Kinsky S C, Loader J E, Hashimoto K
Biochim Biophys Acta. 1987 Feb 14;917(2):211-8. doi: 10.1016/0005-2760(87)90124-x.
Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.
先前的研究表明，非靶向脂质体（其中甲氨蝶呤以甲氨蝶呤-γ-二肉豆蔻酰磷脂酰乙醇胺（methotrexate-γ-DMPE）的形式锚定在脂质双层上）可以抑制体外细胞增殖。为了测试这种抑制作用可能涉及甲氨蝶呤-γ-DMPE的细胞外代谢的可能性，我们对其进行了化学降解（稀碱）或酶促降解（磷脂酶A2、磷脂酶C、磷脂酶C加磷酸酶），并使用人淋巴母细胞样T细胞或具有缺陷甲氨蝶呤转运系统的亚系对产物进行了检测。甲氨蝶呤-γ-（1-肉豆蔻酰）-甘油磷酰乙醇胺、甲氨蝶呤-γ-甘油磷酰乙醇胺、甲氨蝶呤-γ-磷酰乙醇胺或甲氨蝶呤-γ-乙醇胺在阻断氚标记的脱氧尿苷掺入DNA的能力方面，均与甲氨蝶呤-γ-DMPE致敏脂质体或游离衍生物不同。当细胞外添加这些假定的代谢产物时，它们表现出更高的半数抑制浓度（ID50），并且与脂质体或未掺入的甲氨蝶呤-γ-DMPE不同，它们利用甲氨蝶呤转运系统进入细胞。此外，我们合成了甲氨蝶呤-γ-二己adecyl磷脂酰乙醇胺和甲氨蝶呤-γ-己adecyl磷酰乙醇胺，它们是甲氨蝶呤-γ-DMPE的类似物，不能被磷脂酶A2、C和D水解；用这些衍生物制备的脂质体作为细胞毒性剂的效力明显低于甲氨蝶呤-γ-DMPE致敏脂质体。总之，这些结果与甲氨蝶呤-γ-DMPE必须经历细胞内代谢才能发挥最佳抑制作用的结论一致；它们还涉及甲氨蝶呤-γ-DMPE可能进入细胞的潜在机制。
