Atrial natriuretic peptide receptor modulators: effects of disubstituted quinazolines on receptor binding and in vitro biological activity.

Prazosin (25 microM) was found to increase 125I-labeled rat atrial natriuretic peptide ([125I]rANP) receptor binding by 50% (SC50) in bovine adrenal zona glomerulosa membranes. A series of 2,4-disubstituted quinazolines was prepared in order to identify more potent analogues for additional in vitro testing. Compound 7 (N-[3-[[2-(diethyl-amino)-4-quinazolinyl]amino]propyl] guanidine dinitrate) from this series (3 microM) significantly decreased the EC50 for rANP-mediated inhibition of ACTH-stimulated aldosterone synthesis in rat adrenal glomerulosa cells. At a higher concentration (20 microM), compound 7 had no effect on particulate guanylate cyclase from rabbit glomeruli in either the presence or absence of rANP.

Development of dual-acting agents for thromboxane receptor antagonism and thromboxane synthase inhibition. 3. Synthesis and biological activities of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives and related compounds.

A novel series of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives was discovered as potent dual-acting agents to block the TXA2 receptor and to inhibit the thromboxane synthase (TRA/TSI). Synthesis, structure-activity relationship (SAR), and in vitro and in vivo pharmacology of this series of compounds are described. Modification of the series revolved around the oxazole moiety to increase the hydrophilicity of the compounds and to correlate the biological activity with lipophilicity of the compounds. The most potent in the series was (E)-7-[4-[4-[[(4-cyclohexylbutyl)amino]carbonyl]-2-oxazolyl] phenyl]-7 -(3-pyridyl)hept-6-enoic acid (14) with Kd = 9.9 +/- 0.4 nM for the thromboxane receptor antagonism and IC50 = 55.0 +/- 17.9 nM for thromboxane synthase inhibition. The compound 14 was a selective TRA/TSI which exhibited desirable characteristics for oral activity, "shunt" effect to elevate PGI2 level, and absence of agonist activity.

Structure-activity relationships of arylimidazopyridine cardiotonics: discovery and inotropic activity of 2-[2-methoxy-4-(methylsulfinyl)phenyl]-1H-imidazo[4,5-c]pyridine.

Recently several noncatecholamine, nonglycoside cardiotonic drugs have been discovered that possess both inotropic and vasodilator activities in experimental animals and man. Prototypical compounds include amrinone, sulmazole, and fenoximone. We investigated the structural requirements necessary for optimal inotropic activity in a series of molecules containing a heterocyclic ring fused to 2-phenylimidazole and discovered that 2-phenylimidazo[4,5-c]pyridines were generally 5-10-fold more potent than analogous 2-phenylimidazo[4,5-b]pyridines (e.g., sulmazole) or 8-phenylpurines. Furthermore, all imidazo[4,5-c]pyridine analogues we tested were orally active; in contrast, only one of the imidazo[4,5-b]pyridine derivatives, sulmazole, was significantly active. One of several highly active compounds in the [4,5-c] series was 50 (LY175326, 2-[2-methoxy-4-(methylsulfinyl)phenyl]-1H-imidazo[4,5-c]pyridine hydrochloride). The structure-activity relationship of this series is presented and compared to that of the imidazo[4,5-b]pyridine and purine series.

Dihydropyridazinone cardiotonics: the discovery and inotropic activity of 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H -indol-2- one.

We discovered that 6 (N-[4-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)phenyl]acetamide) is a potent positive inotrope in dogs, and we have prepared several lactam analogues of this agent. These included 16 (1,3-dihydro-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one), 32 (the analogous quinolin-2-one), and 37 (the analogous benzazepin-2-one). The inotropic ED50's of these compounds were 24, 3.3, and 5.2 micrograms/kg, respectively, after iv administration to pentobarbital-anesthetized dogs. Compound 20 (LY195115, 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-i ndol-2- one), the geminal dimethyl analogue of 16, was 3.5-fold more potent than 16 when administered iv (ED50 = 6.8 micrograms/kg). However, the most profound effect of the geminal alkyl substitution was on oral activity. The approximate ED50's of 20 and 16 after oral administration to conscious dogs were 25 and 400 micrograms/kg, respectively. The increase in contractility produced by 25 micrograms/kg of 20 was maximally sustained in excess of 8 h. Thus, 20 is one of the most potent and long-acting oral inotropes described to date.

