HUMAN GROWTH HORMONE-RELEASING HORMONE ANALOGS WITH MUCH IMPROVED INVITRO GROWTH HORMONE-RELEASING POTENCIES IN RAT PITUITARY-CELLS

Enhancement of the amphiphilic alpha-helical properties of the central and C-terminal regions of growth hormone-releasing hormone (GRH) by substitution with helix-favouring amino acids, particularly Ala, can result in significant improvements in GH-releasing potencies using monolayer cultures of rat pituitary cells, a system which reflects analogue receptor affinity rather than effects of structural modifications on pharmacokinetic properties. For instance, previously reported, helix-enhanced [Ala15]GRH-(1-29)NH2 was presently 5 times more potent than [Gly15]GRH-(1-29)NH2 in this assay. The extent and importance of alpha-helical character further towards the N-terminus is less clear since Chou-Fasman probability calculations indicate also the possibility of beta-bend formation in the 6-10 region. However, replacement of Asn8 with Ala resulted in a 4-fold improvement in potency and when this was combined with Ala15 to give [Ala8,15]GRH-(1-29)NH2 a 15-fold increase in potency was achieved and combination of D-Ala2, Ala8 and Ala15 gave a 27-fold increase indicating that the effects of all of these modifications were additive. Computer analysis furthermore revealed that substitution of Ala for Ser in position 9 should also increase alpha-helix probability from 0.93 to 1.05. [D-Ala2,Ala8,9,15]GRH-(1-29)NH2 was 49 times more potent than GRH itself making it by far the most potent analogue thus far reported in an in vitro assay system. The Ala8 and Ala9 substitutions were also effective in improving the inhibitory potency of a GRH receptor antagonist, [D-Arg2,Leu27]GRH-(1-29)NH2. [D-Arg2,Ala8,15]GRH-(1-29)NH2 and [D-Arg2,Ala8,9,15]GRH-(1-29)NH2 displayed IC50 values of 5.9 x 10(-8) and 1.7 x 10(-8) M, respectively, against GRH-stimulated GH release compared to an IC50 of 2.2 x 10(-7) M for the unmodified control analogue and are thus commensurate with corresponding agonist analogue potency improvements.