Robin Douglas Clark

The interaction of RS 25259‐197, a potent and selective antagonist, with 5‐HT3 receptors, in vitro

1 A series of isoquinolines have been identified as 5‐HT3 receptor antagonists. One of these, RS 25259‐197 [(3aS)‐2‐[(S)‐1‐azabicyclo[2.2.2]oct‐3‐yl]‐2,3,3a,4,5,6‐hexahydro‐1‐oxo‐1H‐benzo[de]isoquinoline‐hydrochloride], has two chiral centres. The remaining three enantiomers are denoted as RS 25259‐198 (R,R), RS 25233‐197 (S,R) and RS 25233‐198 (R,S). 2 At 5‐HT3 receptors mediating contraction of guinea‐pig isolated ileum, RS 25259‐197 antagonized contractile responses to 5‐HT in an unsurmountable fashion and the apparent affinity (pKB), estimated at 10 nm, was 8.8 ± 0.2. In this tissue, the—log KB values for the other three enantiomers were 6.7 ± 0.3 (R,R), 6.7 ± 0.1 (S,R) and 7.4 ± 0.1 (R,S), respectively. The apparent affinities of RS 25259‐197 and RS 25259‐198, RS 25233‐197 and RS 25233‐198 at 5‐HT3 receptors in membranes from NG‐108‐15 cells were evaluated by a [3H]‐quipazine binding assay. The—log Ki values were 10.5 ± 0.2, 8.4 ± 0.1, 8.6 ± 0.1 and 9.5 ± 0.1, respectively, with Hill coefficients not significantly different from unity. Thus, at these 5‐HT3 receptors, the rank order of apparent affinities was (S,S) > (R,S) > (S,R) = (R,R). 3 RS 25259‐197 displaced the binding of the selective 5‐HT3 receptor ligand, [3H]‐RS 42358‐197, in membranes from NG‐108‐15 cells, rat cerebral cortex, rabbit ileal myenteric plexus and guinea‐pig ileal myenteric plexus, with affinity (pKi) values of 10.1 ± 0.1, 10.2 ± 0.1, 10.1 ± 0.1 and 8.3 ± 0.2, respectively. In contrast, it exhibited low affinity (pKi < 6.0) at 28 other receptors in binding assays, including adrenoceptors (α1A, α1B, α2A, α2B, β1, β2), muscarinic (M1—M4), dopamine (D1, D2), opioid and other 5‐HT (5‐HT1A, 5‐HT1D, 5‐HT2C, 5‐HT4) receptors. 4 RS 25259‐197 was tritium labelled (specific activity: 70 Ci mmol−1) and evaluated in pharmacological studies. Saturation studies with [3H]‐RS 25259‐197 in membranes from NG‐108‐15 and cloned homomeric α subunits of the 5‐HT3 receptor from N1E‐115 cells expressed in human kidney 293E1 cells, revealed an equilibrium dissociation constant (Kd) of 0.05 ± 0.02 and 0.07 ± 0.01 nm, and Bmax of 610 ± 60 and 1068 ± 88 fmol mg−1, respectively. Competition studies in NG‐108‐15 cells indicated a pharmacological specificity entirely consistent with labelling a 5‐HT3 receptor, i.e. RS 25259‐197 > granisetron > (S)‐zacopride > tropisetron > (R)‐zacopride > ondansetron > MDL 72222. 5 In contrast to the majority of radioligands available to label 5‐HT3 receptors, [3H]‐RS 25259‐197 labelled a high affinity site in hippocampus from human post‐mortem tissue with an equilibrium dissociation constant (Kd) of 0.15 ± 0.07 nm and density (Bmax) of 6.8 ± 2.4 fmol mg−1 protein. Competition studies in this tissue indicated a pharmacological specificity consistent with labelling of a 5‐HT3 receptor. 6 Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5‐HT3 receptor sites by [3H]‐RS 25259‐197. High densities of sites were seen in nuclear tractus solitaris and area postrema, a medium density in spinal trigeminal tract, ventral dentate gyrus and basal medial amygdala, and a low density of sites in hippocampal CA1, parietal cortex, medium raphe and cerebellum. 7 In conclusion, the functional, binding and distribution studies undertaken with the radiolabelled and non‐radiolabelled RS 25259‐197 (S,S enantiomer) established the profile of a highly potent and selective 5‐HT3 receptor antagonist.

