PEGYLATED PEPTIDES .4. ENHANCED BIOLOGICAL-ACTIVITY OF SITE-DIRECTED PEGYLATED GRF ANALOGS

Conditions have been developed for the site-specific pegylation (NH2-t erminus, side-chain and carboxy terminus) of a potent analog of growth hormone-releasing factor, [Ala(15)]-hGRF(1-29)-NH2. These pegylated p eptides were prepared by solid-phase peptide synthesis using the Fmoc/ tBu strategy, and were fully characterized by analytical HPLC, amino-a cid analysis, H-1-NMR spectroscopy and laser desorption mass spectrome try. Biological activities of hGRF analogs were determined in vitro ut ilizing stimulation of growth hormone release by cultured rat pituitar y cells as an index. GH-releasing potencies of the pegylated hGRF anal ogs were compared to a series of model analogs of [Ala(15)]-hGRF(1-29) -NH2 that were acetylated or protected as the ethylamides at the pegyl ation sites. It was found that acetylation at the NH2-terminus resulte d in reduced potency, which was not further affected when the NH2-term inus was pegylated, regardless of the size of poly(ethyleneglycol) (PE G) employed (e.g. PEG(2000) or PEG(5000)) Pegylation at Asp(8) or Lys( 12) decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys(21) or Asp( 25) did not significantly affect biological activity. The C-terminal m odel peptide, [Ala(15),Orn(Ac)(30)]-hGRF(1-29)-NH2, was the most poten t analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH 2. The COOH-terminal pegylated analogs retained this increased level o f biological activity independent of PEG molecular weight. These studi es demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biologi cal activity is highly dependent on the site of pegylation and, in som e cases, the molecular weight of PEG (degree of pegylation) moiety use d. (C) Munksgaard 1995.