Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences

Abstract Native human GRF(1–44)-NH 2 (hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala 2 and Asp 3 . The hGRF44 is also subject to chemical rearrangement (Asn 8 → Asp 8 , β-Asp 8 via aminosuccinimide formation) and oxidation [Met 27 → Met(O) 27 ] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop long-acting GRF analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Ala 15 substitution (for Gly 15 ), previously demonstrated to enhance receptor binding affinity, would be predicted to improve GRF analogue potency. Substitution of [His 1 ,Val 2 ]-(from the mouse GRF sequence) for [Tyr 1 ,Ala 2 ]-(human sequence) in [Ala 15 ,Leu 27 ]hGRF(1–32)-OH analogues completely inhibited (24-h incubation) DPP-IV cleavage and greatly increased plasma stability in vitro. Additional substitution of Thr 8 (mouse GRF sequence), Ser 8 (rat GRF sequence), or Gln 8 (not naturally occurring) for Asn 8 (human GRF sequence) resulted in analogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resistant hGRF(1–32)-OH analogues, containing His 1 , Val 2 , Thr/Ser/Gln 8 , Ala 15 , and Leu 27 replacements, were then bioassayed for growth hormone (GH)-releasing activity in vitro (rat pituitary cell culture) and in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1–32)-OH analogues. In vitro, these analogues were approximately threefold more potent than hGRF44, whereas in vivo they were eleven- to thirteenfold more potent. As the in vitro results reflect only receptor affinity and signal transduction, the increment in potency observed in vivo is likely due to the increased biological half-life of these analogues (i.e., the result of decreased enzymatic and chemical decomposition such that more bioactive peptide is available per unit time).