Mi Z, Burke T G
Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus 43210-1291.
Biochemistry. 1994 Aug 30;33(34):10325-36. doi: 10.1021/bi00200a013.
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., & 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 (t1/2 = 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 (t1/2 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.
喜树碱内酯和羧酸盐形式的固有荧光发射已被用于阐明它们与人体血液中各种成分明显不同的相互作用。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，其亲和力比内酯形式高150倍；与不存在蛋白质时相比，这些相互作用导致喜树碱在HSA存在下更快、更完全地开环[Burke, T.G., & Mi, Z. (1993) Anal. Biochem. 212, 285 - 287]。在pH 7.4和37℃的人血浆中，我们观察到喜树碱内酯迅速且完全地开环形成羧酸盐形式（t1/2 = 11分钟；平衡时内酯的百分比为0.2%）。在喜树碱荧光团中引入10 - 羟基部分使该药物的发射光谱对微环境极性高度敏感；我们观察到，无论是与HSA结合还是在低介电强度的有机溶剂中溶解时，羟基取代的羧酸盐的发射光谱都有明显的蓝移（从530纳米移至430纳米）。因此，似乎喜树碱羧酸盐的荧光团位于天然HSA的疏水结合口袋中。离子相互作用似乎也强烈影响喜树碱羧酸盐与HSA结合口袋之间的结合，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变性会消除高亲和力结合，这表明羧酸盐药物形式的相互作用对天然HSA构象具有特异性。有趣的是，在存在其他血液蛋白质（如γ - 球蛋白、α1 - 酸性糖蛋白、纤维蛋白原以及血红蛋白的氧合和脱氧形式）的情况下，羧酸盐似乎不会发生高亲和力结合。与血浆相比，在全血中喜树碱表现出更高的稳定性（t1/2值为22分钟，平衡时内酯浓度为5.3%）。发现喜树碱在人血液中的稳定性增强是由于药物与红细胞的脂质双层结合。喜树碱内酯分配到红细胞的脂质双层中，药物位于受保护不被水解的疏水环境中。
