Increased activity of antagonists of growth

Antagonists of human growth hormone-releasing hormone (hGHRH) with increased potency and improved enzymatic and chemical stability are needed for potential clinical applications. We synthesized 21 antagonistic analogs of hGHRH(1–29)NH2, substituted at positions 8, 9, and 10 of the common core sequence {phenylacetyl-Tyr1, D-Arg2,28, para-chloro-phenylalanine 6, Arg9 homoarginine 9, Tyr10 O-methyltyrosine 10, -aminobutyric acid 15, norleucine 27, Har29} hGHRH(1– 29)NH2. Inhibitory effects on hGHRH-induced GH release were evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinities of the peptides to pituitary GHRH receptors were also determined. Introduction of para-amidinophenylalanine 10 yielded antagonists JV-1–62 and -63 with the highest activities in vitro and lowest receptor dissociation constants (Ki 0.057–0.062 nM). Antagonists JV-1–62 and -63 also exhibited the strongest effect in vivo, significantly (P < 0.05–0.001) inhibiting hGHRH-induced GH release for at least 1 h. Para-aminophenylalanine 10 and O-ethyltyrosine 10 substitutions yielded antagonists potent in vitro, but His10, 3,3 -diphenylalanine 10, 2-naphthylalanine 10, and cyclohexylalanine 10 modifications were detrimental. Antagonists containing citrulline 9 (in MZ-J-7–72), amidinophenylalanine 9 (in JV-1–65), His9, D-Arg9, citrulline 8, Ala8, D-Ala8, or aminobutyric acid 8 substituents also had high activity and receptor affinity in vitro. However, in vitro potencies of analogs with substitution in position 9 correlated poorly with acute endocrine effects in vivo, as exemplified by the weak and or short inhibitory actions of antagonists JV-1–65 and MZ-J-7–72 on GH release in vivo. Nevertheless, antagonist JV-1–65 was more potent than JV-1–63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice. This indicates that oncological activity may be based on several mechanisms. hGHRH antagonists with improved efficacy could be useful for treatment of cancers that depend on insulin-like growth factors or GHRH.