Corticotropin-releasing factor and urocortin belong to a hypothalamic peptide family thought to be important in appetite regulation. The present study compared the appetite-suppressant effect of subcutaneous urocortin in obese mice to its cardiovascular effects. Acutely, urocortin (100 nmol/kg iv) reduced blood pressure and increased heart rate in urethane anesthetized nonobese mice; effects similar to those produced by subcutaneous urocortin (10 and 100 nmol/kg sc) in nonobese and ob/ob mice. Over this same dose range (10–100 nmol/kg sc), urocortin dramatically inhibited food consumption in the ob/ob mouse. To determine if the acute hypotensive effect of urocortin (10 nmol/kg sc) in the ob/ob mouse persisted after repeated urocortin administration, animals were pretreated for 3 days with urocortin (10 nmol/kg sc) or vehicle. Following urocortin pretreatment, urocortin-induced hypotension was similar to the effect in vehicle pretreated mice. However, urocortin-induced appetite suppression was reduced following 3 days of pretreatment with urocortin (10 nmol/kg sc) to ob/ob mice. These data suggest that the hypotensive and appetite-suppressant effects of urocortin are mediated by different mechanisms and tolerance to the hypotension did not readily occur in obese animals.

Pharmacological studies have revealed a non-beta1, beta2 or beta3 adrenergic receptor that mediates tachycardia in rat and human atria. The present studies utilized transgenic mice that lack the rodent beta3 receptor to explore, in a more definitive fashion, whether a non-beta1, beta2 or beta3 receptor can mediate atrial tachycardia. Insofar as the rat stomach fundus possesses a beta3 receptor mediating relaxation, we examined the stomach fundus from beta3 receptor knockout mice for the presence or absence of the beta3 relaxant receptor. Contractile responses to carbamylcholine were similar in potency and magnitude between mouse stomach fundus from wild type and beta3 receptor knockout animals. However, the classical beta3 receptor agonist CL316243, (10(-8)-10(-6)M) relaxed stomach fundus from wild type mice, but not from the beta3 receptor knockout animals. These data provide functional evidence for the absence of the beta3 receptor in beta3 receptor knockout animals and support the role of beta3 receptors mediating relaxation in mouse stomach fundus. Atria from mice lacking the beta3 receptor responded similarly (in potency and maximal increase in heart rate) to isoproterenol (10(-9)-10(-6)M) as atria from wild type mice. Furthermore, propranolol (3 x 10(-7) M) produced a dextral shift in the concentration response to isoproterenol in atria from both the beta3 receptor knockout and wild type mice with negative log K(B) values of 8.03 and 8.09, respectively. Thus, beta receptors mediating tachycardia to isoproterenol are intact and respond similarly in atria from both knockout and wild type mice. Furthermore, CGP12177, a prototypic 'atypical' beta receptor agonist produced tachycardia with a similar EC50 and maximal response in atria from both the wild type and beta3 receptor knockout mice. Cyanopindolol was a partial agonist relative to CGP12177 in both wild type and beta3 receptor knockout mice. Tachycardia to CGP12177 and cyanopindolol was not blocked by propranolol (3 x 10(-7) M) in atria from either group. These data provide definitive evidence that the receptor mediating tachycardia to CGP12177 and to cyanopindolol in atria from the transgenic beta3 receptor knockout mice is neither the beta1, beta2, nor beta3 adrenergic receptor.

1. The in vitro activity of four aryl propanolamines was compared to two prototypic beta3 receptor agonists, CGP 12177 and CL316243 at the human beta3 receptor, the rat beta3 receptor in the stomach fundus and receptors mediating atrial tachycardia. 2. L-739,574 was the most potent (EC50 = 9 nM) and selective agonist at the human beta3 receptor with high maximal response (74% of the maximal response to isoproterenol). 3. A phenol-biaryl ether analogue possessed modest affinity for the human beta3 receptor (EC50 = 246 nM), but was highly efficacious with a maximal response 82% of the maximal response to isoproterenol. The other derivatives were intermediate in potency with low maximal responses. 4. These agonists at the human beta3 receptor did not activate the rat beta3 receptor in the rat stomach fundus. In fact, the aryl propanolamines (10(-6) M) inhibited CL316243-induced activation of the rat beta3 receptor. Thus, agonist activity at the human beta3 receptor translated into antagonist activity at the rat beta3 receptor. 5. L739,574 and the phenol biaryl ether increased heart rate via beta1 receptors. 6. Although CGP12177 produced atrial tachycardia, neither the indole sulphonamide nor biphenyl biaryl ether did, although both had high affinity for the human beta3 receptor. Thus, the atrial tachycardic receptor was not identical to the human beta3 receptor. 7. These studies (a) characterized four aryl propanolamines with high affinity at the human beta3 receptor, (b) found that they were antagonists at the rat beta3 receptor, an observation with profound implications for in vivo rat data, and (c) established that the rodent atrial non-beta1, beta2 or beta3 tachycardic receptor was also unrelated to the human beta3 receptor.