Fibrinogen receptor (GPIIb-IIIa) antagonists derived from 5,6-bicyclic templates. Amidinoindoles, amidinoindazoles, and amidinobenzofurans containing the N-alpha-sulfonamide carboxylic acid function as potent platelet aggregation inhibitors.

A series of highly potent and specific fibrinogen receptor antagonists have been discovered and optimized through structural modification of the novel amidinoindole and benzofuran compounds, I and II. Systematic linker optimization afforded the amidinobenzofuran-containing inhibitor 29, which displayed an IC50 value of 250 nM in platelet aggregation assays. Attempts to enhance activity by modification of the beta-position of the beta-alanyl carboxylate group of 29 had only a modest effect on inhibitory activity in aggregation assays. Analogues prepared to enhance the activity by conformational restriction were also found to be equally or less potent. In contrast, modification at the alpha-position of the beta-alanyl carboxylate group resulted in the identification of extremely potent and novel amidinobenzofuran-containing derivatives 46-49. Reexamination of 5,6-bicyclic aromatic nucleus led to the further identification of amidinoindole- and amidinoindazole-containing derivatives 53-55. These analogues, 46-49 and 53-55, exhibited potent in vitro activity with IC50 values of 25-65 nM in platelet aggregation assays and an IC50 value of 2 nM in fibrinogen binding assays and demonstrated a selectivity of > 50,000-fold for GPIIb-IIIa versus the most closely related integrin, the vitronectin receptor, alpha v beta 3.

Use of conformationally restricted benzamidines as arginine surrogates in the design of platelet GPIIb-IIIa receptor antagonists.

The use of 5,6-bicyclic amidines as arginine surrogates in the design of a novel class of potent platelet glycoprotein IIb-IIIa receptor (GPIIb-IIIa) antagonists is described. The additional conformational restriction offered by the bicyclic nucleus results in 20-400-fold increases in potency compared to the freely flexible, acyclic benzamidine counterpart. The design, synthesis, structure-activity relationships (SAR), and in vitro activity of this novel class of GPIIb-IIIa antagonists are presented.

Cardiovascular effect and stimulus-dependent inhibition of superoxide generation from human neutrophils by tibenelast, 5,6-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt (LY186655).

Tibenelast (LY186655), 5,6,-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt, is an orally active anti-anaphylactic compound in guinea pigs, and has been shown to prevent bronchospasm in moderately severe asthmatic patients. Pharmacological studies with tibenelast demonstrated that it is a selective phosphodiesterase (PDE) inhibitor in that it is moderately active against the lung and stomach enzyme while being a very weak inhibitor of the heart enzyme. The compound was without cardiovascular effects at anti-anaphylactic doses. In contrast to theophylline, tibenelast did not have a direct inotropic effect in the cat papillary muscle system. The concentration that inhibited 50% of the enzymatic activity (IC50) for tibenelast was 20- to 30-fold lower for neutrophil PDE than for PDE of other tissues. It was 100 times more potent than aminophylline in inhibiting superoxide generation from platelet-activating factor (PAF)-primed polymorphonuclear leukocytes (PMNL) challenged with chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. However, tibenelast was less effective in the tumor necrosis factor-primed system, and did not inhibit superoxide generation during phagocytosis or when other soluble stimuli, such as phorbo-12-myristate-13-acetate or the calcium ionophore A23187, were used. Furthermore, tibenelast did not inhibit enzymes involved in arachidonic acid metabolism. These results suggest that tibenelast probably inhibits superoxide release from PMNL via a selective inhibition on PDE.

