Poduslo J F, Curran G L, Gill J S
Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
J Neurochem. 1998 Oct;71(4):1651-60. doi: 10.1046/j.1471-4159.1998.71041651.x.
Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines--putrescine (PUT), spermidine, and spermine--dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine-modified derivatives at different pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT-NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 min and 73-82% intact protein at 15 min. The PS value for PUT-NGF was maximized and the residual plasma volume (Vp) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT-NGF at 15 min showed 22-33% intact protein in different brain regions, which represented 0.4-5.9 ng of PUT-NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine-modified NGF derivative that has enhanced permeability at the blood-brain and blood-nerve barriers with retained bioactivity may obviate the necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases.
我们实验室先前的研究表明，多种蛋白质（包括抗氧化酶）与天然存在的多胺——腐胺（PUT）、亚精胺和精胺——进行共价修饰后，经肠胃外给药，其在血脑屏障和血神经屏障处的通透系数-表面积乘积（PS）会显著增加。在本研究中，我们通过控制羧基的电离程度，利用pH值对其进行分级修饰，从而将PUT与神经生长因子（NGF）进行共价修饰。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白质免疫印迹和等电聚焦分析表明，在不同pH值下，NGF已转化为其多胺修饰衍生物。虽然通过酶联免疫吸附测定（ELISA）和蛋白质免疫印迹分析测定的PUT-NGF免疫反应性随pH值降低而下降，但通过背根神经节和PC12细胞培养的存活及神经突延伸测定，PUT-NGF的生物活性在任何pH值下均不受影响。单次静脉推注给药后的血浆药代动力学显示，10分钟内PUT-NGF保持完整，15分钟时完整蛋白含量为73 - 82%。当修饰反应的pH值>6.4时，PUT-NGF的PS值最大，血管中该蛋白的残余血浆体积（Vp）最小。15分钟时PUT-NGF的生物分布显示，不同脑区中有22 - 33%的完整蛋白，这相当于不同脑区中有0.4 - 5.9 ng的PUT-NGF，这是能够引发生物反应的生理剂量。这种多胺修饰的NGF衍生物在血脑屏障和血神经屏障处具有增强的通透性且保留生物活性，其设计可能无需制备NGF的小分子模拟物，并且可能适用于神经营养因子、工程化多功能嵌合神经营养因子、抗氧化酶以及其他对神经系统疾病具有特定临床应用价值的治疗性蛋白质。
