Development of an in vitro model for vascular injury with human endothelial cells.

The aim of the present work was to establish an in vitro screening assay for drug candidates using human endothelial cells as a model for vascular injury after intravenous application. Different endpoints for viability and functionality of endothelial cells were investigated in human umbilical vein endothelial cells (HUVEC) and in immortalised human endothelial cells (IVEC). Cellular viability was determined by measuring ATP content and by the AlamarBlue assay. For comparison, the toxicity of the selected compounds was also tested in a murine fibroblast cell line (3T3 cells). Selected endpoints for endothelial cell-specific function were vascular permeability, determined by measurement of the transendothelial resistance and the diffusion of tracer molecules (FITC-dextran), and the release of prostaglandin and thromboxane as indicators for prothrombotic or vasoconstrictory action. Five compounds (cyclosporin A, mitomycin C, menadione, amrinone and rolipram) were selected due to their known effects on the vasculature. The cytotoxicity of all compounds was similar in endothelial and 3T3 cells. ATP content and AlamarBlue metabolism did not differ significantly except for amrinone. A dose-dependent decrease of transendothelial resistance and an increase in FITC-dextran permeability could be measured in HUVEC cells for the tested compounds, but the sensitivity was not higher than that of the cytotoxicity assays. Increased prostaglandin or thromboxane release was detected for all compounds at cytotoxic concentrations and for rolipram also at non-toxic concentrations. In conclusion, for a first ranking of drug candidates, cytotoxicity assays on any of the three cell types used are appropriate. For a more detailed characterisation of individual compounds, functional assays on HUVEC cells are proposed.

Arrhythmogenic potencies of amrinone and milrinone in unanesthetized dogs with myocardial infarct.

1. In dogs with a 2-4 day old myocardial infarct and a predominantly sinus heart rhythm, we examine arrhythmogenic potencies of amrinone (0.5 mg/kg/min, 1 and 3 mg/kg) and milrinone (10 micrograms/kg/min, 75 and 100 micrograms/kg). 2. Amrinone and milrinone significantly reinduced ventricular ectopic beats on day 2 after coronary occlusion. 3. These effects were preceded by a cardioacceleration which intensified as the ventricular arrhythmias developed. 4. Over the following days the arrhythmogenic potencies of these inotropic drugs were modest. 5. Thus, amrinone and milrinone can impair heart rhythm chiefly in a recent myocardial infarct.

[Studies on the mutagenicity of amrinone and milrinone].

The mutagenicity of domestic new drugs amrinone and milrinone were studied by Ames test, micronucleus test of mouse bone marrow and chromosome aberration assay in CHO cells. The results were as follows: neither amrinone nor milrinone was mutagenic in the Ames test; in chromosome aberration assay, both gave positive results; in mouse micronucleus test, amrinone gave a positive result when mice were exposed to 0.8 LD50 dose, but milrinone gave a negative result. These results suggested that amrinone and milrinone did not induce gene mutation, but amrinone induced chromosome damage both in vitro and in vivo, while the chromosome-damaging activity of milrinone in vitro may be minimized by biodetoxication in vivo.

The effects of amrinone, cyclophosphamide and anti-platelet serum on platelet production in the Gunn rat.

The effect of amrinone on platelet production was differentiated from that of a known bone-marrow cytotoxic agent (cyclophosphamide) and anti-platelet serum (APS). The rate of platelet production has been observed over a 4-day period in the Gunn rat using [75Se]selenomethionine cohort labelling of platelets following administration of either amrinone, 160 mg kg-1 day-1, cyclophosphamide, 30 mg kg-1 day-1 or APS, 0.75 ml. Platelet numbers were reduced by amrinone, cyclophosphamide and APS. The rate of platelet production was increased following APS and suppressed by cyclophosphamide, but the rate of platelet production when expressed as the selenomethionine incorporation in counts per minute (cpm) per 10(8) platelets appeared to be increased in amrinone-treated animals. When these values are expressed as radioactivity per unit platelet volume the difference between the control and the amrinone-treated group was reduced but the difference between the control, APS- and cyclophosphamide-treated groups remained unchanged. It is concluded that in the Gunn rat amrinone affects platelet production by producing fewer, larger platelets.

The cardiotonic agent amrinone does not increase anatomic infarct size.

The effect of the positive inotropic agent amrinone on anatomic infarct size induced by coronary artery occlusion and reperfusion in dogs is unknown, although previous studies have shown that amrinone increases indices of myocardial ischemia during very brief coronary artery occlusions. Thus, this study was performed to determine if amrinone changes anatomic infarct size and improves hemodynamics induced by 3 h of coronary occlusion and 3 h of reperfusion in anesthetized, open-chest dogs. Amrinone (1-mg/kg bolus followed by 6 mg/kg/h) or an equivalent volume of saline was administered intravenously for 3 h beginning 30 min postocclusion. The area at risk was 19.7 +/- 2.6% in the control group (n = 9) and 20.2 +/- 1.8% in the amrinone-treated group (n = 9; p = NS). The amount of the area at risk that developed infarction was 60.6 +/- 6.1% in the control group and 54.6 +/- 5.6% in the amrinone-treated group (p = NS). Pretreatment left ventricular end-diastolic pressure increased from 11.4 +/- 1.8 to 20.1 +/- 3.0 mm Hg (p less than 0.05) in the control group and from 10.8 +/- 1.3 to 17.3 +/- 1.3 mm Hg (p less than 0.05) in the amrinone-treated group following coronary artery occlusion. During coronary occlusion, amrinone administration significantly increased left ventricular maximum +dP/dt [+483 +/- 85 vs. -11 +/- 53 mm Hg/s (p less than 0.01) in amrinone vs. control group, respectively] and heart rate (+27 +/- 6 vs. -4 +/- 2 beats/min; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)