LY353433 is a selective, potent, and orally active 5-HT4 receptor antagonist with a long duration of pharmacological activity following its oral administration to rats. After oral administration of LY353433 (100 mg/kg) to rats, peak plasma concentration of the parent material was approximately 25 ng/ml and rapidly declined such that 4 h after administration, plasma concentration of the parent material was barely detectable. However, two additional peaks (LY343031 and LY343032) were observed in plasma via HPLC and subsequently identified as hydroxylated metabolites of LY353433. Peak plasma concentrations of LY343031 (approximately 50 ng/ml) and of LY343032 (approximately 150 ng/ml) were achieved within 1 h after the oral administration of LY353433. Furthermore, plasma concentrations of these metabolites were maintained for several hours and declined more slowly than plasma concentrations of the parent material. Both metabolites inhibited esophageal 5-HT4 receptors in a concentration range similar to that observed with LY353433. In addition, the receptor selectivity profiles for LY343031 and LY343032 were similar to that of LY353433 at alpha(1), alpha(2), beta, Dopamine D-1, Dopamine D-2, benzodiazapine, histamine H-1, GABA(A), 5-HT2, and muscarinic receptors. Thus, these hydroxylated derivatives of LY353433 were potent and selective 5-HT4 receptor antagonists in vitro. Lastly, using ex vivo esophageal relaxation to serotonin to assess 5-HT4 receptor antagonism, these compounds were less active after oral administration to rats than LY353433. Thus, the long duration of pharmacological activity observed after oral administration of LY353433 is likely related not only to the 5-HT4 receptor antagonist activity of the parent molecule, but also to the 5-HT4 receptor antagonist activity associated with its two hydroxylated metabolites. (C) 1998 Wiley-Liss, Inc.

Although many 5-HT (serotonin, 5-hydroxytryptamine)(4) receptor antagonists have been described, none possess the requisite oral activity and duration of action for a clinically effective therapeutic agent. The present report identifies LY353433 (1-(1-methylethyl)-N-[2-[4-[tricyclo[3.3.1.1(3,7)]dec-1-ylcarbo nyl) amino]-1-piperidinyl]ethyl]-1H-indazole-3-carboxamide), an indazole amide, as a high affinity antagonist at the 5-HT(4) receptor in the rat esophagus. LY353433 (10(-8), 3 x 10(-8), 10(-7) M) inhibited 5-HT-induced relaxation of carbamylcholine-contracted esophagus with greater potency than cisapride or RS23597-190, a known 5-HT(4) receptor ligand. Furthermore, RS23597-190 possessed marked agonist activity as did cisapride, whereas LY353433 did not relax the rat esophagus in concentrations up to 10(-5) M. LY353433 (up to 10(-5) M) did not possess appreciable affinity for adrenergic, dopaminergic, histaminergic, muscarinic or GABAergic receptors and, thus, was a highly selective 5-HT(4) receptor antagonist. In addition, LY353433 only slowly associated with an dissociated from the 5-HT(4) receptor, an attribute that conferred long-lasting 5-HT(4) receptor antagonist activity, in contrast to RS23597-190, which rapidly dissociated from the 5-HT(4) receptor. LY353433 dose-dependently inhibited the 5-HT(4) receptor-mediated ex vivo relaxation in the rat esophagus after either i.v. (0.1, 0.3 and 1.0 mg/kg) or p.o. (0.1, 0.3, 1.0 and 3.0 mg/kg) administration. Furthermore, the p.o. to i.v. dose ratio was approximately one, suggesting that LY353433 was well absorbed with excellent pharmacodynamics in the rat. LY353433 (0.3 mg/kg p.o.) blocked esophageal 5-HT(4) receptors ex vivo through 6 hr after p.o. dosing with responses returning to control by 16 hr, indicative of long duration receptor blockade. Lastly, in rats LY353433 was exceptionally safe because acute doses up to 300 mg/kg p.o. did not result in either symptoms or deaths. Thus, LY353433 is a potent, selective, orally effective, long-acting and safe 5-HT(4) receptor antagonist that is highly suitable for clinical use.

