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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: pA(2) values were determined on isolated smooth muscle and platelet preparations. KEY RESULTS: RO1138452 antagonized relaxation of human pulmonary artery, guinea-pig aorta and rabbit mesenteric artery induced by the selective IP agonist cicaprost. Schild plots had slopes close to unity, generating pA(2) values of 8.20, 8.39 and 8.12 respectively. Non-surmountable antagonism was sometimes found with the higher concentrations of RO1138452, attributable to the EP(3) contractile action of cicaprost. RO1138452 did not block relaxation of guinea-pig trachea induced by the EP(2)-selective agonist butaprost. In contrast, there was a modest inhibition of butaprost-induced relaxation of human pulmonary artery by RO1138452, implying activation of both EP(2) and IP receptors by butaprost. RO1138452 did not affect relaxation induced by PGE(2) (EP(4) agonist) and substance P (NK(1)/endothelium-dependent agonist) in rabbit mesenteric artery. In human and rat platelet-rich plasmas, RO1138452 antagonized cicaprost-induced inhibition of platelet aggregation in a surmountable manner; pA(2) values may have been affected by binding of RO1138452 to plasma protein. RO1138452 did not affect the inhibitory actions of PGD(2) (DP(1) agonist) and NECA (adenosine A(2A) agonist) in human platelets. CONCLUSIONS AND IMPLICATIONS: The data indicate that RO1138452 is a potent and selective IP receptor antagonist. RO1138452 represents an important addition to our armoury of prostanoid receptor antagonists and a potential clinical agent in situations where prostacyclin has a pathophysiological function.

Cardiovascular effects of nepicastat (RS-25560-197), a novel dopamine beta-hydroxylase inhibitor.

Nepicastat (RS-25560-197) is a novel, selective, and potent inhibitor of dopamine beta-hydroxylase, which modulates catecholamine levels (reduces norepinephrine and elevates dopamine) in cardiovascular tissues. This study was designed to evaluate the cardiovascular effects of nepicastat. Acute oral administration of nepicastat (0.3, 1, 3, 10, and 30 mg/kg) produced attenuation of the pressor and positive chronotropic responses to preganglionic sympathetic nerve stimulation (about twofold to sixfold shift in the frequency-response curve) in pithed spontaneously hypertensive rats (SHRs). In inactin-anesthetized SHRs, the antihypertensive effects of nepicastat (3 mg/kg, i.v.) were accompanied by a significant decrease in renal vascular resistance (38%), a tendency toward an increase in renal blood flow (22%), and no adverse effects on urine output and Na/K excretion. In conscious, unrestrained, telemetry-implanted SHRs, nepicastat (30 and 100 mg/kg/day for 30 days) produced dose-dependent decreases in mean arterial blood pressure (peak decrease of 20 and 42 mm Hg, respectively) without evoking reflex tachycardia. Long-term, concurrent administration of nepicastat (30 mg/kg/day, p.o.) and a subthreshold dose of enalapril (1 mg/kg/day, p.o.) produced greater antihypertensive effects than those produced by nepicastat alone. In normal dogs, nepicastat (5.0 mg/kg, p.o., b.i.d., for 4.5 days) blunted the positive chronotropic and pressor response to tyramine. These findings suggest that nepicastat functionally modulates sympathetic drive to cardiovascular tissues and may be of value in the treatment of cardiovascular disorders associated with overactivation of the sympathetic nervous system such as hypertension and congestive heart failure.

Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel, potent and selective inhibitor of dopamine-beta-hydroxylase.

1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-beta-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study. 2. Nepicastat produced concentration-dependent inhibition of bovine (IC50 = 8.5 +/- 0.8 nM) and human (IC50 = 9.0 +/- 0.8 nM) dopamine-beta-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2-3 fold less potent than nepicastat. Nepicastat had negligible affinity (> 10 microM) for twelve other enzymes and thirteen neurotransmitter receptors. 3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg-1, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg-1, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noadrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg-1, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle. 4. Administration of nepicastat (2 mg kg-1, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively. 5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-beta-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.

The 5-HT3 receptor antagonist, RS-56812, enhances delayed matching performance in monkeys.

