Motoyama Keiichi, Nishiyama Rena, Maeda Yuki, Higashi Taishi, Kawaguchi Yoshimasa, Futaki Shiroh, Ishitsuka Yoichi, Kondo Yuki, Irie Tetsumi, Era Takumi, Arima Hidetoshi
Graduate School of Pharmaceutical Sciences, Kumamoto University.
Biol Pharm Bull. 2016 Nov 1;39(11):1823-1829. doi: 10.1248/bpb.b16-00369. Epub 2016 Sep 6.
Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of β-CyD to NPC cells is expected. In this study, to efficiently deliver β-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended β-CyD with a spacer of γ-aminobutyric acid (R8-β-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-β-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-β-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-β-CyD significantly decreased intracellular cholesterol content compared with HP-β-CyD. These results suggest that R8-β-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.
尼曼-匹克C型病（NPC）是一种常染色体隐性溶酶体贮积症，是一种遗传性疾病，其特征是未酯化胆固醇在内溶酶体中蓄积。最近，2-羟丙基-β-环糊精（HP-β-CyD）已被用于治疗NPC，并改善了患者的肝脾肿大。然而，为了获得治疗效果，需要高剂量的HP-β-CyD。因此，人们期望通过将β-环糊精的活性细胞内递送系统用于NPC细胞来降低剂量。在本研究中，为了将β-环糊精有效地递送至NPC样细胞，我们新合成了带有γ-氨基丁酸间隔区的八聚精氨酸（R8）连接的β-环糊精（R8-β-CyD），并在Npc1陷阱-中国仓鼠卵巢（CHO）细胞（通过NPC1功能受损而积累胆固醇的细胞）中评估了其细胞毒性、细胞内分布、内吞途径和降胆固醇作用。R8-β-CyD在细胞中未显示细胞毒性。此外，Alexa568标记的R8-β-CyD可能通过微胞饮作用被主动内化到Npc1陷阱-CHO细胞中。值得注意的是，与HP-β-CyD相比，R8-β-CyD显著降低了细胞内胆固醇含量。这些结果表明，R8-β-CyD可能是改善NPC中胆固醇蓄积的一种有前景的治疗剂。
