Arylpropanolamines: selective beta3 agonists arising from strategies to mitigate phase I metabolic transformations.

Utilization of N-substituted-4-hydroxy-3-methylsulfonanilidoethanolamines 1 as selective beta(3) agonists is complicated by their propensity to undergo metabolic oxidative N-dealkylation, generating 0.01-2% of a very potent alpha(1) adrenergic agonist 2. A summary of the SAR for this hepatic microsomal conversion precedes presentation of strategies to maintain the advantages of chemotype 1 while mitigating the consequences of N-dealkylation. This effort led to the identification of 4-hydroxy-3-methylsulfonanilidopropanolamines 15 for which the SAR for the unique stereochemical requirements for binding to the beta adrenergic receptors culminated in the identification of the potent, selective beta(3) agonist 15f.

BMS-201620: a selective beta 3 agonist.

A series of N-(4-hydroxy-3-methylsulfonanilidoethanol)arylglycinamides were prepared and evaluated for their human beta3 adrenergic receptor agonist activity. SAR studies led to the identification of BMS-201620 (39), a potent beta3 full agonist (Ki = 93 nM, 93% activation). Based on its favorable safety profile, BMS-201620 was chosen for clinical evaluation.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

Metals, boron, and selenium in Neosho Madtom habitats in the Neosho River in Kansas, U.S.A.

In 1991 and 1992 we determined the levels of metals, arsenic, boron, and selenium in the Neosho River drainage in southeastern Kansas, the primary habitat for the threatened Neosho madtom (Noturus placidus). We evaluated concentrations in sediments, mussels (Quadrula pustulosa and Q. metanevra), and fish (Percina phoxocephala, Cyprinus carpio, and Ictiobus bubalus) from three sites on the Neosho River and one site on the Cottonwood River. We also evaluated contaminant concentrations in C. carpio composite samples collected by the Kansas Department of Health and Environment (KDHE) at two additional locations on the Neosho River in 1990-92. Sediments were contaminated by lead. Concentrations of selenium, boron, and most metals in mussels were low to normal for biota. Arsenic levels in mussels and fish suggest low-level contamination of biota. Aluminum, barium, manganese and strontium concentrations were much higher in mussels than in fish. Five fish composite samples had cadmium concentrations that indicate chronic deleterious effects on biota. Lead concentrations in six fish samples were elevated. Mercury concentrations in most large fish composites exceeded concentration for protection of animals that might consume them. We believe that reductions in cadmium, lead, and mercury contamination, in particular, would benefit aquatic life in the river.

Winter poisoning of coyotes and raptors with Furadan-laced carcass baits.

Three bald eagles (Haliaeetus leucocephalus), a red-tailed hawk (Buteo jamaicensis), and two coyotes (Canis latrans) found in a field in north-central Kansas (USA) in December 1992 were poisoned by flowable carbofuran (Furadan 4F) placed on sheep (Ovis aries) carcasses to kill coyotes. The carbofuran was placed on the carcasses in October 1992, but the coyotes and raptors apparently were killed in late December. Thus, flowable Furadan can cause direct and secondary deaths of wildlife under some circumstances for at least 60 days following placement.

Bradykinin B2 receptor-mediated chronotropic effect of bradykinin in isolated guinea pig atria.

The present study was undertaken to characterize the direct chronotropic effect of bradykinin in isolated spontaneously beating atria of the guinea pig. Bradykinin caused concentration-dependent increases in the beating rate of atria. In contrast, the active metabolite of bradykinin and the typical bradykinin B1 receptor agonist, Des-Arg9-bradykinin, had no effect on the beating rate of atria. Inhibition of converting enzyme or neutral endopeptidase by captopril or SQ-28603, respectively, did not affect beating rate but potentiated bradykinin-induced increase in beating rate. The potent bradykinin B2 receptor antagonist, HOE 140, antagonized bradykinin-induced chronotropic effect. In contrast, the bradykinin B1 receptor antagonist, Lys-[Leu8]Des-Arg9-bradykinin, had no effect. The increase in beating rate caused by bradykinin was not affected by blockade of beta 1-adrenoceptors, cyclooxygenase, or nitric oxide synthesis using atenolol, indomethacin and N omega-nitro-L-arginine, respectively. Unlike bradykinin, angiotensin I and angiotensin II caused very small or no change in beating rate in the presence or absence of captopril and SQ-28603. These results indicate that bradykinin causes a direct positive chronotropic effect which is mediated by activation of bradykinin B2 receptors independently of prostaglandins and beta 1-adrenoceptors.