Substantial evidence indicates that serotonin receptors are involved in the regulation of acetylcholine release in CNS regions important to mnemonic processes, and may thus be exploited pharmacologically as targets for memory improvement. In the present study, the (R) and (S) isomers of a potent serotonin (5-HT3) receptor ligand, RS-56812 were evaluated for potential memory effects in five macaques trained to perform a delayed matching-to-sample (DMTS) task. While both isomers enhanced certain aspects of task performance, the (R) isomer produced more systematic improvements. This differential sensitivity to the isomers in regard to DMTS performance appears to parallel the higher 5-HT3 receptor affinity of the R enanantiomer. The results are consistent with a potential therapeutic role for RS-56812 in disorders involving cognitive decline.

Chronic manipulation of dietary salt modulates renal physiology and kidney dopamine receptor subtypes: functional and autoradiographic studies.

1. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited increased plasma aldosterone and chloride and decreased urinary sodium excretion. 2. Rats maintained on the high NaCl (8%) diet for 4 weeks showed increased systolic blood pressure, water intake, urine volume, sodium and dopamine excretion and decreased plasma aldosterone and glomerular filtration rate. 3. Administration of SCH 23390 (10 mg/kg, po), but not domperidone to the high salt diet rats attenuated the diuretic effect, indicating the involvement of DA1 rather than DA2 receptors. The dopamine decarboxylase inhibitor, carbidopa (30 mg/kg, i.p.), also reduced the high salt-induced diuresis. 4. Kidney sections from rats fed the low NaCl diet showed a 63-100% decrease (P < 0.001-0.02) in cortical and medullary DA1 and DA2 binding sites, while rats fed the high NaCl diet demonstrated only a 70% decrease (P < 0.01-0.02) in cortical DA1 binding, without affecting DA2 binding. 5. These data indicate that chronic modification of dietary salt profoundly affects the sodium, water and dopamine excretion and leads to selective modulation of renal dopamine receptor subtypes.

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 micrograms kg-1, i.v., 3.2 micrograms kg-1, i.d. and 32.8 micrograms 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 micrograms kg-1, i.v. and 3.2 micrograms 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 micrograms kg-1, i.v. and 8.5 micrograms kg-1, p.o.), dacarbazine (ID50 = 4.1 micrograms kg-1, i.v. and 9.7 micrograms kg-1, p.o.), actinomycin D (ID50 = 4.9 micrograms kg-1, i.v. and 2.5 micrograms kg-1, p.o.) and mechlorethamine (ID50 = 4.4 micrograms kg-1, i.v. and 3.0 micrograms 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 microg kg-1, p.o., neither RS25259-197 nor ondansetron was capable of inhibiting apomorphine-induced emesis.5. At doses up to 1000 microg 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.

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 (alpha1A, alpha 1B, alpha2A, alpha 2B ,beta1, beta2), muscarinic (M1-M4), dopamine (D1, D2), opioid and other 5-HT(5-HTlA, 5-HTlD, 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 a subunits of the 5-HT3 receptor from N1E-1 15 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 of610 +/- 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-HT3receptor.6. Quantitative autoradiographic studies in rat brain indicated a differential distribution of 5-HT3receptor 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 CAl, 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 selective5-HT3 receptor antagonist.

Quantitative autoradiography demonstrates selective modulation of rat brain regional dopamine (D1 and D2) receptor subtypes after chronic manipulation of dietary salt.

The effects of chronic dietary sodium chloride (NaCl) consumption on renal function and brain dopamine receptors were studied in adult, male normotensive rats. Compared to rats maintained on the normal NaCl (0.33%) diet, animals maintained on the low NaCl (0%) diet for 4 weeks exhibited significant increases in plasma aldosterone, chloride and changes in urinary electrolyte excretion. In contrast, rats maintained on the high NaCl (8%) diet for 4 weeks demonstrated significant increases in urine volume and urinary sodium, chloride and dopamine excretions and water intake. Rats fed the high NaCl diet displayed a 42-59% decrease (p < 0.001-0.05) in D1 binding in the nucleus accumbens (NA), olfactory tubercle (OT) and the striatum (STM), without any effects on D2 binding in these brain regions. Rats maintained on the low NaCl diet also demonstrated decreased D1 binding in the ventral (24%, p < 0.02) and lateral (29%, p < 0.01) STM, but not in the OT, NA, entopeduncular nucleus and substantia nigra. Rats fed low or high NaCl diets exhibited a 35-180% increase (p < 0.01-0.05) in D2 binding in several mid-brain areas (e.g. hypothalamus, thalamus and hippocampus) and hindbrain regions (e.g. superior colliculus and nucleus tractus solitarius) without affecting the D1 binding. These data indicate that chronic modification of dietary salt intake profoundly affects the renal handling of sodium/water excretion and leads to selective up- and/or down-regulation of DA receptor subtypes in different areas of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

M3 muscarinic receptors on murine HSDM1C1 cells: further functional, regulatory, and receptor binding studies.