Cardiac inotropic responses to calcium and forskolin are not altered by prolonged isoproterenol infusion.

Effects of prolonged isoproterenol infusion upon the density of cardiac calcium channels, calcium-mediated contractile responses, and the ability of forskolin to enhance tension development and cyclic AMP accumulation were studied in ventricular muscle preparations from Sprague-Dawley rats. Isoproterenol infusion (400 micrograms/kg per h s.c., 4 days) significantly decreased calcium channel density (Bmax) in cardiac microsomal membranes as quantified by a 32% decrease in specific [3H]nitrendipine binding sites; binding affinity (KD) was unchanged. A 57% decrease of beta-adrenoceptors confirmed homologous down regulation. To examine functional effects of decreased [3H]nitrendipine binding sites, responses to calcium, BAY K8644 and nifedipine were determined in isolated right ventricular strips. Significant decreases in basal developed tension were observed in muscles from isoproterenol-infused rats. However, concentration-dependent increases in contractility in response to CaCl2 or BAY K8644 were comparable, and the negative inotropic effect of nifedipine was unchanged. Whereas isoproterenol infusion was associated with significantly decreased basal cardiac cyclic AMP concentrations, exposure of ventricular strips from either vehicle- or isoproterenol-infused rats to 10 microM forskolin resulted in comparable increases in cyclic AMP and in developed tension. Cumulative, submaximal concentrations of forskolin also produced similar increases in contractility with maximum responses in ventricular strips from vehicle-infused animals attained at 4.4 microM forskolin. Higher concentrations resulted in automaticity. By contrast, ventricle from isoproterenol-infused animals responded to 14.4 microM forskolin with maximal increases in force of contraction.

Tick-borne pathogens in the blood of wild and domestic ungulates in South Africa: interplay of game and livestock.

We screened for tick-borne pathogens blood samples from 181 wild and domestic ungulates belonging to 18 host species in 4 South African Provinces. Polymerase chain reaction followed by reverse line blotting and sequencing allowed detecting 16 tick-borne pathogen species belonging to the genera Babesia, Theileria, Anaplasma, and Ehrlichia. Ten pathogen species were involved in 29 new host-pathogen combinations. Most infections (77.9%) involved more than one pathogen species. Principal component analysis (PCA) assigned the 163 infections, identified to species level, to 4 groups. Three groups were associated with sheep, cattle, and horse and their respective wild counterparts. Each group was characterised by high homogeneity in pathogen assemblage and host phylogenetic status. These groups characterised the most privileged transmission routes between and among wild and domestic ungulates. The 4th group showed high heterogeneity in pathogen assemblage and host phylogenetic status. This group seems to indicate frequent spill over events in impala of pathogens that usually circulate among cattle- or sheep-related species. Within 6 localities, we sampled an equal number of wild and domestic animals (n=128). On this dataset once having controlled for the significant variation among localities, the infection prevalence and intensity of infection did not differ significantly between wild and domestic hosts. This suggests that both animal types, domestic and wild hosts, could act as evenly efficient sources of infection for themselves and for each other. Overall, this study shed new light on the pathogen circulation naturally achieved at the interplay between wild and domestic ungulates.

Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa.