Activation of beta-adrenergic receptors is known to have profound cardiovascular effects. Recently, selective ligands for the beta(3)-adrenergic receptor have been identified permitting an exploration of the role for beta(3) receptors in cardiovascular homeostasis. Isoproterenol, (0.01-1 mu g/kg iv) a non-selective beta agonist with relatively low beta(3)-receptor affinity, similarly decreased blood pressure and increased heart rate in pithed and conscious rats; effects that could be antagonised by propranolol (1mg/kg iv). Like isoproterenol, BRL 37,344 (0.3-100 mu g/kg iv), on agent with higher affinity for beta(3) receptors, but also Possessing beta(1)- and beta(2)-adrenergic receptor affinity, lowered blood pressure and increased heart rate in pithed and conscious rots. BRL 37,344 which has lower beta(1)- and beta(2)-receptor affinity than isoproterenol, was also less potent than isoproterenol end, like isoproterenol, its effects were inhibited by propranolol which has only minimal beta(3)-receptor affinity. In contrast, the most selective beta(3) agonist CL 316,243 in doses up to 100 mu g/kg iv did not effect either heart rate or blood pressure in the pithed or conscious rat These data with isoproterenol, BRL 37,344 and CL 316,243 suggest that in the rat, beta(1)- and beta(2)-receptor activation ploys a major role in cardiovascular homeostasis whereas beta(3)-receptor activation does not modify heart rate or blood Pressure. Thus, it is unlikely that beta(3) receptors in the rot contribute in any important fashion to either vascular tone or cardiac contractility, in contrast to previously published data in dogs suggesting a role for vascular beta(3) receptors in modulating blood pressure and heart rate.

1. Marked heterogeneity among species exists in the esophageal response to pharmacological agents. The present study compared the response to serotonin in esophagus from the rat, guinea pig, rabbit and dog. 2. The esophagus from all four species contracted to carbamylcholine and to PGF2 alpha; responses to serotonin were the most variable among species. 3. Serotonin contracted the guinea pig and rabbit esophagus; an effect blocked by LY53857 (10(-7 M) and ketanserin (10(-7) M), consistent with 5-HT2 receptor activation mediating this contraction. 4. Serotonin neither contracted nor relaxed the canine esophagus and relaxed the rat esophagus via 5-HT4 receptor activation as determined by antagonism with ICS 205-930 (-log KB = 6.4), metoclopramide (-log KB = 6.7) and its ester congener SDZ 205-557 (-log KB = 7.9). Two methylene homologs of SDZ 205-557 also had high 5-HT4 receptor affinity (-log KB = 7.7). 5. Thus, in guinea pig and rabbit esophagus, serotonin induced a contraction mediated by 5-HT2 receptors; and serotonin neither contracted nor relaxed the canine esophagus. In rat esophagus, serotonin induced a relaxation mediated by activation of 5-HT4 receptors.

Amesergide and LY215840 are potent and long‐lasting 5‐HT2 receptor antagonists after oral administration to animals. In animals, these ergolines are metabolized to their desisopropyl ergoline congeners which have lower affinity (40–60 nM) at 5‐HT2 receptors in the rat relative to higher 5‐HT2 receptor affinity (2–3 nM) at the cloned human 5‐HT2 receptor. Because amesergide and LY215840 are effective in rabbit models of thrombosis, we asked whether their efficacy in the rabbit was related in part to the activity of both the parent and desisopropyl metabolites at rabbit platelet 5‐HT2 receptors. Platelet aggregation responses were first optimized to ADP and the combination of ADP and serotonin with regard to platelet number (300,000 platelets/μl of plasma) and time (70 to 140 min after platelet harvest). In ex vivo studies, both amesergide and LY215840 (3.0 mg/kg p.o.) showed similar and marked antagonism of rabbit platelet 5‐HT2 receptors at 1 and 24 h after their oral administration to rabbits. Furthermore, the desisopropyl ergoline metabolites of both amesergide and LY215840 inhibited serotonin‐amplified platelet aggregation responses in vitro as did amesergide and LY215840. Thus, these studies add support to the hypothesis that the desisopropyl metabolites of amesergide and LY215840 may contribute to the oral antithrombotic efficacy of the parent molecules in rabbits.

