DIFFERENTIAL INTERACTIONS OF CAMPTOTHECIN LACTONE AND CARBOXYLATE FORMS WITH HUMAN BLOOD COMPONENTS
DIFFERENTIAL INTERACTIONS OF CAMPTOTHECIN LACTONE AND CARBOXYLATE FORMS WITH HUMAN BLOOD COMPONENTS
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DOI：
10.1021/bi00200a013
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发表时间：
1994-08-30
期刊：
影响因子：
2.9
通讯作者：
BURKE, TG
中科院分区：
文献类型：
--
作者：
MI, ZH;BURKE, TG
人们利用喜树碱的内酯和羧酸盐形式的固有荧光发射来阐明它们与人类血液的各种成分之间显着不同的相互作用。在 pH 7.4 的磷酸盐缓冲盐水 (PBS) 中，人血清白蛋白 (HSA) 优先结合羧酸盐形式，亲和力比内酯形式高 150 倍；这些相互作用导致喜树碱在 HSA 存在的情况下比在蛋白质不存在的情况下更快、更完全地打开 [Burke, T. G., and Mi, Z. (1993) Anal.生物化学。 212、285-287]。在人血浆中，在 pH 7.4 和 37 摄氏度下，我们观察到喜树碱内酯快速且完全打开为羧酸盐形式（t(1/2) = 11 分钟；平衡状态下的内酯百分比为 0.2%）。将 10-羟基部分替换为喜树碱荧光团，使得该试剂的发射光谱对微环境极性高度敏感；我们在 HSA 缔合以及药物在低介电强度的有机溶剂中溶解时，观察到羟基取代的羧酸盐的发射光谱出现明显的蓝移（从 530 到 430 nm）。因此，看来喜树碱羧酸盐的荧光团位于天然 HSA 的疏水结合袋中。离子相互作用似乎也强烈影响喜树碱羧酸盐和 HSA 结合袋之间的结合，因为溶液盐浓度增加 6 倍会使喜树碱羧酸盐的结合减少 10 倍。我们发现 HSA 变性消除了高亲和力结合，这表明羧酸盐药物形式的相互作用对于天然 HSA 构象具有特异性。有趣的是，在存在其他血液蛋白（例如丙种球蛋白、α(1)-酸性糖蛋白、纤维蛋白原以及血红蛋白的氧和脱氧形式）的情况下，似乎不会发生羧酸盐的高亲和力结合。与血浆相比，在全血中，喜树碱表现出增强的稳定性（t(1/2) 值为 22 分钟，内酯浓度为平衡值 5.3%）。人们发现喜树碱在人体血液中稳定性的增强是由于药物与红细胞脂质双层的结合。喜树碱内酯分配到红细胞的脂质双层中，药物位于防止水解的疏水环境中。
The intrinsic fluorescent emissions from the lactone and carboxylate forms of camptothecin have been exploited in order to elucidate their markedly different interactions with the various components of human blood. In phosphate-buffered saline (PBS) at pH 7.4, human serum albumin (HSA) preferentially binds the carboxylate form with a 150-fold higher affinity than the lactone form; these interactions result in camptothecin opening more rapidly and completely in the presence of HSA than in the protein's absence [Burke, T. G., and Mi, Z. (1993) Anal. Biochem. 212, 285-287]. In human plasma, at pH 7.4 and 37 degrees C, we have observed camptothecin lactone to open rapidly and fully to the carboxylate form (t(1/2) = 11 min; % lactone at equilibrium, 0.2%). Substitution of a 10-hydroxy moiety into the camptothecin fluorophore makes the agent's emission spectrum highly sensitive to microenvironment polarity; we have observed pronounced blue shifting (from 530 to 430 nm) in the emission spectra of the hydroxy-substituted carboxylate both upon HSA association as well as upon drug dissolution in organic solvents of low dielectric strength. Hence, it appears that camptothecin carboxylate's fluorophore locates in a hydrophobic binding pocket in native HSA. Ionic interactions also appear to strongly affect binding between camptothecin carboxylate and the HSA binding pocket, since a 6-fold increase in solution salt concentration diminished camptothecin carboxylate binding by 10-fold. Our findings that HSA denaturation abolishes high-affinity binding indicate that interactions of the carboxylate drug form are specific for the native HSA conformation. Interestingly, high-affinity binding of the carboxylate appeared not to occur in the presence of other blood proteins, such as gamma-globulin, alpha(1)-acid glycoprotein, fibrinogen, and the oxy and deoxy forms of hemoglobin. In whole blood versus plasma, camptothecin was found to display enhanced stability (t(1/2) value of 22 min and a lactone concentration at equilibrium value of 5.3%). The enhanced stability of camptothecin in human blood was found to be due to drug associations with the lipid bilayers of red blood cells. Camptothecin lactone partitions into the lipid bilayers of erythrocytes, with the drug locating in a hydrophobic environment protected from hydrolysis.