ENHANCED STABILITY AND POTENCY OF NOVEL GROWTH HORMONE-RELEASING FACTOR (GRF) ANALOGS DERIVED FROM RODENT AND HUMAN CRF SEQUENCES

Native human GRF(1-44)-NH2 (hGRF44) is subject to biological inactivat ion by both enzymatic and chemical routes. In plasma, hGRF44 is rapidl y degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residu es Ala(2) and Asp(3). The hGRF44 is also subject to chemical rearrange ment (Asn(8) --> Asp(8), beta-Asp(8) via aminosuccinimide formation) a nd oxidation [Met(27) --> Met(O)(27)] in aqueous environments, greatly reducing its bioactivity. It is therefore advantageous to develop lon g-acting GRF analogues using specific amino acid replacements at the a mino-terminus (to prevent enzymatic degradation): residue 8 (to reduce isomerization) and residue 27 (to prevent oxidation). Inclusion of Al a(15) substitution (for Gly(15)), previously demonstrated to enhance r eceptor binding affinity, would be predicted to improve GRF analogue p otency. 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 o f Thr(8) (mouse GRF sequence), Ser(8) (rat GRF sequence), or Gln(8) (n ot naturally occurring) for Asn(8) (human GRF sequence) resulted in an alogues with enhanced aqueous stability in vitro (i.e., decreased rate of isomerization). These three highly stable and enzymatically resist ant 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 h ormone (GH)-releasing activity in vitro (rat pituitary cell culture) a nd in vivo (SC injection into pigs). Enhanced bioactivity was observed with all three hGRF(1-32)-OH analogues. In vitro, these analogues wer e approximately threefold more potent than hGRF44, whereas in vivo the y were eleven- to thirteenfold more potent. As the in vitro results re flect only receptor affinity and signal transduction, the increment in potency observed in vivo is likely due to the increased biological ha lf-life of these analogues (i.e., the result of decreased enzymatic an d chemical decomposition such that more bioactive peptide is available per unit time).