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50 = 9 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) were full agonists which stimulated PI turnover by 13.3 +/- 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin (5-2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 microM) PI turnover. The half-maximal effect of TPA was produced at 1.8 +/- 0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd = 0.19 +/- 0.04 nM) and moderate capacity (Bmax = 201 +/- 22 fmol/mg protein). The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

(R) and (S) RS 56532: mixed 5-HT3 and 5-HT4 receptor ligands with opposing enantiomeric selectivity.

The pharmacological properties of the (R) and (S) enantiomers of RS 56532 have been studied in vitro and in vivo. In radioligand binding studies at 5-HT4 receptors in guinea-pig striatum, (S) RS 56532 exhibited a higher affinity than (R) RS 56532 (-log Ki = 7.6 and 6.5, respectively). (S) RS 56532 acted as a potent agonist at 5-HT4 receptors mediating relaxation of rat oesophageal muscularis mucosae (-log EC50 = 7.9) while (R) RS 56532 acted as a weaker agonist at this receptor (-log EC50 < 6.0). These data suggest that at 5-HT4 receptors, the enantiomeric selectivity of RS 56532 was (S) > (R). In binding studies at 5-HT3 receptors in rat cortex, (R) RS 56532, conversely, exhibited a higher affinity than (R) RS 56532 (-log Ki = 9.1 and 8.0, respectively). At 5-HT3 receptors in guinea-pig isolated ileum, (R) RS 56532 exhibited an affinity (-log KB) of 7.9, whereas (S) RS 56532 (1 nM-1 microM) was inactive. No agonism was observed at ileal 5-HT3 receptors with either enantiomers. These data suggest that at 5-HT3 receptors in rat and guinea-pig, both enantiomers acted as antagonists, with (R) > (S) RS 56532. At the non-5-HT3, high affinity '(R) zacopride' site, (R) RS 56532 exhibited a higher affinity than (S) RS 56532 (-log Ki = 6.1 and 4.9). This site was insensitive to potent 5-HT3 antagonists such as (R) YM 060 or ondansetron. However, it was recognized with relatively high affinity (-log Ki = 7.5) by the (R), but not (S) enantiomer, of RS 42358 (-log Ki = 4.7). Since (S) RS 42358 is a high affinity 5-HT3 receptor antagonist, these data further highlight the dissimilarity between the 5-HT3 receptor and the '(R) zacopride' site. The '(R) zacopride' site also appeared to be pharmacologically distinct from the 5-HT4 receptor, since 5-HT4 ligands such as renzapride, SDZ 205,557 or RS 23597-190 exhibited low affinities. The enantiomeric selectivity of (R) and (S) RS 56532 in vivo was consistent with findings in vitro. At 5-HT4 receptors mediating tachycardia in the pig, 5-HT induced a dose-dependent tachycardia (ED50 = 3 micrograms kg-1, i.v.; maximum response = 90-100 beats min-1). (S) RS 56532 increased heart rate by 88 min-1 with a potency of (ED50) of 3 micrograms kg-1, i.v.(ABSTRACT TRUNCATED AT 400 WORDS)

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies.

Bradykinin (BK) and its analogs (1 nM-100 microM) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC50) were: BK = 48 +/- 4 nM; Lys-BK = 39 +/- 3 nM; Met-Lys-BK = 158 +/- 33 nM, Des-Arg9-BK = 2617 +/- 598 nM (means +/- SEM, n = 3-14). All these analogs were full agonists and they produced up to 5.4 +/- 0.4-fold stimulation of PI turnover at the highest concentration (10-100 microM) of the peptides. In contrast, the analogs [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (HYP3-antagonist), [D-Arg0-HYP3-Thienyl,5,8-D-Phe7]-BK (Thienyl antagonist) and Des-Arg9-Leu8-BK were inactive, as agonists, at 0.1 nM-1 microM in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B2-type of BK receptors. This was confirmed further by the fact that both the B2-selective Hyp3- and Thienyl-antagonists inhibited BK-induced PI turnover with KBS of 369 +/- 51 nM and 368 +/- 118 nM respectively while the B1-selective antagonist, Des-Arg9-Leu8-BK, was inactive at 1 microM. [3H]BK receptor binding studies revealed two binding sites, one with high affinity (Kd = 0.24 +/- 0.06 nM; Bmax = 1.4 +/- 0.4 pmol/g tissue) and the other with low affinity (Kd = 18.5 +/- 0.95 nM; Bmax = 25.1 +/- 0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [3H]BK binding was similar to that found for PI turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

RS 42358-197, a novel and potent 5-HT3 receptor antagonist, in vitro and in vivo.

