Previously we reported that site-specific modification of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) A3C analog with polyethylene glycol (PEG) dramatically improved the pharmacokinetic properties of the protein in rats. However, we could not evaluate the hematological properties of the PEG-A3C protein in rats because human GM-CSF is inactive in rodents. To study the biological effects of PEGylated GM-CSF analogs in rodents we created a homologous site-specific PEGylated murine (mu) GM-CSF (T3C) protein. muGM-CSF and the T3C protein were expressed in Escherichia coli and purified by column chromatography. The purified T3C protein was covalently modified with a linear 20 kDa- or a branched 40 kDa-maleimide-PEG, and the monoPEGylated proteins purified by column chromatography. muGM-CSF, T3C and the two PEG-T3C proteins had comparable in vitro biological activities, as measured by stimulation of proliferation of the murine FDC-P1 cell line. The PEG-T3C proteins had 10- to 25-fold longer circulating half-lives than muGM-CSF and stimulated greater and longer lasting increases in neutrophils and white blood cells than muGM-CSF following a single intravenous or subcutaneous administration to rats. Treatment of rats made neutropenic with cyclophosphamide with the PEG-T3C proteins shortened the time for recovery of neutrophils to normal levels from 9 or 10 days to 5 or 6 days, whereas muGM-CSF showed no benefit versus vehicle solution. Acceleration of neutrophil recovery in cyclophosphamide-treated rats required a minimum of three PEG-T3C treatments over five days. The PEG-T3C proteins should prove useful for evaluating the potential therapeutic benefits of GM-CSF and long-acting GM-CSF proteins in rodent disease models.

中文翻译：

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在正常和环磷酰胺治疗的中性粒细胞减少性大鼠中，长效特定部位的聚乙二醇化小鼠GM-CSF类似物的表征及其造血特性分析。

以前我们报道过用聚乙二醇（PEG）对人粒细胞-巨噬细胞集落刺激因子（GM-CSF）A3C类似物进行位点特异性修饰可大大改善该蛋白在大鼠中的药代动力学特性。但是，我们无法评估PEG-A3C蛋白在大鼠中的血液学特性，因为人GM-CSF在啮齿动物中没有活性。为了研究聚乙二醇化GM-CSF类似物在啮齿动物中的生物学效应，我们创建了一个同源位点特异性聚乙二醇化鼠类（μ）GM-CSF（T3C）蛋白。muGM-CSF和T3C蛋白在大肠杆菌中表达并通过柱色谱法纯化。纯化的T3C蛋白用线性20kDa-或支链40kDa-马来酰亚胺-PEG共价修饰，并且单PEG化的蛋白通过柱色谱法纯化。如通过刺激鼠FDC-P1细胞系的增殖所测量的，muGM-CSF，T3C和两种PEG-T3C蛋白具有相当的体外生物活性。在对大鼠单次静脉内或皮下给药后，PEG-T3C蛋白的循环半衰期比muGM-CSF长10至25倍，并且刺激中性粒细胞和白细胞的增长与muGM-CSF相比更大，更持久。用PEG-T3C蛋白用环磷酰胺治疗中性白细胞减少症的大鼠将中性粒细胞恢复至正常水平的时间从9天或10天缩短到5天或6天，而muGM-CSF与车辆解决方案相比没有优势。加速环磷酰胺治疗的大鼠中性粒细胞的恢复需要在五天内至少进行三种PEG-T3C治疗。PEG-T3C蛋白应被证明可用于评估GM-CSF和长效GM-CSF蛋白在啮齿动物疾病模型中的潜在治疗效果。