In vitro assessment of oral delivery for hexapeptide endothelin antagonists

Abstract Endothelin (ET-1) is a 21-amino acid, vasoconstrictive peptide originally isolated from endothelial cells. It is one member of a class of potent, purportedly paracrine substances that act at receptors in multiple target organs. Antagonists to the receptor subtypes, ET A and ET B , have been designed around the hydrophobic carboxy-terminus of ET-1. The resulting hexapeptides possess low nanomolar receptor affinity, but face formidable challenges to oral delivery, given their peptidic nature. Hence, it was important to discriminate between analogs, as well as to optimize structural features combining binding potency with stability in intestinal fluids and permeability across biological membranes. PD 142893 (Ac-DDip 16 -Leu-Asp-Ile-Ile-Trp 21 ) and PD 145065 (Ac-DBhg 16 -LeuAsp-Ile-Ile-Trp 21 ), as well as the N-methyl-isoleucine 20 analogs were studied, where DDip = 3,3-diphenylalanine and DBhg = 10,11-dihydro-5H-dibenzo[a,d]cycloheptene glycine. Analyses were conducted with specific HPLC methods. Permeabilities across CACO-2 cell monolayers ranged from 2.0 × 10 −4 to 6.3 × 10 −4 cm/mm. The results suggested that these compounds can be absorbed in vivo , based on comparison of permeabilities with those obtained with reference compounds. Much greater differences were observed between the analogs when stability half-lives were compared after incubation in rat intestinal perfusate. The parent peptides, PD 142893 and PD 145065, were unstable, with half-lives less than 20 min. N-Methylation of He 20 resulted in large increases in stability half-lives to greater than 500 min. Enzyme inhibition studies demonstrated the involvement of carboxypeptidase A in production of the primary metabolite, the des-Trp derivative. Identification of the primary metabolite of the parent peptide was made by differential UV scanning at 214/280 nm and mass spectral analyses.