Pharmacological characterization of two novel and potent 5-HT4 receptor agonists, RS 67333 and RS 67506, in vitro and in vivo.

1 The pharmacology of two novel 5‐HT4 receptor agonists, RS 67333 (1‐(4‐amino‐5‐chloro‐2‐methoxy phenyl)‐3‐[1(n‐butyl)‐4‐piperidinyl]‐1‐propanone HC1) and RS 67506 (1‐(4‐amino‐5‐chloro‐2‐methoxy‐phenyl)‐3‐[1‐(2‐methyl sulphonylamino)ethyl‐4‐piperidinyl]‐1‐propanone HC1) have been assessed in vitro and in vivo. 2 RS 67333 and RS 67506 exhibited affinities (pK1 = 8.7 and 8.8, respectively) for the 5‐HT4 binding sites, labelled with [3H]‐GR 113808, in guinea‐pig striatum. The Hill coefficients from these displacement curves were not significantly different from unity. The compounds exhibited lower affinities (<6.0) at several other receptors including 5‐HT1A, 5‐HT1D, 5‐HT2A, 5‐HT2C, dopamine D1, D2 and muscarinic Mr‐M3 receptors. However, RS 67333 and RS 67506 did exhibit affinities for the σ (pK1 = 8.9 and 7.9, respectively) and a2 (pK1 = 8.0 and 7.3, respectively) binding sites. 3 At the 5‐HT4 receptor mediating relaxation of the carbachol‐precontracted oesophagus, RS 67333 and RS 7506 acted as potent (pEC50 8.4 and 8.6, respectively), partial agonists (intrinsic activities, with respect to 5‐HT were 0.5 and 0.6, respectively) with respect to 5‐HT. Relaxant responses to RS 67333 or RS 67506 were surmountably antagonized by GR 113808 (10 nM), with apparent affinities (pKB) of 9.1 and 9.0, respectively. RS 67333 and RS 67506 induced dose‐dependent increases in heart rate of the anaesthetized micropig (ED50 4.9 and 5.4 ug kg−1, i.v.), with maximal increases of 35 and 47 beats min−1, respectively. 4 RS 67333 and RS 67506, therefore, acted as potent, partial 5‐HT4 receptor agonists in vitro and in vivo. These compounds, by virtue of their high potency and selectivity, may have some utility in elucidating the physiological role of 5‐HT4 receptors.

Pharmacological characterization of RS 25259‐197, a novel and selective 5‐HT3 receptor antagonist, in vivo

1 The pharmacological effects in vivo, of RS 25259‐197, a selective 5‐HT3 receptor antagonist, have been investigated. 2 In anaesthetized rats, RS 25259‐197, administered by the intravenous, intraduodenal or transdermal route, dose‐dependently inhibited the von Bezold‐Jarisch reflex induced by 2‐methyl 5‐HT (ID50 = 0.04 μg kg−1, i.v., 3.2 μg kg−1, i.d. and 32.8 μg per chamber, respectively). In this regard, when administered intraduodenally, RS 25259‐197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. 3 In conscious ferrets, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin. The ID50 estimates of RS 25259‐197 were 1.1 μg kg−1, i.v. and 3.2 μg kg−1, p.o. In this respect, RS 25259‐197 was more potent than ondansetron and equipotent with granisetron. 4 In conscious dogs, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin (ID50 = 1.9 μg kg−1, i.v. and 8.5 μg kg−1, p.o.), dacarbazine (ID50 = 4.1 μg kg−1, i.v. and 9.7 μg kg−1, p.o.), actinomycin D (ID50 = 4.9 μg kg−1, i.v. and 2.5 μg kg−1, p.o.) and mechlorethamine (ID50 = 4.4 μg kg−1, i.v. and 3.0 μg kg−1, p.o.). Against each of the emetogenic agents, RS 25259‐197 was very much more potent than ondansetron. When tested at equi‐effective intravenous doses against cisplatin‐induced emesis in dogs, RS 25259‐197 had a longer duration of anti‐emetic activity (7 h) than ondansetron (4 h). At doses up to and including 1000 μg kg−1, p.o., neither RS 25259‐197 nor ondansetron was capable of inhibiting apomorphine‐induced emesis. 5 At doses up to 1000 μg kg−1, i.v., RS 25259‐197 produced no meaningful haemodynamic changes in anaesthetized dogs. 6 In summary, RS 25259‐197 is a novel, highly potent and orally active 5‐HT3 receptor antagonist in vivo. With respect to its anti‐emetic activity, RS 25259‐197 appears to be a significant improvement over ondansetron in terms of potency and duration of action.

