Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc.,South San Francisco, CA 94080, USA.
Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc.,South San Francisco, CA 94080, USA.
J Pharm Sci. 2023 Sep;112(9):2561-2569. doi: 10.1016/j.xphs.2023.05.006. Epub 2023 May 13.
The confidence in fraction unbound (ƒ) 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 ƒ measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒ 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). ƒ 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 ƒ 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, ƒ values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.
由于不确定是否达到真正的平衡，使用平衡透析（ED）来确定游离分数（ƒ）的置信度常常受到质疑（例如，高度结合、不稳定的化合物）。已经开发了不同的方法来提高ƒ测量的置信度，例如预饱和、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，ƒ测量的置信度仍然可能受到影响。为了解决这个问题，我们引入了一种称为反平衡透析（CED）的正交方法，其中非标记和同位素标记的化合物在快速平衡透析（RED）中反向给药。在同一次运行中同时测量非标记和标记化合物的ƒ值。这些策略不仅最大限度地减少了非特异性结合和运行间变异性，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，则非标记化合物和标记化合物的ƒ值将收敛。该改进的方法学已通过具有不同物理化学性质和血浆结合特征的各种化合物进行了广泛测试。我们的结果表明，通过使用 CED 方法，可以准确确定广泛化合物的ƒ值，并且置信度显著提高，包括具有挑战性的高度结合和不稳定的化合物。