A total of 7364 ticks belonging to 13 species was collected from 64 game animals (belonging to 11 species) and from 64 livestock animals (cattle and sheep) living in close vicinity at 6 localities in 3 South African Provinces (Free State, Mpumalanga, and Limpopo). The geographic distribution of all tick species was congruent with the literature except for Haemaphysalis silacea. From each infested host, a maximum of 10 males and 10 females of each tick species were dissected to isolate the salivary glands. Salivary glands were screened for tick-borne pathogens using polymerase chain reaction followed by reverse line blotting and sequencing. This approach allowed us to evaluate the exposure of wild and domestic hosts to tick-borne pathogens in their respective environments. Among the 2117 examined ticks, 329 (15.5%), belonging to 8 species, were infected and harboured 397 infections. Among those, 57.7% were identified to species level and were assigned to 23 pathogen species of the genera Babesia, Theileria, Anaplasma, and Ehrlichia. In 3 out of 6 localities, salivary glands from ticks infesting wild ruminants displayed significantly higher infection prevalence and pathogen mean density than salivary glands from ticks infesting livestock animals. Four piroplasm species [Theileria bicornis, Babesia sp. (sable), Theileria sp. (giraffe), and Theileria sp. (kudu)] were detected for the first time in ticks. The tick species Rhipicephalus evertsi evertsi, Rhipicephalus (Boophilus) decoloratus, Hyalomma rufipes, Rhipicephalus appendiculatus, and Amblyomma hebraeum were associated with a broader pathogen range than previously known, and thus new vector-pathogen combinations are described. In addition, previously unknown coinfection patterns in tick salivary glands are reported.

[Applied physiology: the work of driving an industrial heavy-duty truck on the highway]. / Fisiologia applicata: il lavoro di guida su strada di un veicolo industriale di grande portata.

Scope of this work was the investigation of the following parameters: energetic metabolism, by O2 consumption measurement using the open-circuit technique; pulmonary ventilation; cardio-circulatory activity behaviour, by recording heart rate, E.C.G., arterial blood pressure and plethysmography of lower limbs; body temperature pattern; visual function, through the determination of the visual field; kidney function through urine analysis; body joints flexibility, by proper test. The subjects chosen for this investigation are three experienced heavy-duty vehicle drivers that where submitted to proper checks before, during and after having driven a 190.38 Model truck fitted with spoiler and an S.R. Viber, high cargo body tarpaulin, PTT, 43.2 tons overall weight truck, over 9 different courses including town traffic, level highway, up and downhill highway with different gradients, in a period from 9 a.m. to 8 p.m. Along the total run of 404 kilometres, covered in 364 minutes, recording of the parameters under investigation was carried out over 249 kilometres covered 240 minutes (72% of overall driving time). Energy expenditure (1.20 +/- .19 Kcal/min at rest) resulted higher on steep uphill stretches with many bends (2.28 +/- .44 Kcal/min) than on less steep up- and downhill stretches and in city traffic (1.82 +/- .21 Kcal/min). The lowest energy expenditure--in some cases lower than the value found in the experimental rest--was recorded during motorway driving (1.28 +/- .19 Kcal/min). Pulmonary ventilation was characterized by frequent changes in rate and tidal air, in strict relationship with driving work bio-mechanical requirements (operation of the steering wheel). Also heart rate underwent ariations well in line with the energy expenditure pattern: of limited magnitude in uphill driving (+33% max over the value at rest), up to values identical with those at rest in the level highway drive. No appreciable variations were recorded in the respiratory quotient, calories/ventilation ratio and oxygen pulse. No significant changes occurred in arterial blood pressure, body temperature, auditory, visual and kidney functions, and in flexibility. Conversely, slight swelling (5%) of lower limbs was noticed at the end of the driving day. It was hence proven that--from the standpoint of energy expenditure--the work performed in driving a vehicle is not too exacting and does not induce particularly high nervous stresses. However, under given conditions, such as steady speed driving in level highway, a pre-sleep state occurs in all cases approximately after the first 30 minutes of driving, which in most cases the driver is not even aware of.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of L-carnitine administration on VO2max and the aerobic-anaerobic threshold in normoxia and acute hypoxia.