The pharmacological activity of RS 42358-197, a novel 5-HT3 receptor antagonist has been evaluated in vitro and in vivo. In functional experiments in vitro, RS 42358-197 behaved as a competitive antagonist against 5-HT-induced contractions in the guinea pig ileum (low-potency phase), yielding a pA2 estimate of 8.1. RS 42358-197 was devoid of any agonistic or antagonistic activity at 5-HT1-like receptors (contraction of canine saphenous vein), 5-HT2 receptors (contraction of rabbit aorta) or 5-HT4 receptors (contraction of guinea pig ileum, high-potency phase). RS 42358-197 failed to affect the concentration-effect curve to substance P in guinea pig ileum. In anesthetized rats. RS 42358-197, administered by the intravenous, intraduodenal or transdermal route, dose-dependently inhibited the Bezold-Jarisch reflex induced by 2-methyl 5-HT (ID50:0.05 micrograms/kg; i.v., 5.7 micrograms/kg; i.d., and 11.6 micrograms/chamber, respectively). In this regard, when administered intraduodenally, RS 42358-197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron. In dogs, RS 42358-197, administered either intravenously or orally, dose-dependently inhibited the emesis induced by cisplatin, actinomycin and cyclophosphamide, but not that induced by apomorphine. When tested at maximally effective doses against cisplatin-induced emesis in dogs, RS 42358-197 had a longer duration of antiemetic activity (> 6 h) than ondansetron (2 h). RS 42358-197, administered orally, also afforded protection against cisplatin-induced emesis in ferrets. At doses that showed marked anti-emetic activity in dogs (10-100 micrograms/kg; i.v. and 100-1000 micrograms/kg; i.d.), RS 42358-197 did not produce any hemodynamic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the 5-HT3 receptor antagonist, RS-42358-197, in animal models of anxiety.

The S-isomer of the novel 5-HT3 receptor antagonist RS-42358 ((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1-H- benzo[de]isoquinolin-1-one, RS-42358-197) disinhibited behaviour in the mouse suppressed by the aversive situation of the light/dark test box. RS-42358-197 was effective at sub-ng/kg dose levels and the efficacy was maintained over a 100 million-fold dose range. In contrast, the R-isomer was ineffective at all doses studied. The S-isomer also disinhibited a suppressed behaviour in social interaction and elevated X-maze tests in the rat and reduced anxiety-related behaviours in a marmoset human threat test. RS-42358-197 prevented the exacerbation of the suppression of behaviour in the mouse light/dark test following withdrawal from treatment with alcohol, nicotine, cocaine and diazepam. Thus, the S-isomer of RS-42358 has a consistent non-sedating anxiolytic profile in rodent and primate models. It is exceptionally potent and a maintained efficacy at high doses distinguishes its actions from many other 5-HT3 receptor antagonists.

Agonist action of indole derivatives at 5-HT1-like, 5-HT3, and 5HT4 receptors in vitro.

