Abstract：
The confidence in fraction unbound (ƒu) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒu measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒu measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒu values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒu for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒu values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.
摘要：
使用平衡透析(ED)的未绑定分数(fu)的置信度经常受到质疑(例如，高度绑定，不稳定化合物)，由于是否实现真正平衡的不确定性。已经开发了不同的方法来增加fu测量的置信度，比如预饱和，稀释，和双向ED方法。然而,由于在平衡和分析过程中引入的非特异性结合和运行间变化，对fu测量的信心仍然会受到影响。为了解决这一问题，我们引入了一种称为反平衡透析（CED）的正交方法，其中在快速平衡透析（RED）中，将非标记和同位素标记的化合物反向给药。在同一运行中同时测量非标记和标记化合物的fu值。这些策略不仅使非特异性结合和运行间可变性最小化，而且还能够确认真正的平衡。如果在两个透析方向都达到平衡，非标记化合物和标记化合物的fu将收敛。使用具有不同理化性质和血浆结合特性的各种化合物对精制方法进行了广泛测试。我们的研究结果表明,通过使用CED方法，大范围的化合物的fu值可以准确地确定，具有显著提高的置信度，包括具有挑战性的高度结合和不稳定的化合物。
