E. Leung

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.

Anandamide hydroxylation by brain lipoxygenase:metabolite structures and potencies at the cannabinoid receptor

Anandamide (arachidonyl ethanolamide) is a compound that was identified from porcine brain lipids by its ability to bind to the brain cannabinoid receptor. This study assessed anandamide as a substrate for a brain lipoxygenase and characterised the brain metabolite 12-hydroxyanandamide. Anandamide was also compared with arachidonic acid as a lipoxygenase substrate by examining enzyme kinetics in the presence of either of the two compounds. In addition, a non-mammalian enzyme was used to generate 11- and 15-hydroxy-anandamide in order to compare the cannabinomimetic properties of a range of anandamide derivatives. A ligand displacement assay indicated a large variation in the affinity of anandamide metabolites for the brain cannabinoid receptor. The brain metabolite, 12-hydroxyanandamide had an affinity twice that of anandamide, although the 11- and 15- hydroxy-metabolites were considerably poorer ligands of this receptor. Consistent with the receptor binding data, 12-hydroxyanandamide (unlike 15-hydroxyanandamide) inhibited forskolin-stimulated cAMP synthesis, indicating it to be a functional agonist at the brain cannabinoid receptor. Pharmacological studies of the capacity of anandamide and its metabolites to inhibit the murine vas deferens twitch response indicated the 12-hydroxy-metabolite to be less active than the parent compound, but a better cannabinomimetic than 15-hydroxyanandamide.

RS-127445: a selective, high affinity, orally bioavailable 5-HT2B receptor antagonist.

Efforts to define precisely the role of 5‐HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5‐HT2B receptor antagonists. RS‐127445 (2‐amino‐4‐(4‐fluoronaphth‐1‐yl)‐6‐isopropylpyrimidine) was found to have nanomolar affinity for the 5‐HT2B receptor (pKi=9.5±0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. In cells expressing human recombinant 5‐HT2B receptors, RS‐127445 potently antagonized 5‐HT‐evoked formation of inositol phosphates (pKB=9.5±0.1) and 5‐HT‐evoked increases in intracellular calcium (pIC50=10.4±0.1). RS‐127445 also blocked 5‐HT‐evoked contraction of rat isolated stomach fundus (pA2=9.5±1.1) and (±)α‐methyl‐5‐HT‐mediated relaxation of the rat jugular vein (pA2=9.9±0.3). RS‐127445 had no detectable intrinsic activity in these assays. In rats, the fraction of RS‐127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS‐127445 (5u2003mgu2003kg−1) produced plasma concentrations predicted to fully saturate accessible 5‐HT2B receptors for at least 4u2003h. In conclusion, RS‐127445 is a selective, high affinity 5‐HT2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated.

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.

Characterization of putative 5‐ht7 receptors mediating direct relaxation in Cynomolgus monkey isolated jugular vein

1 5‐Hydroxytryptamine (5‐HT) receptors mediating contraction and relaxation are present in Cynomolgus monkey isolated jugular vein denuded of endothelium. 2 In the absence of spasmogen, α‐methyl‐5‐HT and sumatriptan contracted the tissues with potency values (pEC50) of 6.8 (n=2) and 6.4 ± 0.1 (mean ± s.e. mean, n=3), respectively. In contrast, 5‐HT caused an initial contraction (10 nM‐1 μm), followed by relaxation (1 μm‐32 μm). The contractile effect of α‐methyl‐5‐HT was antagonized by ketanserin with a pKB value of 8.1 (n=2). 5‐Carboxamidotryptamine (5‐CT), 5‐methoxytryptamine (5‐MeOT) and 8‐OH‐DPAT did not contract or relax the tissues in the absence of spasmogen. 3 In tissues precontracted with U46619 (10 nM) and in the presence of 5‐HT1A, 5‐HT1B, 5‐HT2A, 5‐HT3, and 5‐HT4 receptor blockade, 5‐CT and 5‐MeOT caused endothelium‐independent relaxation with potency values of 7.5±0.1 (n=21) and 5.7±0.1 (n=4), respectively. The potency of 5‐HT was 7.2 (n=2) while α‐methyl‐5‐HT did not start to relax the tissues below a concentration of 10 μm. 4 Relaxations elicited by 5‐CT were antagonized by the following compounds (with pKB values in parentheses): methiothepin (9.7), mesulergine (8.1), metergoline (8.0), clozapine (7.8), mianserin (7.7), spiperone (7.3), ritanserin (7.1), methysergide (7.0) and ketanserin (5.7). 5 It is concluded that the 5‐HT receptor mediating endothelium‐independent relaxation may be a functional correlate of the putative 5‐ht7 receptor.

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.