RS 39604 : a potent, selective and orally active 5-HT4 receptor antagonist

1 Selective antagonism of 5‐HT4 receptors may provide therapeutic benefit in certain disorders of the myocardium, alimentary and lower urinary tract. We now report on RS 39604, a novel and selective 5‐HT4 receptor antagonist and compare its pharmacological properties with those of SB 204070. 2 In guinea‐pig striatal membranes, both RS 39604 and SB 204070 inhibited specific binding of [3H]‐GR 113808 in a concentration‐dependent manner yielding p Ki estimates of 9.1 and 10.9, respectively. RS 39604 displayed a low affinity (pKi <6.5) for 5‐HT1A, 5‐HT2C, 5‐HT3, α1c, D1? D2, M1 M2, AT1 B1 and opioid u receptors and moderate affinity for σ1 (pK1 = 6.8) and σ2 (pK1 = 7.8) sites. 3 In the rat isolated oesophagus, precontracted with carbachol, RS 39604 (30–300 nM) behaved as a competitive antagonist towards 5‐HT‐induced relaxation (pA2 = 9.3; Schild slope =1.0). We and others have shown previously that SB 204070 behaves as an unsurmountable antagonist in this preparation (pA2‐10.5). In the guinea‐pig isolated ileal mucosa, RS 39604 (30 nM) antagonized 5‐MeOT‐induced increase in short‐circuit current (pA2 = 9.1). 4 In anaesthetized vagotomized micropigs, RS 39604, administered by the i.v. or intraduodenal (i.duod.) route, produced dose‐dependent inhibition of 5‐HT‐induced tachycardia (ID50 = 4.7 μg kg−1, i.v. and 254.5 ug kg−1, i.duod). At maximal doses of 30 μg kg−1, i.v. and 6 mg kg−1, i.duod., the inhibitory effects of RS 39604 lasted for more than 6 h. In this preparation, SB 204070 was as potent as RS 39604 by the i.v. route but was inactive by the intraduodenal route at doses up to 3 mg kg−1. 5 In conscious mice, RS 39604, administered by the i.p. or p.o. route, produced dose‐dependent inhibition of 5‐hydroxytryptophan (5‐HTP)‐induced diarrhoea (ID50 = 81.3 ug kg−1, i.p. and 1.1 mg kg−1, p.o.). In this assay, SB 204070 was inactive by the oral route at doses up to 30 mg kg−1. 6 In anaesthetized guinea‐pigs, RS 39604 antagonized the contractile effect of 5‐HT in the proximal colon by producing parallel, dextral displacement of the dose‐response curve to 5‐HT. The mean dose‐ratios to 5‐HT at 0.1 mg kg−1, i.v., 1 mg kg−1, i.v. and 10 mg kg−1, i.duod. were 4.6, 30.7 and 10.8, respectively. SB 204070 behaved as an unsurmountable antagonist in this assay. 7 In a model of visceral pain in conscious rats, RS 39604 (0.01‐1 mg kg−1, i.v.) did not affect colorectal distension‐induced increases in arterial pressure whereas morphine (1 mg kg−1, i.v.) produced significant inhibition of the response, implying that 5‐HT4 receptors are not involved in nociception in this model. 8 The data suggest that RS 39604 is a high affinity and selective 5‐HT4 receptor antagonist that is orally active and long‐lasting in vivo. It is concluded that RS 39604 may be the preferable probe to use for investigating the physiological and pathophysiological role of 5‐HT4 receptors in vivo.

