title: Methods for assessing endothelin ETA-receptor antagonists in preclinical studies.
abstract: Endothelin-1 (ET-1) has been found to be one of the most potent vasoconstrictive peptides known, and is therefore considered to be an important factor in diseases such as hypertension, heart failure, pulmonary hypertension, renal diseases, etc. Thus, the development of ET-receptor antagonists may offer a new therapeutic strategy in these fields. In this article, we summarize the method for assessing our compound as a selective ETA-receptor antagonist. Binding assays and in vitro function assays (isolated vessels) were examined for the assessment of in vitro potency, selectivity of the ETA receptor against the ETB receptor, specificity for ET receptors, agonistic activities for ET receptors and the blocking manner of the compound on ET receptors. Chinese hamster ovary (CHO) cells expressing human ET receptors and tissue membrane preparations from both human and animals were used for the binding assays. The specificity of the compound against ET receptors was demonstrated using 116 and 9 receptor binding and enzyme assays, respectively. The agonistic activity and potency of the compound at tissue levels were examined using isolated vessels. We also demonstrated the effect of protein binding on the potency of the compound by adding a physiological concentration of serum albumin to the tissue baths. In vivo potency and features of the compound as a selective ETA-antagonist were confirmed using mice, rats and dogs exogenously treated with ET-1 or big ET-1. We also demonstrated the compound's duration of action and pharmacokinetics in animal models and intact animals, respectively. From these experiments, we found a nonpeptide, potent, orally active and long-lasting, highly selective ETA-receptor antagonist.
authors.affiliation: Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Ibaraki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Okada
authors.forename: P K S
authors.initials: PK
authors.lastname: Siegl
authors.forename: M
authors.initials: M
authors.lastname: Nishikibe
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001808:Blood Vessels / Q000187:drug effects / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D004285:Dogs; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D004987:Ethers / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000493:pharmacokinetics / Q000494:pharmacology*; D006973:Hypertension / Q000503:physiopathology; D066298:In Vitro Techniques; D051381:Rats; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B
chemical_list: D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D004987:Ethers; D006845:Hydrocarbons, Fluorinated; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; C078742:fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether