Burke T G, Mi Z
Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus 43210-1291.
J Med Chem. 1994 Jan 7;37(1):40-6. doi: 10.1021/jm00027a005.
The intense intrinsic fluorescence emissions from several clinically relevant camptothecin drugs have been exploited in order to study the structural basis of drug binding to human serum albumin. Both HPLC and time-resolved fluorescence spectroscopic methodologies were employed to characterize the associations of camptothecins with HSA in phosphate-buffered saline (pH 7.4) at 37 degrees C. The alpha-hydroxy delta-lactone ring moiety of camptothecin (C), 10-hydroxycamptothecin (HC), 10,11-(methylenedioxy)camptothecin (MC) and 9-chloro-10,11-(methylenedioxy)camptothecin (CMC) was in each case observed to hydrolyze more rapidly and completely in the presence of HSA than in the protein's absence. Binding isotherms constructed by the method of fluorescence lifetime titration showed that HSA bound preferentially the carboxylate forms of C, HC, MC, and CMC over their lactone forms, thereby providing an explanation for the shift to the right in the lactone-carboxylate equilibrium observed for each compound upon HSA addition. In marked contrast, three analogues (SN-38, CPT-11, and topotecan) all displayed enhanced stabilities in the presence of HSA. While the lifetimes of CPT-11, topotecan, and the carboxylate forms of both drugs were insensitive to the addition of HSA, the lifetimes of both SN-38 and its carboxylate form did titrate upon HSA addition. Analysis of binding isotherms constructed for the albumin interactions of SN-38 and its carboxylate form demonstrated a higher overall association constant for the lactone form [640 (M amino acid (aa) residues)-1] relative to the carboxylate form [150 (M aa)-1]. Our studies indicate that specific modifications at the 7- and 9-positions of the quinoline nucleus, such as those contained in CPT-11, topotecan, and SN-38, enhance drug stability in the presence of HSA. In the case of SN-38, the enhanced stability was shown to be due to preferential associations between the drug's lactone form and the blood protein.
几种临床相关的喜树碱类药物强烈的固有荧光发射已被用于研究药物与人血清白蛋白结合的结构基础。采用高效液相色谱法（HPLC）和时间分辨荧光光谱法来表征喜树碱类药物与在37℃的磷酸盐缓冲盐水（pH 7.4）中的人血清白蛋白（HSA）的结合情况。喜树碱（C）、10-羟基喜树碱（HC）、10,11-（亚甲二氧基）喜树碱（MC）和9-氯-10,11-（亚甲二氧基）喜树碱（CMC）的α-羟基δ-内酯环部分在每种情况下均被观察到，相较于不存在蛋白质时，在HSA存在下更快速且更完全地水解。通过荧光寿命滴定法构建的结合等温线表明，HSA优先结合C、HC、MC和CMC的羧酸盐形式而非其内酯形式，从而为添加HSA后每种化合物内酯-羧酸盐平衡向右移动提供了解释。与之形成显著对比的是，三种类似物（SN-38、CPT-11和拓扑替康）在HSA存在下均表现出增强的稳定性。虽然CPT-11、拓扑替康以及两种药物的羧酸盐形式的寿命对添加HSA不敏感，但添加HSA后SN-38及其羧酸盐形式的寿命均发生了滴定。对SN-38及其羧酸盐形式与白蛋白相互作用构建的结合等温线分析表明，内酯形式[640（M氨基酸（aa）残基）-1]相对于羧酸盐形式[150（M aa）-1]具有更高的总体缔合常数。我们的研究表明，喹啉核7位和9位的特定修饰，如CPT-11、拓扑替康和SN-38中所含的修饰，可增强在HSA存在下药物的稳定性。就SN-38而言，稳定性增强被证明是由于药物内酯形式与血液蛋白之间的优先缔合。
