Experimental Validation of Mass Balance Models for in Vitro Cell-Based Bioassays

The freely dissolved concentration in the assay medium (Cfree) and the total cellular concentration (Ccell) are essential input parameters for quantitative in vitro-to-in vivo extrapolations (QIVIVE), but available prediction tools for Cfree and Ccell have not been sufficiently validated with experimental data. In this study, medium–water distribution ratios (DFBS/w) and cell–water distribution ratios (Dcell/w) for four different cells lines were determined experimentally for 12 neutral and five ionizable chemicals. Literature data for seven organic acids were added to the dataset, leading to 24 chemicals in total. A mass balance model based on bovine serum albumin–water (DBSA/w) and liposome–water distribution ratios (Dlip/w) of the chemicals was used to calculate DFBS/w and Dcell/w. For all neutral and basic test chemicals, the mass balance model predicted DFBS/w and Dcell/w within a factor of 3 and 3.4, respectively, indicating that existing models can reliably predict Cfree and Ccell for these chemicals. For organic acids, a further refinement of the model will be required as large deviations between modeled and measured binding to assay medium and cells of up to a factor of 370 were found. Furthermore, saturation of medium proteins should be further explored for organic acids and neutral chemicals with moderate hydrophobicity.实验介质中的游离溶解浓度（Cfree）与细胞内总浓度（Ccell）是定量体外-体内外推（QIVIVE）的核心输入参数，但当前可用的Cfree与Ccell预测工具尚未通过实验数据获得充分验证。本研究通过实验测定了12种中性化合物与5种可电离化合物针对4种不同细胞系的介质-水分配比（DFBS/w）与细胞-水分配比（Dcell/w）。本数据集还补充了7种有机酸的文献数据，最终累计涵盖24种化合物。本研究基于受试化合物的牛血清白蛋白-水分配比（DBSA/w）与脂质体-水分配比（Dlip/w）构建质量平衡模型，用于计算DFBS/w与Dcell/w。针对所有中性与碱性受试化合物，该质量平衡模型对DFBS/w与Dcell/w的预测偏差分别控制在3倍与3.4倍以内，表明现有模型可可靠预测这类化合物的Cfree与Ccell。但对于有机酸，研究发现模型预测值与实测的化合物结合实验介质及细胞的结果间存在最高达370倍的显著偏差，因此需对模型开展进一步优化。此外，针对有机酸与中等疏水性的中性化合物，还需进一步探究介质蛋白的饱和结合效应。