1. The potency of indole analogues has been studied, in vitro, at 5-hydroxytryptamine4 (5-HT4) receptors mediating contractions of guinea-pig ileum and relaxation of rat oesophagus. These have been compared to other 5-HT receptors in canine saphenous vein (5-HT1-like), rabbit aorta (5-HT2), and guinea-pig ileum (5-HT3). 2. At receptors mediating 5-HT4 responses in ileum and oesophagus, the rank orders of potency were similar. These rank orders differed from those observed at 5-HT1-like, 5-HT2, and 5-HT3 receptors. In particular, 5-hydroxy N,N, dimethyltryptamine but not 5-methoxy N,N, dimethyltryptamine acted as agonists at 5-HT4 receptors. At 5-HT1-like, 5-HT2 and 5-HT3 receptors these compounds were both active. 3. The 5-HT receptors mediating contractions of canine cephalic vein exhibited a rank order profile similar to that observed at receptors mediating contractions of canine saphenous vein, suggesting stimulation of a 5-HT1-like receptor. 4. The rank order of potency of the substituted indoles differed at 5-HT receptors mediating responses in canine saphenous vein, rabbit aorta and guinea-pig ileum (determined in the presence of 5-methoxytryptamine to desensitize 5-HT4 receptors), suggesting the presence of three distinct receptors. Indeed, at 5-HT3 receptors in the ileum, only three agonists (5-HT, 2-methyl-5-HT and 5-hydroxy N,N, dimethyltryptamine) elicited a response, while all remaining compounds were inactive. 5. It is concluded that rank orders of indole potency can prove useful in the delineation of 5-HT subtypes and together with differential antagonist affinities support the existence of four 5-HT receptor subtypes.

Characterization of the interaction of the cervane alkaloid, imperialine, at muscarinic receptors in vitro.

The action of the cervane alkaloid, imperialine, has been assessed at M1, M2 and M3 receptors in functional assays and at M1, M2, M3 and putative M4 sites in binding studies. In functional studies, imperialine acted as a selective surmountable antagonist at M2 receptors in guinea-pig isolated atria and uterus (-log KB = 7.7 and 7.4, respectively), in comparison to M1 receptors in canine isolated saphenous vein (-log KB = 6.9) or M3 receptors in a range of guinea-pig isolated smooth muscles including ileum, trachea, fundus, seminal vesicle or oesophagus (-log KB = 6.6-6.8). In rat aorta, the -log KB value at the M3 receptor (5.9) was slightly, but significantly, lower. In competition radioligand binding studies, imperialine was also selective toward to M2 sites in rat myocardium (-log Ki = 7.2) with respect to M1 and M3 sites (rat cerebral cortex, rat submaxillary gland; -log Ki = 6.1 and 5.7, respectively). However, it did not significantly discriminate between rat cardiac M2 sites and putative M4 sites in rabbit lung (-log Ki = 6.9). Imperialine resembles the alkaloid himbacine in terms of its pharmacological profile at muscarinic receptor subtypes in that it acts as an M2 selective antagonist with respect to M1 or M3 sites. It may also provide a second, commercially available, antagonist with which to discriminate between M1 and M4 receptors.

A pharmacological comparison of [3H]GBR12935 binding to rodent striatal and kidney homogenates: binding to dopamine transporters?

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.

Acute desensitization of muscarinic receptors in the isolated guinea-pig ileal longitudinal muscle.

1. The effects of acute desensitization of muscarinic receptors mediating contractile responses of the guinea-pig ileal longitudinal muscle were studied in vitro, using similar conditions for both functional and radioligand binding studies. 2. The pA2 values for a number of muscarinic antagonists (pirenzepine, methoctramine, (+/-)para-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methyl piperidine-methiodide) indicated that the contractile response to carbachol was mediated through an M3 muscarinic receptor. In binding experiments the muscarinic receptor subtype population in ileal longitudinal muscle was found to be heterogeneous, consisting of approximately 77% M2 and 23% M3 receptors. 3. Pre-exposure of ileal longitudinal muscle to 10 microM carbachol for 30 min produced a reduction (28 +/- % of control maximum) in the maximum contractile response and a dextral shift in the concentration-effect curve to carbachol. Prior equilibration (60 min) with (+/-)p-F-HHSiD (1 microM), but not with methoctramine (1 microM) or pirenzepine (0.3 microM), prevented the desensitization. Desensitization under these conditions did not alter either the apparent affinity, the total number of binding sites or the relative, proportions of M2 and M3 muscarinic receptors, as determined in radioligand binding studies. Desensitization did not cause any meaningful change in either the apparent affinity of carbachol or the proportion of the high and low affinity binding sites. 4. It is concluded that desensitization of the contractile responses of the guinea-pig ileal longitudinal muscle is a result of M3 but not M2 muscarinic receptor desensitization. Acute desensitization, therefore, is not accompanied by meaningful changes in the total number of both M2 and M3 receptors or by alterations in the affinity of the receptor to ligands.

The action of (+/-)L-660,863 [(+/-)3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] at muscarinic receptor subtypes in vitro.