Beta 1- and beta 2-adrenoceptor antagonist activities of ICI-215001, a putative beta 3-adrenoceptor agonist.

1. The present study was undertaken to characterize the beta 3-adrenoceptor agonist activity of ICI-215001 and to determine whether it exhibits additional activities on beta 1- and beta 2-adrenoceptors in isolated spontaneously beating atrium, trachea and ileum of guinea-pig. 2. In guinea-pig atrium, isoprenaline, a non-selective beta-adrenoceptor agonist, caused concentration-dependent, positive chronotropic effects that were inhibited by atenolol, a selective beta 1-antagonist. ICI-215001 also competitively antagonized the increase in heart rate caused by isoprenaline. 3. ICI-215001 exhibited low intrinsic activity at increasing the beating rate of atrium and no activity on resting or induced tone of tracheal strips. 4. In strips of guinea-pig trachea, contracted submaximally with carbachol, isoprenaline, caused concentration-dependent relaxations. Both ICI-118551, a selective beta 2-adrenoceptor antagonist, and ICI-215001 competitively inhibited the relaxations caused by isoprenaline. 5. In isolated strips of guinea-pig ileum longitudinal smooth muscle contracted with histamine, isoprenaline and ICI-215001 caused relaxations which were inhibited by alprenolol, a beta-adrenoceptor antagonist with modest affinity for beta 3-adrenoceptors, but were resistant to ICI-118551 and atenolol. 6. These results indicate that ICI-215001 exhibits beta 3-adrenoceptor agonist activity as demonstrated by relaxations mediated via atypical beta-adrenoceptors in the longitudinal smooth muscle of guinea-pig ileum. Further, the studies demonstrate that ICI-215001 can act as an antagonist at beta 1- and beta 2-adrenoceptors in situations where its intrinsic agonist activity is low.

Involvement of the "tethered ligand" receptor in thrombin-induced endothelium-mediated relaxations.

The mechanisms by which the serine protease, alpha-thrombin, mediates relaxations were examined in isolated dog and pig coronary arteries and dog saphenous veins. In rings of coronary arteries and saphenous veins contracted submaximally with prostaglandin F2 alpha or U46619, alpha-thrombin (0.1-10 nM) caused relaxations that were abolished by treatment with N omega-nitro-L-arginine (L-NNA) or removal of the endothelium, indicating that the relaxations were mediated by endothelium-derived nitric oxide. These relaxations were blocked by the thrombin active site inhibitor, MD-805, indicating the requirement of thrombin's catalytic site to induce the relaxations. The thrombin exosite inhibitor, BMS-180742, decreased the sensitivity to alpha-thrombin without altering maximal relaxations. Indomethacin, a cyclooxygenase inhibitor, had no inhibitory effect on the relaxations caused by alpha-thrombin, indicating that the relaxations were not mediated by cyclooxygenase products. Similar to alpha-thrombin, the thrombin receptor activating peptide (human sequence: SFLLRNP, 1-100 microM) caused relaxations in pig coronary artery and dog saphenous vein but not in dog coronary artery. These relaxations were blocked by L-NNA but not by indomethacin. The results indicate that alpha-thrombin induces endothelium-dependent relaxations by a novel signaling mechanism that involves proteolytic cleavage of the thrombin receptor to expose a new amino terminus that functions as a "tethered peptide ligand" to activate thrombin receptors on the endothelial cells and release nitric oxide.

Isolation and identification of lysophosphatidylcholines as endogenous modulators of thromboxane receptors.

Inhibition of thromboxane receptor radioligand binding to human platelet membranes has been employed as the basis for a radioreceptor assay designed to measure thromboxane receptor binding activity in samples of biological fluids. This method was used during phase 1 clinical evaluation of the thromboxane receptor antagonist SQ 30,741. Frequently, baseline plasma samples as well as plasma samples from placebo-treated subjects showed significant inhibition of radioligand binding in the radioreceptor assay, suggesting the presence of endogenous thromboxane receptor ligands. This receptor binding activity was stable and could be monitored in blood from normal volunteers using a modification of the radioreceptor assay. In order to identify the substance responsible for the observed activity, the activity present in pooled bovine blood was isolated and evaluated by a combination of FAB/MS, 1H-NMR, 13C-NMR and co-injection with reference standards on HPLC. Several endogenous thromboxane receptor ligands were identified as L-alpha-lysophosphatidylcholine (LPC) species. One major species, palmitoyl-LPC, contracted isolated rat aortic spirals, and these contractions could be delayed or prevented, but not reversed by the thromboxane receptor antagonist SQ 29,548. Palmitoyl-LPC slightly potentiated aortic contractions induced by the thromboxane receptor agonist, U-46,619, and diminished in a concentration-dependent manner the antagonism by SQ 29,548 of contractile responses to U-46,619. These findings are consistent with a potential for LPC species to bind and activate thromboxane receptors.