Seven healthy young male adults were subjected to a total of 56 tests to ascertain the effects of L-carnitine (L-C) and a placebo (P) on ventilation, O2 intake (VO2), CO2 output, heart rate, blood pressure and serum lactic acid, non-esterified fatty acid, glycerol and glucose during strenuous and aerobic/anaerobic threshold-level treadmill exercise. The tests were made in conditions of normoxia (O2 = 20.9%) and hypoxia (O2 = 13.0%, equivalent to 3,500 m above sea level). The only clear difference was in the respiratory quotient (RQ = 0.883, SD 0.025 vs 0.904, SD 0.035) after L-C and P administration respectively (P less than 0.01), under normal oxygenation and 0.861, SD 0.052 following L-C vs 0.926, SD 0.040 after P (P less than 0.01) in acute hypoxia at VO2 levels around the anaerobic threshold. The lower RQ values of the L-C-treated subjects during hypoxia indicate a lower rate of carbohydrate transformation.

A comparison of the cardiotonic effects of AR-L115 and AR-L57: evidence for distinct inotropic mechanisms.

AR-L57 and AR-L115 have been of interest as inotropic agents for management of heart failure. Although their physiological effects are well documented, their mechanism(s) of action are unclear. Both AR-L57 and AR-L115 increased contractile force of cat papillary muscles in concentration-dependent manners; these effects were independent of either alpha- or beta-adrenoceptor stimulation. To determine if these effects occurred via a cAMP-dependent mechanism, cardiotonic actions were studied in the presence of carbachol. Muscarinic stimulation of papillary muscles attenuated contractile responses to AR-L115 thus implying a cAMP-mediated response. By contrast, carbachol did not alter the dose-response profile to AR-L57. In addition, AR-L115 potentiated the inotropic actions of isoproterenol whereas AR-L57 was ineffective. Both AR-L57 and ouabain increased diastolic resting tension in papillary muscles--a phenomenon associated with a state of Ca2+ overload; AR-L115 was without effect. In anesthetized dogs, i.v. AR-L57 and AR-L115 increased contractility and heart rate while reducing mean arterial blood pressure. Both agents had similar rates of onset (10-15 s) and durations of action (40-60 min). Although in vitro studies clearly indicate that AR-L57 and AR-L115 enhance inotropic state by distinct mechanisms, their in vivo cardiovascular profiles are comparable.

The relation between vascular relaxant and cardiac electrophysiological effects of pinacidil.

Pinacidil may represent an example of a new class of vasodilators that act by increasing membrane permeability to potassium ions. In the present study, the cardiac electrophysiological and venorelaxant effects of a series of pinacidil analogs in canine tissues in vitro were examined. Piacidil (3 x 10(-5) M) markedly reduced action potential duration in Purkinje fibers (82 +/- 3% decrease) and ventricular muscle (54 +/- 2% decrease) without significantly affecting maximal upstroke velocity of the action potential or conduction time. The EC50 for the reduction in Purkinje fiber action potential duration was 2.6 +/- 0.5 microM. Pinacidil also decreased barium-induced automaticity in Purkinje fibers; the concentration that decreased the rate of firing by 50% was identical to the EC50 for decreasing action potential duration. In some preparations, high concentrations of pinacidil (greater than or equal to 3 x 10(-5) M) were associated with the appearance of spontaneous action potentials that were closely coupled to the preceding driven action potential. The EC50 for pinacidil in relaxing phenylephrine-contracted cephalic veins was 0.43 +/- 0.09 microM, and in isolated cat papillary muscle, pinacidil had a direct negative inotropic effect with an EC50 of 4.1 +/- 0.7 microM. Thus, pinacidil was 6 and 10 times more potent in relaxing phenylephrine-contracted veins than in shortening action potential or decreasing cardiac contractility. There was an excellent correlation (r = 0.933, p = 0.002) between decreases in action potential duration and venorelaxation for all pinacidil analogs, as well as for BRL 34915 and nicorandil, two purported potassium channel openers. Significant correlations were also obtained between negative inotropic effects and reductions in action potential duration for the pinacidil series. Pinacidil (10(-5) M) also inhibited the venoconstrictor responses to the selective alpha 2 agonist, B-HT 920, to a greater extent than the alpha 1 agonist, methoxamine. Since a good correlation exists in vitro among all the compounds studied in reducing action potential duration, relaxing vascular tissue, and decreasing cardiac contractility, it is concluded that pinacidil as well as nicorandil and BRL 34915 affect vascular and cardiac tissues by similar mechanisms, possibly by increases in potassium ion permeability, although other mechanisms may also play a role.

