Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs.

A M Felix, Y A Lu, R M Campbell
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Abstract

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.

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聚乙二醇缩氨酸。IV.位点定向聚乙二醇化GRF类似物的生物活性增强。
生长激素释放因子[Ala15]-hGRF(1-29)-NH2的强效类似物的位点特异性聚乙二醇化(nh2端,侧链和羧基端)的条件已经开发出来。采用Fmoc/tBu固相合成方法制备了聚乙二醇化肽,并通过HPLC、氨基酸分析、1H-NMR和激光脱附质谱等手段对其进行了表征。以体外培养的大鼠垂体细胞刺激生长激素释放为指标,测定hGRF类似物的体外生物活性。将聚乙二醇化的hGRF类似物的gh释放能力与一系列[Ala15]-hGRF(1-29)-NH2的模型类似物进行了比较,这些模型类似物在聚乙二醇化位点被乙酰化或作为乙胺保护。研究发现,nh2末端的乙酰化会导致效力降低,而当nh2末端被聚乙二醇化时,无论使用的聚乙二醇(PEG)大小(例如PEG2000或PEG5000),效力都不会受到进一步影响。在Asp8或Lys12位点的聚乙二醇化会降低生物效力,这种情况会随着PEG分子量的增加而加剧。Lys21或Asp25的聚乙二醇化对生物活性没有显著影响。c端模型肽[Ala15,Orn(Ac)30]-hGRF(1-29)-NH2是该系列中发现的最有效的类似物(约为hGRF(1-44)-NH2的4-5倍)。cooh末端聚乙二醇化类似物保持了这种独立于PEG分子量的生物活性水平。这些研究表明,生物活性肽可以聚乙二醇化,并保留肽的全部体外效力。然而,生物活性高度依赖于聚乙二醇化的位点,在某些情况下,使用的PEG(聚乙二醇化程度)片段的分子量。
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