Brain-derived neurotrophic factor (BDNF) bound with lecithin derivative in order to prolong plasma-half and permeate through blood brain barrier (BBB)

我们合成了卵磷化脑源性神经营养因子，其中卵磷脂衍生物的几个分子与重组人脑源性神经营养因子结合。我们在C57BL/KSJ-db/db小鼠身上评价了它的药理活性，并评价了它对神经系统的靶向性和亲和力。皮下注射卵磷脂BDNF可显著降低C57BL/KSJ-db/db糖尿病小鼠的血糖水平、摄食量和体重。其效价是未经修饰的BDNF的20多倍。并对其药理活性显著增强的机制进行了研究。用四甲基偶氮唑盐比色法测定卵磷脂化的BDNF的体外细胞生长活性低于未修饰的BDNF，空间位阻卵磷脂部分。此外，皮下注射磷脂化BDNF后的血浆BDNF水平并不高于未修饰的BDNF。磷脂化BDNF在大脑、小脑和脊髓中的蓄积量高于未修饰的BDNF。我们最终发现，磷脂化的BDNF与PC-pAB1神经细胞的体外结合力明显高于未修饰的BDNF。此外，PC-pAB1细胞结合的磷脂化BDNF不随未修饰的BDNF或卵磷脂BDNF的过量而改变。卵磷脂化BDNF处理的PC-pAB1细胞表现出持续的MAPK(ERK1/2)激活。这些数据表明，卵磷脂化的BDNF与靶细胞的高亲和力以及随后长时间的MAPK激活将在其更有效的药理活性中发挥重要作用。

We synthesized lecithinized brain-derived neurotrophic factor (lecithinized-BDNF), in which several molecules of a lecithin derivative were bound to recombinant human BDNF. We evaluated its pharmacological activity in C57BL/KsJ-db/db mice, and assessed its targetability and affinity for the nervous system. When administered subcutaneously, lecithinized-BDNF markedly reduced the plasma glucose level, food intake, and body weight in C57BL/KsJ-db/db diabetic mice. Its potency was over 20 times greater than that of unmodified BDNF. We studied the mechanism of marked enhancement of pharmacological activity. In vitro cell growth activity of lecithinized-BDNF using MTT assay was lower than unmodified BDNF, steric hindrance of lecithine moieties. Moreover, the plasma BDNF level after subcutaneous administration of lecithinized-BDNF was not higher, compared with unmodified BDNF. The accumulated lecithinized-BDNF in the cerebrum, cerebellum, and spinal cord were higher than that of unmodified BDNF. We found finally that in vitro binding of lecithinized-BDNF for PC-pAB1 neural cells was much higher than that of unmodified BDNF. Moreover, lecithinized-BDNF bound to PC-pAB1 cells didn't change with even excess unmodified BDNF or even excess lecithinized-BDNF. PC-pAB1 cells treated with lecithinized-BDNF showed a sustained MAP kinase (ERK1/2) activation. These data would indicate that the high affinity of lecithinized-BDNF for the target cells followed by prolonged MAPK activation would play an important role on its more potent pharmacological activity.