Effects of prolonged phenylephrine infusion on cardiac adrenoceptors and calcium channels.

Effects of prolonged in vivo infusion of phenylephrine upon receptor binding and cardiac contractility were studied in Sprague-Dawley rats. A 1-hr i.v. infusion of phenylephrine (3 mg/kg/hr) resulted in a sustained 50% increase in diastolic blood pressure and 5% increase in heart rate. Chronic (6-day) infusion (3 mg/kg/hr) utilizing Alzet mini-osmotic pumps maintained plasma concentrations of phenylephrine at 1.0 microgram/ml, depleted myocardial norepinephrine stores 8-fold and resulted in a modest cardiac hypertrophy. Density and affinity of myocardial adrenoceptors and calcium channels were quantified by analyzing saturation isotherms of radioligand binding. [3H]Prazosin, [3H]dihydroalprenolol and [3H]nitrendipine bound specifically and with high affinity to cardiac alpha-1 and beta adrenoceptors and calcium channels, respectively. As measured by Scatchard analyses, phenylephrine infusion significantly decreased the maximum number (Bmax) of specific [3H]prazosin binding sites by 39% (430 +/- 20 vs. 263 +/- 16 fmol/mg of protein; P less than .05). Chronic phenylephrine treatment also decreased the Bmax for [3H]dihydroalprenolol binding by 31% (124 +/- 3.3 vs. 86 +/- 6.6 fmol/mg of protein; P less than .05) and the Bmax for [3H]nitrendipine binding by 32% (342 +/- 8.8 vs. 235 +/- 6.7 fmol/mg of protein; P less than .05). Binding affinities (Kd) of [3H]prazosin, [3H]dihydroalprenolol and [3H]nitrendipine remained unchanged. Administration of vehicle alone or surgical manipulation due to osmotic pump implantation did not affect either the density or affinity of [3H]prazosin, [3H]dihydroalprenolol or [3H]nitrendipine binding. Contractile responses to phenylephrine were studied in isolated ventricular strips to determine the functional significance of alpha-1 adrenoceptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolonged isoproterenol infusion on cardiac and vascular responses to adrenoceptor agonists.

In vitro responses of cardiac and vascular smooth muscle to both adrenoceptor agonists and phosphodiesterase inhibitors were studied in tissues from either saline- or isoproterenol-infused rats. After chronic isoproterenol infusion the sigmoidal relationship between concentration of acutely administered isoproterenol and inotropic response of cardiac muscle was shifted to the right; the maximum response was decreased by approximately 40%. Inotropic responses were attenuated further by the beta adrenoceptor antagonist, propranolol. By contrast, quantitatively comparable inotropic responses to phenylephrine were not altered after isoproterenol infusion. However, they were blocked by the selective alpha adrenoceptor antagonist, prazosin, but were not affected by propranolol. Inotropic effects of the phosphodiesterase inhibitor, isobutylmethylxanthine, were comparable in tissues from either saline- or isoproterenol-infused rats. Similar results were obtained in vascular tissues. Portal veins and aortas from isoproterenol-infused rats were less responsive to the acute relaxant properties of the beta adrenoceptor agonists, isoproterenol and salbutamol. However, as in cardiac muscle, relaxant effects to phosphodiesterase inhibitors (isobutylmethylxanthine and papaverine) were not attenuated. In addition, contraction to norepinephrine was comparable in tissues from either saline- or isoproterenol-infused rats. These data indicate that isoproterenol infusion attenuates beta adrenoceptor-mediated responses of vascular and cardiac muscle to similar degrees but does not alter responses to either alpha adrenoceptor agonists or phosphodiesterase inhibitors.