Analysis of concentration-response relationships by Seemingly Unrelated Nonlinear Regression (SUNR) technique

In performing statistical evaluation of concentration-response relationship in pharmacological studies, all the commercially available statistical packages assume each data point is an independent measure of the drug response, and do not account for the dependence between the multiple measurements taken from the same subject (tissue, animal, or sample). Seemingly unrelated nonlinear regression (SUNR) is a statistical technique that takes into account both within-and between-subject variance. This technique has been implemented in an SAS-based interactive program called SUNR. The statistical analyses are based upon the original work by Gallant (Gallant, 1975, J Econometrics 3:35-50; Gallant and Goebel, 1976, JASA 71:961-967), which has been further developed by Muller and Helms (1984) (Presented at ASA meeting in Washington, D.C.) To test this program, we have analyzed both simulated and actual data with SUNR, comparing our results to those of several popular statistical programs. All the programs yielded essentially the same estimates for the EC50, minimum and maximum response in both the simulated and experimental data sets. However, our results differed markedly from the commercial packages in the estimates of standard errors associated with the estimated maxima. When analyzing simulated data, which were far less noisy than the experimental data, differences between the analyses were minimal. However, in the analyses of experimental data, the standard errors calculated by the commercial programs appear to significantly underestimate the standard error. Using SUNR, however, the 95% confidence limits on the maxima are markedly wider, and, importantly, always cover the observed actual data range.

Functional interactions between muscarinic M2 receptors and 5‐hydroxytryptamine (5‐HT)4 receptors and β‐adrenoceptors in isolated oesophageal muscularis mucosae of the rat

1 Relaxations of isolated oesophageal muscularis mucosae of rat are mediated by 5‐hydroxytryptamine (5‐HT), acting at 5‐HT4 receptors, and isoprenaline, principally acting via β3‐adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M2 receptors, also present in this tissue, functionally oppose 5‐HT and β‐adrenoceptor‐relaxant effects in this preparation. 2 Contractions of rat oesophageal muscularis mucosae were induced, in a concentration‐dependent manner, by the muscarinic receptor agonist, oxotremorine m (pEC50 = 6.7 ± 0.1). The contractile responses to oxotremorine m were surmountably antagonized by the following compounds, (pKB values in parentheses): atropine (9.1 ± 0.2), 4‐DAMP (4‐diphenylacetoxy‐N‐methyl piperidine methiodide, 8.7 ± 0.1), p‐F‐HHSiD (para‐fluoro‐hexa‐hydro‐siladifenidol, 7.5 ± 0.1), zamifenacin (8.6 ± 0.3), himbacine (7.2 ± 0.2), pirenzepine (6.8 ± 0.3) and methoctramine (6.2 ± 0.2). These data are consistent with a role for muscarinic M3 receptors mediating contractions to oxotremorine M. The contractile response was associated with a low receptor reserve, since the responses were shifted to the right and virtually abolished by the alkylating agent, 4‐DAMP mustard (4‐diphenylacetoxy‐N‐(2‐chloroethyl) piperidine, 40 nM; 60 min equilibration). 3 In tissues precontracted with U46619 (0.7 μm; approx. EC90), isoprenaline (pEC50 = 8.0 ± 0.1) and 5‐HT (pEC50 = 7.5 ± 0.2) induced concentration‐dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine m (isoprenaline, pEC50 = 7.4 ± 0.2). In contrast, the potency of 5‐HT (pEC50 = 7.5 ± 0.2), in tissues that were precontracted with 1 μm (EC90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M2 receptor antagonist, methoctramine (1 μ), there was no effect on the relaxant potencies to either 5‐HT or isoprenaline. Collectively, these data suggest that muscarinic M2 receptors do not, under these conditions, modulate relaxant potencies to either 5‐HT or isoprenaline. 4 In a second protocol, tissues were pre‐contracted with U46619 (0.7 μm) and relaxed with either 5‐HT (0.1 μm) or isoprenaline (0.1 μm). In these tissues (in which the muscarinic M3 receptor population was extensively depleted by alkylation), oxotremorine m caused concentration‐dependent re‐contractions (i.e. reversal of relaxations). In tissues relaxed with 5‐HT, the potency of oxtremorine m was 5.9 ± 0.2, while in tissues relaxed with isoprenaline, the potency (pEC50) = 5.6 ± 0.3. These re‐contractions were antagonized, in a surmountable fashion, by methoctramine (1 μm; pKB = 7.6 ± 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 μm; pKB = 7.5 ± 0.2). Under these conditions, therefore, the pKB values are consistent with activation of muscarinic M2 receptors, and inconsistent with activation of M3 receptors. 5 It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M3 receptors mediate direct contractions and are associated with a low receptor reserve. When this population is depleted, and the tissues relaxed via activation of receptors that augment adenylyl cyclase activity, a functional role for muscarinic M2 receptors is revealed.

Rs-100235: A high affinity 5-HT4 receptor antagonist

Abstract The 1,4-benzodioxanyl ketone 14 (RS-100235) was found to be high affinity 5-HT4 antagonist with potent in vivo activity and a sustained duration of action in the anesthetized pig.

(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.