Zatosetron is a potent, orally active 5‐HT3 receptor antagonist with a long duration of activity in laboratory animals and humans. Several metabolites have been detected in plasma and urine of humans and experimental animals receiving zatosetron. The present study was designed to explore the pharmacological activity of the detected metabolites, 3‐hydroxyzatosetron, 3‐ketozatosetron, and N‐desmethylzatosetron, relative to the parent molecule. These three metabolites had relatively high affinity at 5‐HT3 receptors based on in vitro radioligand binding and inhibited serotonin‐induced bradycardia in urethane‐anesthetized rats after intravenous administration. However, these metabolites had lower affinity and were less potent than zatosetron. Of these metabolites, 3‐hydroxyzatosetron (ED50 = 4.0 μg/kg iv) was approximately 5‐fold less potent than zatosetron (ED50 = 0.8 μg/kg iv) in vivo and had approximately 10‐fold lower affinity at 5‐HT3 receptors in vitro relative to zatosetron. N‐desmethylzatosetron and 3‐ketozatosetron were approximately 15‐fold less potent than zatosetron in vivo as 5‐HT3 receptor antagonists. With regard to duration of activity in vivo, after intravenous administration, 3‐hydroxyzatosetron and 3‐ketozatosetron blocked 5‐HT3 receptors longer than zatosetron, whereas N‐desmethylzatosetron showed a duration of pharmacological activity similar to zatosetron. A fourth metabolite, zatosetron‐N‐oxide can exist in two isomeric forms, with stereoselective N‐oxidation of zatosetron resulting in formation of only one isomer in humans, zatosetron‐β‐N‐oxide. Zatosetron‐β‐N‐oxide had approximately 100‐fold lower 5‐HT3 receptor affinity relative to zatosetron and was approximately 150‐fold less active as an antagonist at 5‐HT3 receptors in vivo (ED50 = 115 μg/kg iv). Thus, although pharmacological activity was observed with all four metabolites, they were all less active in vivo than zatosetron. Therefore, these metabolites would contribute significantly to the activity of zatosetron only if plasma (and tissue) levels greatly exceeded those of zatosetron. © 1993 Wiley‐Liss, Inc.

A series of tricyclic (nor-D) partial ergolines were synthesized via a highly convergent enantiospecific strategy, ultimately arising from a racemic tricyclic ketone. Michael addition to an acrylamide, followed by reductive methylation, afforded the key intermediate. Selective deprotection and oxidation provided the tricyclic ergoline. Vascular 5HT2 receptor interactions for the partial ergolines were dramatically reduced compared to the parent ergoline, amesergide, as determined in vivo by activation of a pressor response or blockade of 5HT-induced pressor responses in pithed rats. The desisopropyl tricyclic ergolines possessed some modest pressor activity that was unlikely to be related to 5HT2 receptor activation since these compounds did not inhibit the pressor response to serotonin. In contrast, the isopropyl tricyclic ergolines exhibited no agonist activity, but inhibited the pressor response to serotonin at 1 mg/kg i.v. The ergoline amesergide inhibited the pressor response to serotonin in doses of 0.01-0.1 mg/kg i.v. The homochiral isopropyl tricyclic ergoline was more potent as a 5HT2 receptor antagonist than the epimeric (unnatural stereochemistry) analogue. Thus, the isopropyl moiety on the indole nitrogen is important for vascular 5HT2 receptor affinity in the rat. Most importantly, these data suggest that conformational rigidity of the ergoline D-ring is required for optimal 5HT2 receptor interactions in the rat.

