Vol. 281, Issue 2, 791-798, 1997

Endothelin Receptor Antagonists: Effect of Serum Albumin on Potency and Comparison of Pharmacological Characteristics

Jinshyun R. Wu-Wong, Douglas B. Dixon, William J. Chiou and Terry J. Opgenorth

Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois

Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. Two types of ET receptors, ET_A and ET_B, have been identified. Various ET receptor antagonists are being developed as therapeutic agents. This report examines the effects of bovine serum albumin (BSA) on the potency of ET receptor antagonists and compares five ET receptor antagonists. Competition studies show that in the absence of BSA, A-127722 and L-749329 inhibited ET-1 binding to ET_A receptor with the same IC₅₀ value of 0.09 nM. Addition of increasing concentrations of BSA incrementally decreased the potency of the antagonists: in the presence of 5% BSA, the IC₅₀ values increased to 4.3 and 820 nM, respectively. Similarly, addition of BSA decreased the potency of antagonists in inhibiting ET-1-stimulated phosphatidylinositol hydrolysis. These results suggest that serum albumin has profound effects on the potencies of ET receptor antagonists. FR139317, PD-156707, L-749329, Ro-47-0203 and A-127722 were then selected for direct comparison under identical experimental conditions with 0.2% BSA. The potency of antagonists was assessed by binding studies for the determination of IC₅₀ and K_i values and by ET-1-stimulated phosphatidylinositol hydrolysis and arachidonic acid release for the determination of IC₅₀ and pA₂ values. All five antagonists inhibited ET binding and the biological effects exerted by ET in a competitive mode. The K_i values for A-127722, PD-156707, FR139317, Ro-47-0203 and L-749329 for the ET_A receptor were 0.07, 0.38, 0.80, 3.67 and 33.6 nM, respectively. A similar hierarchy was revealed by the functional assays. Our results suggest that the rank order of potency of the antagonists is A-127722  PD-156707  FR139317 > Ro-47-0203 > L-749329.