RADIORECEPTOR ASSAY OF AN ENDOTHELIN-A RECEPTOR ANTAGONIST IN PLASMA AND URINE

Orally active nonpeptide antagonists of endothelin (ET) receptors may prove beneficial in the treatment of cardiovascular and renal disease. The pharmacodynamics and pharmacokinetics of these drugs are not suff iciently known, and practical methods for their analysis have not been developed. We describe a simple, sensitive, and reproducible radiorec eptor assay (RRA) for LU135252, a selective antagonist of the ET, rece ptor, using porcine aortic smooth muscle membranes as the acceptor and I-125-endothelin-1 as the ligand. With methanol extraction of plasma and urine samples, recovery of LU135252 ranged from 79% to 91% at 60-1 000 nmol/L. The legit-log transformed calibration curves constructed w ith LU135252 added to plasma or to urine were linear (r = 0.993 +/- 0. 005, n = 11) in the range from 18.7 to 2400 nmol/L. The detection limi t with plasma- and urine-based calibration curves was 19 nmol/L. The i nterassay coefficient of variation was 12.6% at 70 nmol/L (n = 9) and 6.5% at 590 nmol/L (n = 9). Endothelin-1 did not interfere in the RRA at pathophysiologically and clinically relevant concentrations [up to 15 pmol/L (40 pg/mL)]. When LU135252 was added to plasma, the signal w as completely stable after storage for 1 week at 4 degrees C, although there was a modest loss of the signal after 24 h at room temperature. The practical performance of this RRA was then tested in plasma sampl es obtained from (n) rats after a single oral administration of LU1352 52, (b) from coronary-ligated rats chronically treated with LU135252, and (c) in plasma and urine samples obtained from dogs during intraren al infusion of LU135252.