Antide is the decapeptide N-Ac-D-Nal(2),DPhe(pCl),D-Pal(3),Ser,Lys(Nic),D-Lys(Nic),LeuLys(iPr),Pro, D-Ala-NH2 [Nal(2) represents 3-(2-naphthyl)alanine; Phe(p-Cl) represents 3-(4-chlorophenyl)alanine; Pal(3) represents 3-(3-pyridyl)alanine; Lys(Nic) represents Ne-nicotinoyllysine; Lys-(iPr) represents N6-isopropyllysine], which is an antagonist of luteinizing hormone-releasing hormone (LHRH), which has high antiovulatory activity, releases negligible histamine, and is scheduled for scale-up, safety testing, and evaluation in the experimental primate and in clinical medicine. Thirty-five more peptides were synthesized, designed on antide with variations in positions 5-8. Antide showed oral antiovulatory activity at 600 pg (73%) and at 1200 pg (100%) with negligible difference between water and corn oil oral formulations.We have synthesized and bioassayed antagonists of luteinizing hormone-releasing hormone (LHRH) with high potency and diminished activity to release histamine (1). Our designs featured N6-nicotinoyl-D-lysine [D-Lys(Nic)] in position 6 and NE-isopropyllysine [Lys(iPr)] in position 8. We avoided the basic D-Arg6, which, in combination with Arg8 and a cluster of hydrophobic aromatic amino acid residues at the N terminus, has been implicated in the release of histamine (2-4). Herein are results from analogs with acylated aminocyclohexylalanine residues in position 6, from analogs in which Leu' has been substituted with other neutral residues, from a comparison of Lys(iPr)8 vs. N8-isopropylornithine at position 8 [Orn(iPr)8], and from tests on oral activity and duration of activity, oral and s.c.
MATERIALS AND METHODSSynthesis. The peptides were synthesized by the solidphase method (1, 11).Purification. The peptides were purified by silica gel chromatography with the solvent system 1-butanol/acetic acid/water, 4:1:2 (vol/vol), or 4:1:5 (vol/vol), upper phase followed by gel filtration on Sephadex G-25. Alternatively, purification was achieved by gel filtration on Sephadex G-25 followed by chromatography on Sephadex LH-20 with the solvent system water/1-butanol/acetic acid/methanol, 90: 10:10:8 (vol/vol).The purity was checked by HPLC, TLC, and amino acid analysis. HPLC was performed on a Waters Associates instrument equipped with a model 660 solvent programer and either a uBondapak C18 or a Vydac C18 analytical column. Single peaks were obtained at flow rates of 2 or 1.5 ml/min using linear gradients of increasing acetonitrile in 0.01 M KH2PO4 (pH 3).