Antagonists of 5HT3 receptors are clinically effective in treating nausea and emesis associated with certain oncolytic drugs, including cisplatin. Moreover, these agents may be useful in pharmacological management of several central nervous system disorders, including anxiety, schizophrenia, dementia, and substance abuse. Our studies on aroyltropanamides led to the discovery that dihydrobenzofuranyl esters and amides are potent 5HT3 receptor antagonists. Simple benzoyl derivatives of tropine and 3 alpha-aminotropane possessed weak 5HT3 receptor antagonist activity, as judged by blockade of bradycardia produced by iv injection of serotonin (5HT) to anesthetized rats. Within this series, use of benzofuran-7-carboxamide as the aroyl moiety led to a substantial increase of 5HT3 receptor affinity. The optimal 5HT3 receptor antagonist identified via extensive SAR studies was endo-5-chloro-2,3-dihydro-2,2-dimethyl-N-(8-methyl-8-azabicyclo[3.2.1]oc t- 3-yl)-7-benzofurancarboxamide (Z)-2-butenedioate (zatosetron maleate). The 7-carbamyl regiochemistry, dimethyl substitution, chloro substituent, and endo stereochemistry were all crucial elements of the SAR. Zatosetron maleate was a potent antagonist of 5HT-induced bradycardia in rats (ED50 = 0.86 micrograms/kg i.v.). Low oral doses of zatosetron (30 micrograms/kg) produced long-lasting antagonism of 5HT3 receptors, as evidenced by blockade of 5HT-induced bradycardia for longer than 6 h in rats. Moreover, this compound did not produce hemodynamic effects after i.v. administration to rats, nor did it block carbamylcholine-induced bradycardia in doses that markedly blocked 5HT3 receptors. Thus, zatosetron is a potent, selective, orally effective 5HT3 receptor antagonist with a long duration of action in rats.

The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in [3H]-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of [3H]-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

Several 5-hydroxytryptamine (5-HT3) receptor antagonists have been described. In addition to 5-HT3 receptor antagonist activity, many of these agents also possess gastroprokinetic activity. In the present report, we identify compound LY277359 maleate as a potent, p.o. active, highly selective 5-HT3 receptor antagonist lacking gastroprokinetic effects. LY277359 maleate was a potent and selective antagonist of 2-methyl 5-HT-induced contraction in the guinea pig ileum (KB = 1.6 nM), a response mediated by activation of 5-HT3 receptors. Given both i.v. (0.0003, 0.001 and 0.003 mg/kg) and p.o. (0.01 and 0.03 mg/kg), LY277359 maleate inhibited the bradycardic response to i.v. administered 5-HT in urethane-anesthetized rats. The duration of antagonism of 5-HT-induced bradycardia persisted beyond 6 hr after p.o. administration of LY277359 maleate (0.03 mg/kg p.o.). In contrast to its potent 5-HT3 receptor antagonist activity, LY277359 maleate, in doses up to 1 mg/kg p.o., did not affect gastric emptying in rats, suggesting minimal, if any, gastroprokinetic activity of LY277359 maleate. This is in contrast to another 5-HT3 receptor antagonist, zacopride, which did produce a marked increase in gastric emptying in rats at doses of 0.1 mg/kg p.o. and higher. LY277359 maleate (0.03 and 0.1 mg/kg i.v. and 0.07, 0.1, 0.3 and 1.0 mg/kg p.o.) did have effects consistent with other 5-HT3 antagonists, to inhibit cisplatin-evoked emesis in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

The well-documented 5-HT3 receptor antagonists, ICS 205-930 and GR38032F, have been compared with regard to their inhibitory activity at 5-HT3 receptors to another gastrokinetic agent, zacopride. Zacopride and ICS 205-930 showed similar affinity (-log kB approximately 8.0), whereas GR38032F showed lower affinity (-log ka approximately 7.0) at 5-HT3 receptors in the guinea pig ileum. After i.v. administration to anesthetized rats, zacopride was approximately 10-fold more potent than either ICS 205-930 or GR38032F, which were equipotent as inhibitors of serotonin-induced bradycardia (5-HT3-mediated activation of the von Bezold Jarisch reflex). After oral administration to anesthetized rats, zacopride remained approximately 10-fold more potent than ICS 205-903, which was approximately 2-fold more potent than GR38032F as an inhibitor of serotonin-induced bradycardia. Furthermore, the inhibitory effectiveness of GR38032F persisted for less than 3 hr after oral administration and for less than 15 min after intravenous administration. ICS 205-930 produced maximal inhibition of serotonin-induced bradycardia for over 3 hr with heart rate returning to control values 6 hr after oral administration. Zacopride possessed the longest duration of inhibitory effectiveness in urethane-anesthetized rats with maximal inhibition still apparent 6 hr after oral administration. All three agents inhibited cisplatin-induced emesis after i.v. administration in dogs with zacopride being 10-fold more potent than ICS 205-930 or GR38032F, which were equipotent. These comparative data with three 5-HT3 receptor antagonists indicate that in animals, zacopride was more potent and longer acting than either ICS 205-930 or GR38032F. Furthermore, after oral administration to rats, GR38032F was slightly less potent than ICS 205-930 and possessed the shortest duration of action.