Pharmacological characterization of potent, long-acting thromboxane receptor antagonists, SQ 33,261 and SQ 33,552.

SQ 33,261 ([1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-6-[3-[[2- [(phenylamino)carbonyl]hydrazono]methyl]-7-oxabicyclo[2.2.1]hept-2 - yl]-4-hexenoic acid) and SQ 33,552 ([1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-6-[3-[[[[(4- chlorophenyl)amino]carbonyl]hydrazono]methyl]-7- oxabicyclo[2.2.1]hept-2-yl]-4-hexenoic acid) are potent thromboxane (Tx) A2 receptor antagonists. They inhibited platelet aggregation in platelet-rich plasma induced by the TxA2 mimetic, U-46,619 (10 microM), with IC50 values of 200 and 70 nM, respectively. Neither compound inhibited ADP (20 microM)-induced platelet aggregation (IC50 greater than 1000 microM). SQ 33,261 and SQ 33,552 competitively antagonized U-46,619-induced contraction of rat aortic strips with respective pA2 values of 9.0 and 10.1 and KB values of 1.2 and 0.1 nM. They also competitively antagonized U-46,619-induced contraction of guinea pig tracheal strips with pA2 values of 8.9 and 9.9 and KB values of 1.9 and 0.4 nM, respectively. SQ 33,261 and SQ 33,552 (p.o.) were potent inhibitors of U-46,619 (2 mg/kg i.v.)-induced death in mice with ID50 values of 8 and 1 micrograms/kg, respectively. SQ 33,261 and SQ 33,552 (0.2 mg/kg p.o.), also had long duration of action in this assay with 50% survival times of 7 and 15 hr, respectively. SQ 33,261 at 0.01 and 1.0 mg/kg i.v., inhibited arachidonic acid-induced bronchoconstriction and reversed arachidonic acid-induced hypertension to a hypotensive response. SQ 33,552 inhibited TxA2 synthase at high concentrations (IC50 = 307 microM), whereas SQ 33,261 was inactive. Neither compound inhibited cyclooxygenase or caused an elevation of platelet cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Interspecies differences in thromboxane receptors: studies with thromboxane receptor antagonists in rat and guinea pig smooth muscles.

To investigate possible subclasses of thromboxane receptors in vascular and airways smooth muscles, we evaluated activities of five structurally different competitive thromboxane receptor antagonists (i.e., SQ 29,548 [( 1S-[1 alpha, 2 alpha(5Z), 3 alpha, 4 alpha]]-7-[3- [2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid), L655,240 (3-[1-(4-chlorobenzyl)-5- fluoro-3-methylindol-2yl]2,2-dimethyl propanoic acid), BM 13,505 (4-[2-[[(4-chlorophenyl)sulfonyl]amino]ethyl]benzeneacetic acid), GR 32,191 [( 1R-[1 alpha (Z), 2 beta, 3 beta, 5 alpha]]-(+)-7- [5-[[(1, 1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1- piperidinyl)cyclopentyl]-4-heptenoic acid hydrochloride] and SQ 30,741 [1S- [1 alpha,2 alpha(5Z),3 alpha,4 alpha]]- 7[[[[[( oxaheptyl)amino]acetyl]amino]methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid)] in trachea, aorta and portal vein from both rats and guinea pigs. Schild plots and drug receptor dissociation constants (KB) were determined for each antagonist in each tissue using U-46,619 as the agonist. Rank orders of potency were identical in rat aorta, rat trachea and rat portal vein (SQ 29,548 greater than L 655,240 greater than BM 13,505 greater than GR 32,191 greater than SQ 30,741), with calculated KB values ranging from 0.5 to 20 nM. Rank orders of potency in guinea pig trachea and guinea pig portal vein were the same (GR 32,191 greater than SQ 29,548 greater than SQ 30,741 greater than L 655,240 greater than BM 13,505), with KB values ranging from 0.1 to 30 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of thromboxane receptor activation in the bronchospastic response to endothelin.

