The in vitro pharmacological profile of SK&F 106760, a novel GPIIB/IIIA antagonist.

The properties of SK&F 106760 [N alpha-acetyl-cyclo(S,S)-cysteinyl-N alpha-methyl-arginyl-glycyl-aspartyl-penicillamine-amide] as a GPIIb/IIIa antagonist have been studied in vitro and compared with those of the parent molecule, Ac-RGDS-NH2. Ac-RGDS-NH2 inhibited biotinylated fibrinogen binding to purified human GPIIb/IIIa immobilized on plastic microtitre plates with a Ki of 530 +/- 73 nM. In canine platelet rich plasma Ac-RGDS-NH2 produced a concentration related inhibition of adenosine diphosphate-induced platelet aggregation following preincubation for 3 min with an IC50 of 91 +/- 1 microM. However, incubation in platelet rich plasma for 3 hr abolished the activity of Ac-RGDS-NH2. SK&F 106760 inhibited biotinylated fibrinogen binding to purified human GPIIb/IIIa immobilized on plastic microtitre plates with a Ki of 477 +/- 57 pM. SK&F 106760 inhibited adenosine diphosphate-induced platelet aggregation in human platelet rich plasma with an IC50 of 230 +/- 60 nM but did not inhibit the von Willebrand Factor receptor (GPIb/IX)-mediated platelet agglutination produced by ristocetin. In canine platelet rich plasma SK&F 106760 inhibited aggregation produced by adenosine diphosphate, collagen and epinephrine/U-46619 with IC50 values of 355 +/- 35, 260 +/- 20, and 490 +/- 90 nM, respectively and in gel filtered platelets inhibited thrombin-mediated aggregation with an IC50 of 188 +/- 10 nM. Preincubation of SK&F 106760 in platelet rich plasma for three hours had no significant effect on its ability to inhibit adenosine diphosphate-induced platelet aggregation. SK&F 106760 produced insurmountable inhibition of adenosine diphosphate-induced platelet aggregation in the presence of constant fibrinogen concentrations, but produced competitive inhibition of the concentration-response curve to fibrinogen in adenosine diphosphate-activated platelets with a Kb of 8.0 +/- 1.0 nM. Thus, SK&F 106760 is a potent, stable competitive GPIIb/IIIa antagonist with no detectable activity at the von Willebrand Factor receptor (GPIb/IX).

Coronary thrombolysis with intravenous streptokinase in the anesthetized dog: a dose-response study.

In order to study the in vivo coronary thrombolytic dose-response effectiveness of i.v. streptokinase, pentobarbital-anesthetized, open chest dogs were instrumented for the measurement of lead II ECG, systemic arterial blood pressure, left ventricular developed pressure, left ventricular end-diastolic pressure, left ventricular dP/dt and left anterior descending (LAD) coronary artery blood flow. In some animals, two pairs of ultrasonic dimension gauges were positioned in 1) an area of the left ventricle which would become ischemic after thrombotic occlusion of the LAD and 2) a reference area which would remain unaffected by LAD occlusion. After making an occlusive thrombus by injecting thrombin (50-200 U) and calcium chloride (50-100 mumols) into a cannulated and mechanically occluded side-branch of an isolated segment of the mid- to distal LAD, animals were given either streptokinase (5,000, 10,000, 20,000 or 40,000 U of bolus, followed by 500, 1000, 2000 or 4000 U/min of infusion, respectively) or saline, i.v. At the termination of the experiment, thrombus wet weight and, in some animals, infarct size (measured by tetrazolium staining) were determined. In control animals, thrombus wet weight was 26.7 +/- 5.4 mg (n = 8) and infarct size, as a percentage of the total left ventricle, was 30.1 +/- 4.7% (n = 6). Spontaneous reperfusion (return of LAD blood flow) was not observed in control animals that received saline. Intravenous administration of streptokinase produced a dose-dependent increase in the percentage of animals reperfusing, and a dose-dependent decrease in both thrombus wet weight and the time to reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha adrenoceptor regulation of coronary artery blood flow in normal and stenotic canine coronary arteries.

