine release from rat striatal slices is modulated by D1 dopamine receptors

Dopamine release is regulated by presynaptic dopamine receptors and interactions between adenosine and dopamine receptors have been well documented. In the present study, dopamine release from isolated striatal slices from Wistar rats was measured using fast cyclic voltammetry. Single‐pulse stimulation (0.1 ms, 10 V) was applied every 5 min over a 2‐h period. Superfusion with the adenosine (A)1 receptor agonist N6‐cyclopentyladenosine (CPA), but not the A2 receptor agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino]ethyl] phenyl]propanoic acid (CGS 21680), inhibited dopamine release in a concentration‐dependent manner (IC50 3.80 × 10−7 m; n = 10). The dose–response curve to CPA was shifted to the right (IC50 6.57 × 10−6 m; n = 6, P < 0.05 vs. control) by the A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Neither the D1 agonist 6‐chloro‐APB nor the D1 antagonist R‐(+)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1H‐3‐ benzazepine‐7‐ol (SCH 23390) altered dopamine release on their own. However, SCH 23390 (3 µm) significantly attenuated the response to CPA (IC50 1.44 × 10−5 m; n = 6, P < 0.01 vs. control). Furthermore, the inhibitory effect of CPA was significantly increased in the presence of 6‐chloro‐APB (1 µm). In radioligand binding experiments, CPA interacted with high‐ and low‐affinity states of [3H]DPCPX‐lableled A1 receptors. The high‐affinity agonist binding to A1 receptors was inhibited by the stable guanosine triphosphate analogue Gpp(NH)p. In contrast, neither the proportion nor the affinity of high‐affinity A1 receptors was altered by dopamine or SCH 23390. These results provide evidence that the inihibition of dopamine release by adenosine A1 receptors is dependent, at least in part, on the simultaneous activation of D1 dopamine receptors. While the mechanism underlying this interaction remains to be determined, it does not appear to involve an intramembrane interaction between A1 and D1 receptors.

[3H]SCH 23390 Binding to Human Putamen D-1 Dopamine Receptors: Stereochemical and Structure-Affinity Relationships Among l-Phenyl-1H-3-Benzazepine Derivatives as a Guide to D-l Receptor Topography

Abstract: A series of l‐phenyl‐1H‐3‐benzazepine analogues were assessed for enantiomeric and structure‐affinity relationships at human putamen D‐1 dopamine receptors labelled with [3H]SCH 23390. Substitution at the 7‐position of both 3‐H and 3‐methyl benzazepine molecules critically affected affinity for these receptors over a 500‐fold range. The general rank order of potency of 7‐substituents was Cl = Br ≫ CH3 > OH ≥ H. 3‐Methyl substituents increased the affinity of 7‐H and 7‐OH compounds two‐ to fivefold compared to desmethyl counterparts. The displacement of [3H]SCH 23390 binding showed substantial enantioselec‐tivity; the R‐enantiomer of SKF 83566 was 500‐fold more potent that its S‐antipode. However, the displacement of [3H]spiperone binding from D‐2 sites in the same tissue showed negligible enantioselectivity. Through such structure‐affinity relationships, these studies may help to define the topography of the human brain D‐1 dopamine receptor and guide the design of more selecive agents for functional studies.

Discovery of a dual action first-in-class peptide that mimics and enhances CNS-mediated actions of thyrotropin-releasing hormone

Thyrotropin-releasing hormone (TRH) displays multiple CNS-mediated actions that have long been recognized to have therapeutic potential in treating a wide range of neurological disorders. Investigations of CNS functions and clinical use of TRH are hindered, however, due to its rapid degradation by TRH-degrading ectoenzyme (TRH-DE). We now report the discovery of a set of first-in-class compounds that display unique ability to both potently inhibit TRH-DE and bind to central TRH receptors with unparalleled affinity. This dual pharmacological activity within one molecular entity was found through selective manipulation of peptide stereochemistry. Notably, the lead compound of this set, L-pyroglutamyl-L-asparaginyl-L-prolyl-D-tyrosyl-D-tryptophan amide (Glp-Asn-Pro-D-Tyr-D-TrpNH(2)), is effective in vivo at producing and potentiating central actions of TRH without evoking release of thyroid-stimulating hormone (TSH). Specifically, this peptide displayed high plasma stability and combined potent inhibition of TRH-DE (K(i) 151 nM) with high affinity binding to central TRH receptors (K(i) 6.8 nM). Moreover, intraperitoneal injection of this peptide mimicked and augmented the effects of TRH on behavioural activity in rat. Analogous to TRH, it also antagonized pentobarbital-induced narcosis when administered intravenously. This discovery provides new opportunities for probing the role of TRH actions in the CNS and a basis for development of novel TRH-based neurotherapeutics.