The selective TxA2/PGH2 (TP) receptor antagonist, SQ 30,741, was used to test the hypothesis that TP-receptor activation contributes to the reactivity of airways and isolated trachea to endothelin-1 (ET-1). Dose-dependent contractions of guinea pig tracheal strips to ET-1 in vitro were unaffected by either SQ 30,741 (1 microM) or indomethacin (2.8 microM). In contrast, maximal bronchospastic responses (increases in airways resistance and decreases in dynamic lung compliance) of anesthetized guinea pigs to ET-1 (0.5 and 1.5 nmole/kg i.v.) in vivo were blocked greater than 90% by SQ 30,741 (1 mg/kg i.v.). Concurrent increases in arterial blood pressure and decreases in leukocyte counts induced by ET-1 were unaffected by SQ 30,741. In rats, ET-1 (1.5 nmole/kg i.v.) did not affect lung mechanics, but did cause biphasic blood pressure and leukopenia responses which were unaltered by SQ 30,741. These data demonstrate that there is considerable species variability in the bronchospastic response to ET-1, and that in guinea pigs, this response is caused predominantly by the activation of TP-receptors.

Leukotrienes cause mesenteric vasoconstriction and hemoconcentration in rats without activating thromboxane receptors.

Thromboxane A2/prostaglandin H2 (TP)-receptor activation has been reported to participate in some of the responses to peptide leukotrienes (LT). We examined the effect of TP-receptor antagonism on LT-induced mesenteric vasoconstriction and hemoconcentration in anesthetized rats. The antagonist used in these studies, SQ 30,741, was shown to have high selectivity and potency for vascular TP-receptors in the rat. Arterial (i.a.) injection of LTC4 and D4 elicited dose-dependent and transient reductions in mesenteric blood flow without changes in arterial blood pressure. These responses were unaffected by a dose of SQ 30,741 which produced approximately 99% inhibition of similar responses to U-46,619. In contrast, LT-induced mesenteric vasoconstriction was inhibited approximately 90% by two LT antagonists, LY 171,883 and SKF 104,353. In other experiments i.v. infusion of LTD4 caused increases in hematocrit and reductions in arterial blood pressure that were not influenced by SQ 30,741. These data suggest that increases in mesenteric vascular resistance and hemoconcentration in response to LTs are not the result of TP-receptor activation.

Role of thromboxane receptors in Forssman shock in guinea pigs.

Forssman shock is a bronchospastic reaction mounted in guinea pigs on intravenous administration of an antiserum obtained from rabbits immunized against sheep erythrocytes. The involvement of thromboxane receptors in Forssman shock was determined with SQ 30,741, which was characterized as a selective antagonist of these receptors in guinea pig airways in vitro and in vivo. A volume of antiserum producing consistent, sublethal bronchoconstriction was given either alone (control) or 3 min after SQ 30,741 (0.03, 0.3, or 1.0 mg/kg iv) to urethan-anesthetized guinea pigs. In controls, maximum reductions in dynamic compliance (-59 +/- 6%, P less than 0.01) and increases in airways resistance (383 +/- 97%, P less than 0.01) were detected 1 min after antiserum. Both responses were significantly inhibited by SQ 30,741, either partially at 0.03 mg/kg or completely at 0.3 mg/kg. An accompanying thrombocytopenia was not abated by SQ 30,741. In separate experiments, bronchospasm was reduced by aerosol administration of 0.1% SQ 30,741 and completely prevented by aspirin (10 mg/kg iv). When Forssman antiserum was injected in lethal quantities to other guinea pigs, SQ 30,741 (1 mg/kg iv) attentuated only the resistance component of bronchospasm and did not prevent death. These data demonstrate that thromboxane receptor stimulation is a pivotal step in the pulmonary manifestations of sublethal Forssman shock but is less crucial in more severe forms of the reaction.

Parasitism in llamas.

Llamas in North America are infected with a number of helminth parasites, including: gastrointestinal nematodes, lungworms, meningeal worms, tapeworms, and flukes. Most of these helminths can be treated with the anthelmintic currently used to treat cattle and sheep. At least two protozoan parasites, coccidia and toxoplasma, have been reported in the llamas in North America. Coccidia can be controlled with the anticoccidial drugs used to control these organisms in other domestic animals. Toxoplasma has been reported uncommonly in llamas in North America and control is very difficult. Several external parasites occur in llamas in North America, including lice, mites, ticks, and the deer nasal bot. Except for the deer nasal bot, the other external parasites in the llama can be treated with pesticides approved for use in cattle.