The response of systemic blood pressure, heart rate, lead II ECG and left circumflex (LCX) coronary artery blood flow to left cardiac sympathetic nerve stimulation was measured in pentobarbital-anesthetized, open chest, spinal transected and vagotomized dogs. After beta adrenoceptor blockade, left cardiac sympathetic nerve stimulation produced frequency dependent decreases in LCX blood flow. Selective alpha-2 adrenoceptor blockade with idazoxan produced a greater inhibition of this decrease in LCX blood flow than did selective alpha-1 adrenoceptor blockade with prazosin. In an additional population of dogs which were similarly prepared but were not spinally transectioned or pretreated with a beta adrenoceptor antagonist, left cardiac sympathetic nerve stimulation produced an increase in LCX blood flow in all animals which reached a maximum within 40 sec, and then began to decline slowly. However, after beta adrenoceptor blockade, identical stimulation parameters produced only a decline in LCX blood flow which returned to the level of control resting blood flow by the end of the stimulation period. Both selective alpha-2 adrenoceptor blockade with idazoxan and selective alpha-1 adrenoceptor blockade with prazosin produced an inhibition of the LCX blood flow decrease provoked by left cardiac sympathetic nerve stimulation in dogs pretreated with beta adrenoceptor antagonists. Idazoxan produced a slightly greater inhibition of the LCX blood flow decrease than did prazosin, suggesting a greater role for postjunctional vascular alpha-2 adrenoceptors in LCX blood flow regulation during cardiac sympathetic nerve stimulation. The presence of a severe coronary artery stenosis reduced, but did not inhibit, the increase in LCX blood flow in response to cardiac sympathetic nerve stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A new model of myocardial infarction in Yucatan minipigs.

Myocardial infarction studies in pigs have been complicated by the use of antiarrhythmic drugs or the high incidence of ventricular fibrillation. We report on a new model of experimental myocardial infarction in thiamylal-anesthetized Yucatan minipigs. These studies were performed in the absence of intravenous antiarrhythmic drugs. No animals required resuscitation during either surgery or reperfusion and only 19% were resuscitated during occlusion. Extensive systemic hemodynamic, regional contractility and coronary blood flow measurements were continuously obtained during left anterior descending coronary artery (LAD) occlusion (45 min) and reperfusion (240 min). Mean arterial blood pressure and left ventricular + dP/dt decreased during occlusion, and both declined further upon reperfusion. Persistent dysfunction (segmental shortening from 24.8 +/- 1.3 to 3.9 +/- 0.9% (p less than 0.001); pre-occlusion and 5 min post-occlusion, respectively) occurred immediately after occlusion in the myocardium perfused by the LAD, while late declines in segmental shortening (19.6 +/- 0.9 to 17.2 +/- 1.2%; pre-occlusion and 240 min post-reperfusion, respectively) were observed in myocardium perfused by the left circumflex coronary artery. While heart rate did not change during occlusion, tachycardia occurred at the onset of reperfusion. Although initial reactive hyperemia following reperfusion was manually inhibited, high LAD blood flow following reperfusion occurred early (0 to 60 min) but returned below pre-occlusion values late (180 to 240 min). The area at risk represented 23.1 +/- 0.9% (n = 34) of the left ventricle and 39.0 +/- 3.2% of this area was infarcted. Therefore, 9.2 +/- 0.9% of the left ventricle was infarcted. These data suggest that myocardical infarction in anesthetized minipigs can be achieved without the aid of intravenous antiarrhythmic drugs and reduced cardioversion. Therefore, this new model can be utilized in the evaluation of therapeutic compounds focused on altering the detrimental consequences of myocardical infarction.

Evaluation of the acute hemodynamic effects and pharmacokinetics of coronary thrombolysis produced by intravenous tissue-type plasminogen activator in the anesthetized dog.

The thrombolytic dose-response effectiveness and pharmacokinetics of tissue-type plasminogen activator (rt-PA) was evaluated in anesthetized, open-chest dogs instrumented for the measurement of systemic hemodynamics. Intracoronary thrombi were formed by injecting thrombin (100 U) and CaCl2 (50 microM) into a cannulated, isolated segment of the left anterior descending coronary artery (LAD). Coronary blood flow was measured by placing an electromagnetic flow probe proximal to the LAD thrombus. Thirty minutes after formation of a stable LAD thrombus, intravenous infusion of rt-PA was given at rates of 0.5, 1, 2, 4, or 8 micrograms/kg/min (n = 8/dose) for 60-90 min, and the animals were followed for an additional 30 min. In vehicle-treated animals, residual thrombus wet weight, determined at the end of the experiment, was 30 +/- 4 mg (mean +/- SEM, n = 8) and spontaneous reperfusion did not occur. The rt-PA produced a dose-related increase in the number of animals reperfusing, a decrease in the time to reperfusion, and a decrease in residual thrombus weight, but had no effect on systemic hemodynamics. The increase in infusion rate from 0.5 to 4 micrograms/kg/min resulted in a linear increase in both the steady-state rt-PA plasma concentration and the area under the rt-PA plasma concentration versus time curve (n = 3-5 animals/dose); between the infusion rates of 4 and 8 microgram/kg/min there was a disproportionate increase in both these parameters that was due to a decrease in the total systemic clearance of rt-PA. The postdosing elimination half-life (t1/2 alpha) did not differ significantly at any dose of rt-PA, and the pooled half-life (t1/2 alpha) for all doses of rt-PA was 2.36 +/- 0.12 min (n = 19).(ABSTRACT TRUNCATED AT 250 WORDS)

