Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran.
Int J Nanomedicine. 2011;6:1217-27. doi: 10.2147/IJN.S19081. Epub 2011 Jun 15.
Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.
A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr(-) cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.
The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.
This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.
由于重组人红细胞生成素（rhEPO）在全球范围内用于治疗与贫血相关的疾病，因此被认为是市场上最重要的药物之一。然而，与其他治疗性蛋白质一样，它的药代动力学特性并不适合每周至少给药两次或三次。利用计算机模拟中的分子动力学和图形学进行化学选择性半胱氨酸聚乙二醇化，可以解决这个问题。
根据文献综述、同源建模、分子动力学模拟以及影响聚乙二醇化反应的因素，设计出一种特殊的 EPO 类似物。然后，通过定点突变生成所选类似物的 cDNA，随后将其克隆到表达载体中。将构建体转染到中国仓鼠卵巢/dhfr（-）细胞中，通过甲氨蝶呤扩增选择高表达的克隆。使用离子固定亲和和尺寸排阻（SE）色谱技术来纯化表达的类似物。然后进行化学选择性聚乙二醇化，并通过透析获得纳米聚乙二醇化 EPO。研究了体外生物学测定和体内药代动力学参数。最后，对 E31C 类似物傅里叶变换红外光谱、分析 SE-高效液相色谱、ζ电位以及聚乙二醇化前后的大小进行了表征。
研究结果表明，与未经修饰的 rhEPO 相比，新型纳米 EPO31-PEG 在增殖细胞测定中具有五倍更长的末端半衰期，且具有相似的生物活性。结果还表明，EPO31-PEG 的大小和电荷相对于未经修饰的蛋白质在纳米谱中增加，这可能是增强 EPO 生物学效力的一个标准。
这种新型工程纳米聚乙二醇化 EPO 具有优于 rhEPO 的显著优势。