1. The muscarinic pharmacology of a novel oxadiazole muscarinic agonist, (+/-) L-660,863, [+/-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] has been studied using pharmacological, radioligand binding and biochemical techniques, in vitro. 2. In isolated tissue experiments, (+/-)L-660,863 was a more potent agonist than carbachol in all preparations studied, being most potent at muscarinic receptors mediating negative chronotropy in guinea-pig right, spontaneously beating atria and least potent at receptors mediating contractions in canine saphenous vein and endothelial denuded rabbit aorta (-log EC50 values were 8.8, 6.6 and 6.3, respectively. The apparent affinities (-log KA) of (+/-)L-660,863) estimated by receptor inactivation, showed some selectivity toward the atrial M2 muscarinic receptor (-log KA = 7.6) in comparison to the M1 or M3 muscarinic receptors (-log KA = 5.4 and 6.2) respectively. This degree of selectivity was also observed in competition radioligand binding studies. 3. At M3 muscarinic receptors mediating inositol phosphates (IPs) accumulation in longitudinal muscle of guinea-pig ileum, the potency of (+/-)L-660,863 (-log EC50 value = 6.2) was similar to the apparent affinity calculated at M3 muscarinic receptors in the functional studies (see above). In contrast, at muscarinic receptors mediating IPs accumulation in guinea-pig atria and ventricles, the potency for (+/-)L-660,863 (-log EC50 = 6.2 and 6.4, respectively) was lower than the apparent affinity calculated at M2 muscarinic receptors from inotropic and binding studies in cardiac tissue (see above).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of muscarinic cholinoceptors mediating phosphoinositide hydrolysis in guinea pig cardiac muscle.

The muscarinic receptor mediating stimulation of PI hydrolysis in guinea pig atria and ventricles has been studied. The non-selective muscarinic agonist (+)-cis-dioxolane elicited this response, concentration-dependently, with a potency indicative of a low receptor reserve. The potency of a novel, M2-selective agonist, L-660,863 (-log EC50 = 6.3, atria; 6.0, ventricles) was observed to be lower than its apparent affinity (-log KA = 7.6) for M2 receptors, indicating an action probably mediated by a population distinct from that producing negative inotropy in the same tissue. The inhibition of the response to (+)-cis-dioxolane by several muscarinic antagonists (atropine, pirenzepine, AF-DX 116, methoctramine, HHSiD and pFHHSiD) generated an affinity profile for this receptor also dissimilar to that described for the receptor mediating the classical cardiac 'M2' response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M2 receptor diversity.

Dopamine D2-receptors mediate hypothermia in mice: ICV and IP effects of agonists and antagonists.

The effect of centrally and peripherally administered dopamine D1 and D2 specific compounds on core body temperature in mice was investigated. Quinpirole (LY-17155), a D2 agonist, induced a dose-dependent fall in body temperature (2.4-11.6%; p less than 0.003) when injected intraperitoneally (ip, 0.3-3.0 mg/kg) and intracerebroventricularly (icv, 0.1 mg/kg). This quinpirole-induced (1.0 mg/kg, ip) hypothermia was reversed by the central and peripheral administration of the D2 antagonists S-(-)-sulpiride (3.0-30.0 mg/kg, ip; 0.1-3.0 mg/kg, icv) and spiperone (0.03 and 0.1 mg/kg, ip; 0.03-3.0 mg/kg, icv). Domperidone, a D2 antagonist which does not cross the blood brain barrier, had no effect on quinpirole-induced hypothermia (1.0-10.0 mg/kg, ip). Domperidone partially reversed quinpirole-induced hypothermia at 0.1-30.0 mg/kg, icv. The D1 agonist, SKF-38393 at a high dose of 10.0 mg/kg, ip mildly attenuated quinpirole-induced hypothermia (a 1.8% increase in temperature). SKF-38393 at 10.0 mg/kg, icv potentiated quinpirole-induced hypothermia. SCH-23390 (0.1-3.0 mg/kg, ip), a D1 antagonist, had no effect on quinpirole-induced hypothermia and potentiated the hypothermia when administered icv. An ineffective icv dose of spiperone (0.01 mg/kg) in reversing quinpirole-induced hypothermia was rendered effective by prior administration of SCH-23390 (0.1-3.0 mg/kg, icv) but not by SKF-38393 (1.0-10.0 mg/kg, icv). These data suggest a central D2 receptor mechanism mediating hypothermia in mice which is capable of being modulated by the D1 receptor.