Combination of the thromboxane receptor antagonist, sulotroban (BM 13.177; SK&F 95587), with streptokinase: demonstration of thrombolytic synergy.

We examined the ability of the prostaglandin endoperoxide/thromboxane A2 antagonist, sulotroban (BM 13.177; SK&F 95587) to enhance the thrombolytic efficacy of a minimally effective thrombolytic dose of streptokinase. A critical stenosis sufficient to just abolish the hyperemic response to a 20-sec total occlusion was placed on the left circumflex coronary artery of anesthetized open chest dogs using an adjustable screw occluder clamp. Thrombi were formed by applying a 150 microA anodal current to a wire placed within the lumen of the left circumflex just proximal to the screw occluder clamp. After thrombus formation, animals were given either streptokinase (20,000 I.U. bolus + 2,000 I.U./min x 180 min, N = 10), streptokinase + sulotroban (5 mg/kg bolus + 5 mg/kg/hr, N = 10), streptokinase + heparin (300 I.U./kg bolus + 100 I.U./kg/hr, N = 9) or streptokinase + heparin + sulotroban (N = 9). Plasma thromboxane A2 level (as measured by the metabolite, thromboxane B2) was not significantly reduced by any treatment, whereas the dose of sulotroban used completely abolished U46619-induced ex vivo platelet aggregation. Of 10 animals receiving streptokinase alone, only 1 reperfused at 55 min after the start of the streptokinase infusion. Conversely, 9 of 10 animals receiving streptokinase + sulotroban reperfused at 79.4 +/- 10.5 min poststreptokinase (P less than .05). When animals were treated with heparin before streptokinase administration, 8 of 9 animals receiving the streptokinase + heparin combination reperfused in an average of 66.8 +/- 8.6 min after the start of streptokinase infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of sympathetic ganglionic neurotransmission by the selective dopamine-1 receptor agonist fenoldopam (SK&F 82526) in the anesthetized dog.

This study was designed to determine the role of dopamine (DA) receptors in modulation of sympathetic ganglionic neurotransmission utilizing the selective DA-1 agonist fenoldopam. Preganglionic stimulation of cardiac sympathetic nerves (0.5-2.0 Hz), in pentobarbital anesthetized dogs, resulted in frequency-dependent tachycardia. Fenoldopam (10 and 30 micrograms/kg/min i.v.) suppressed the tachycardic response 45% at 0.5 Hz with no significant effect at higher stimulation frequencies. In the presence of SK&F 83566 (10 micrograms/kg/min i.v.), a selective DA-1 receptor antagonist, fenoldopam no longer elicited significant inhibition of the preganglionic response. When postganglionic cardiac nerves were stimulated (0.5 Hz), fenoldopam (100 micrograms/kg/min i.v.) inhibited the response 55% with no significant effect at lower doses. Stimulation of sympathetic preganglionic fibers in the autoperfused hindlimb of the dog induced vasoconstriction. Fenoldopam (3 micrograms/kg/min i.a.) produced marked inhibition of nerve-induced constriction that was partially antagonized by SK&F 83566 (3 micrograms/kg/min i.a.). Complete inhibition of the effect of fenoldopam on sympathetic nerve stimulation in the hindlimb could not be achieved, as a component of this action was apparently due to postjunctional alpha-2 adrenoceptor blockade. This was evidenced by a reduction in the pressor response to the selective alpha-2 adrenoceptor agonist B-HT 920 by fenoldopam. These data indicate that fenoldopam stimulates DA-1 receptors in sympathetic ganglia to inhibit neurotransmission and this effect can be reversed by a selective DA-1 receptor antagonist.