RS 23597-190: a potent and selective 5-HT4 receptor antagonist.

1 The pharmacological properties of RS 23597‐190 (3‐(piperdine‐1‐yl)‐propyl‐4‐amino‐5‐chloro‐2‐methoxy benzoate hydrochloride) have been studied in vitro and in vivo. 2 RS 23597‐190 competitively antagonized 5‐HT4 receptor‐mediated relaxations of rat, carbachol precontracted oesophageal muscularis mucosae, (pA2 = 7.8 ± 0.1; Schild slope = 1.2 ± 0.2). Affinity estimates (−log KB) at 5‐HT4 receptors using either renzapride or SC‐53116 as agonists yielded a −log KB value of 8.0 ± 0.01. In contrast, RS 23597‐190 failed to antagonize contractile responses to 5‐HT of guinea‐pig ileal 5‐HT3 receptors, even at concentrations up to 10 μm. 3 Increases in short‐circuit current, induced by 5‐HT, were studied in guinea‐pig ileal mucosal sheets. Concentration‐response curves to 5‐HT were biphasic, with the high potency phase to 5‐HT inhibited by RS 23597‐190 and mimicked by 5‐methoxytryptamine. The −log KB value for RS 23597‐190 at the high potency phase was 7.3 confirming that 5‐HT4 receptors mediated the high potency phase. 4 In rat isolated vagus nerve, 5‐HT elicited a slow, maintained depolarization at low concentrations and a rapid, transient depolarization at higher concentrations. The high potency, slow depolarizing phase to 5‐HT was abolished selectively in the presence of 1 μm RS 23597‐190 and the low potency phase was abolished selectively in the presence of 1 μm ondansetron. These data confirm that 5‐HT4 and 5‐HT3 receptors mediated slow and fast depolarization responses, respectively. 5 At 5‐HT3 binding sites in membranes from NG 108‐15 cells, labelled by [3H]‐quipazine, RS 23597‐190 exhibited an apparent affinity (−log Ki) of 5.7 ± 0.1. At 5‐HT3 receptors in membranes from rat cerebral cortex, labelled by [3H]‐RS 42358‐197, the apparent affinity (−log Ki) of RS 23597‐190 was also 5.7 ± 0.1. In both studies, Hill coefficients were not significantly different from unity. At 5‐HT1A, 5‐HT2, muscarinic M15 M2, M3, M4 and dopamine D1 and D2 receptors, RS 23597‐190 exhibited low apparent affinities, with all −log Ki values less than 5.5. 6 Intravenous infusion of RS 23597‐190 in the conscious, restrained rat antagonized the von Bezold Jarisch reflex induced by 2‐methyl 5‐HT, with an ID50 of 300 μg kg−1 min−1, i.v. In the anaesthetized, bilaterally vagotomized micropig, RS 23597‐190 (6 mg kg−1, i.v.) antagonized 5‐HT‐induced tachycardia with a half‐life of 77 (63–99) min. Transient arrhythmic effects were noted after administration of the compound. 7 In conclusion, RS 23597‐190 acts as a high affinity, selective competitive antagonist at 5‐HT4 receptors. Thus, the compound appears to be a useful tool for 5‐HT4 receptor identification in vitro. In vivo, the compound is rapidly metabolized in pigs such that 5‐HT4 blockade is not maintained. However, in the rat, when given by infusion, RS 23597‐190 antagonizes 5‐HT3 mediated responses, at doses consistent with a low affinity 5‐HT3 receptor. These data suggest that, under appropriate experimental conditions, RS 23597‐190 may also be used in vivo to characterize further 5‐HT4 receptor function.

