Heinle Lance, Peterkin Vincent, de Morais Sonia M, Jenkins Gary J, Badagnani Ilaria
Department of Drug Metabolism, AbbVie Inc., North Chicago, IL 60064, USA.
Comb Chem High Throughput Screen. 2015;18(5):442-52. doi: 10.2174/1386207318666150401101737.
A high throughput, semi-automated clearance screening assay in hepatocytes was developed allowing a scientist to generate data for 96 compounds in one week. The 384-well format assay utilizes a Thermo Multidrop Combi and an optimized LC-MS/MS method. The previously reported LCMS/ MS method reduced the analytical run time by 3-fold, down to 1.2 min injection-to-injection. The Multidrop was able to deliver hepatocytes to 384-well plates with minimal viability loss. Comparison of results from the new 384-well and historical 24-well assays yielded a correlation of 0.95. In addition, results obtained for 25 marketed drugs with various metabolism pathways had a correlation of 0.75 when compared with literature values. Precision was maintained in the new format as 8 compounds tested in ≥39 independent experiments had coefficients of variation ≤21%. The ability to predict in vivo clearances using the new stability assay format was also investigated using 22 marketed drugs and 26 AbbVie compounds. Correction of intrinsic clearance values with binding to hepatocytes (in vitro data) and plasma (in vivo data) resulted in a higher in vitro to in vivo correlation when comparing 22 marketed compounds in human (0.80 vs 0.35) and 26 AbbVie Discovery compounds in rat (0.56 vs 0.17), demonstrating the importance of correcting for binding in clearance studies. This newly developed high throughput, semi-automated clearance assay allows for rapid screening of Discovery compounds to enable Structure Activity Relationship (SAR) analysis based on high quality hepatocyte stability data in sufficient quantity and quality to drive the next round of compound synthesis.
开发了一种用于肝细胞的高通量半自动清除筛选测定法，使科学家能够在一周内生成96种化合物的数据。这种384孔板测定法采用了赛默飞世尔的Multidrop Combi和优化的液相色谱-串联质谱（LC-MS/MS）方法。先前报道的LC-MS/MS方法将分析运行时间缩短了3倍，降至每次进样1.2分钟。Multidrop能够将肝细胞输送到384孔板中，且活力损失最小。新的384孔板测定法与历史24孔板测定法的结果比较显示，二者的相关性为0.95。此外，对25种具有不同代谢途径的上市药物的结果与文献值比较时，相关性为0.75。新方法保持了精度，因为在≥39次独立实验中测试的8种化合物的变异系数≤21%。还使用22种上市药物和26种艾伯维化合物研究了使用新的稳定性测定法预测体内清除率的能力。通过结合肝细胞（体外数据）和血浆（体内数据）对内在清除率值进行校正，在比较人类的22种上市化合物（0.80对0.35）和大鼠的26种艾伯维发现化合物（0.56对0.17）时，体外与体内的相关性更高，这表明在清除率研究中校正结合的重要性。这种新开发的高通量半自动清除测定法能够快速筛选发现化合物，以便基于高质量的肝细胞稳定性数据进行构效关系（SAR）分析，这些数据在数量和质量上足以推动下一轮化合物合成。
