Growth hormone-releasing factor analogs with hydrophobic residues at position 19. Effects on growth hormone releasing activity in vitro and in vivo, stability in blood plasma in vitro, and secondary structure

To test the hypothesis that replacing Ala19 in growth hormone-releasing factor (GRF) with more hydrophobic residues will increase growth hormone releasing activity, four GRF analogs were prepared and tested. The molecules were made by substituting Val, Ile, or Leu at position 19 of [Thr2,Ala15,Leu27]bGRF(1-29)NH2. The compounds were evaluated for growth hormone (GH) releasing activity in vitro (rat anterior pituitary cells) and in vivo (steers). Additionally, their half-life in vitro was determined in bovine plasma, and their secondary structure was examined by circular dichroism. In pituitary cells, peptides with substitutions at position 19 had the following potencies: Ala (native), 0.37; Val, 1.16; Ile, 0.37; Leu, 0.043. When assayed in steers as a single iv bolus, over a 2-h period, the compounds gave the following integrated GH response: Ala, 2.75; Val, 2.67; Ile, 2.57; Leu, 1.55. Only the Leu analog was statistically different from the other three (p = 0.05). In bovine plasma, the half-lives (hours) were as follows: Ala, 4.9; Val, 6.6; Ile, 12.3; Leu, 14.7. In phosphate buffer the compounds were calculated to have the following percent helical content: Ala, 26; Val, 21; Ile, 27; Leu, 32. For these analogs, helicity in aqueous buffer is inversely related to their in vitro activity. Using a linear multiple regression model, the plasma half-life of the analogs positively correlated (r2 = 0.999) with both the hydrophobicity of the residue at position 19 and the helicity of the analog. Although the Val analog had both increased inherent activity and increased plasma stability in vitro compared to the Ala analog, in this study we were unable to demonstrate an increase in activity in vivo. The in vivo GH releasing activity of the analogs was not simply related to a combination of their intrinsic GH releasing activity and their in vitro plasma half-life. This suggests that in vivo additional factors are moderating the expression of activity.