Investigation of the prostacyclin (IP) receptor antagonist RO1138452 on isolated blood vessel and platelet preparations

The current study examined the utility of the recently described prostacyclin (prostanoid IP) receptor antagonist RO1138452 (2‐(4‐(4‐isopropoxybenzyl)‐phenylamino) imidazoline) as a tool for classifying prostanoid receptors.

Evaluation in rat of RS-79948-197 as a potential PET ligand for central α2-adrenoceptors

Tritium-labelled RS-79948-197 {(8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine} was evaluated in rat brain as an in vivo ligand for central α2-adrenoceptors, as a preliminary step in the development of a radioligand for positron-emission tomography (PET) studies. The maximal receptor-specific signal was achieved within 90–120 min after i.v. injection of [ethyl-3H]RS-79948-197 and was selective for the α2-compared with the α1-adrenoceptor, with no detectable binding to the imidazoline-I2 site. Estimates for binding potential (approximating to BmaxKd) ranged between 3.4 in entorhinal cortex and 0.5 in medulla oblongata. The results, which indicate a similarly localised but 2-fold increase in specific binding compared with that previously demonstrated using [3H]RX 821002 (2-methoxy-idazoxan), are sufficiently encouraging as to support further investment in the development of 11C-labelled RS-79948-197, or a close structural analogue, as a ligand for clinical PET.

Evaluation of [O-methyl-11C]RS-15385-197 as a positron emission tomography radioligand for central α2-adrenoceptors

Abstract.Carbon-11 labelled RS-15385-197 and its ethylsulphonyl analogue, RS-79948-197, were evaluated in rats as potential radioligands to image central α2-adrenoceptors in vivo. The biodistributions of both compounds were comparable with that obtained in an earlier study using tritiated RS-79948-197 and were consistent with the known localisation of α2-adrenoceptors. The maximal signals (total to non-specific binding) were, however, reduced, in the order [11C]RS-79948-197 < [11C]RS-15385-197 < [3H]RS-79948-197, primarily due to the difference in radiolabel position (O-methyl for carbon-11 compared with S-ethyl for tritium). This resulted in the in-growth of radiolabelled metabolites in plasma, which, in turn, contributed to the non-specific component of brain radioactivity. Nonetheless, the signal ratio of ∼5 for a receptor-dense tissue compared with the receptor-sparse cerebellum, at 90–120 min after radioligand injection, encouraged the development of [O-methyl-11C]RS-15385-197 for human positron emission tomography (PET). Unfortunately, in two human PET scans (each of 90 min), brain extraction of the radioligand was minimal, with volumes of distribution more than an order of magnitude lower than that measured in rats. Following intravenous injection, radioactivity was retained in plasma and metabolism of the radiolabelled compound was very low. Retrospective measurements of in vitro plasma protein binding and in vivo brain uptake index (BUI) in rats demonstrated a higher protein binding of the radioligand in human compared with rat plasma and a lower BUI in the presence of human plasma. It is feasible that a higher affinity of RS-15385-197 for human plasma protein compared with receptor limited the transport of the radioligand. Although one of the PET scans showed a slight heterogeneity in biodistribution of radioactivity which was consistent with the known localisation of α2-adrenoceptors in human brain, it was concluded that [O-methyl-11C]RS-15385-197 showed little promise for routine quantification of α2-adrenoceptors in man.

(R)-3-(6-CHLORO-1-ISOPROPYLBENZIMIDAZOLE-4-CARBOXAMIDO)QUINUCLIDINE : A HIGH AFFINITY LIGAND FOR THE (R)-ZACOPRIDE BINDING SITE

Abstract The (R)-3-amido quinuclidine 6 (RS-16566) was found to be a high affinity ligand for the (R)-zacopride binding site.