Department of Biology, Drexel University, Philadelphia, PA, USA.
Drug Metab Dispos. 2013 Jul;41(7):1347-66. doi: 10.1124/dmd.112.050500. Epub 2013 Apr 25.
A P-glycoprotein (P-gp) IC₅₀ working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC₅₀ determinations. Each laboratory followed its in-house protocol to determine in vitro IC₅₀ values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC₅₀ values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC₅₀ values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC₅₀ values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC₅₀ values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC₅₀ values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC₅₀ determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.
一个 P-糖蛋白（P-gp）IC₅₀ 工作组由 23 家制药和合同研究实验室以及一家学术机构组成，旨在评估 P-gp IC₅₀ 测定中的实验室间变异性。每个实验室都遵循其内部方案，使用四种不同的测试系统来确定 16 种抑制剂的体外 IC₅₀ 值：人结肠腺癌细胞（Caco-2；11 个实验室）、转染 MDR1 cDNA 的 Madin-Darby 犬肾细胞（MDCKII-MDR1；6 个实验室）、转染 MDR1 cDNA 的 Lilly 实验室细胞-猪肾 1 号细胞（LLC-PK1-MDR1；4 个实验室）和含有人 P-糖蛋白的膜囊泡（5 个实验室）。对于细胞模型，还评估了计算剩余转运活性的各种方程（例如，外排比、单向通量、净分泌通量）。所有测试系统和方程中，每种抑制剂的 IC₅₀ 值差异最小为最低和最高 IC₅₀ 值的 20-24 倍，最大为替米沙坦和维拉帕米的 407-796 倍。对于替米沙坦和维拉帕米，每个实验室的数据对变异性的影响都很大。排除这两个数据集后，替米沙坦和维拉帕米的 IC₅₀ 值范围缩小到 69-159 倍。基于外排比的方程通常导致 IC₅₀ 值比单向或净分泌通量方程低几个数量级。统计分析表明，IC₅₀ 值的变异性主要是由于实验室间的变异性，而不是测试系统之间隐含的系统差异。讨论了变异性的潜在原因，并提出了最简单、最稳健的 P-gp IC₅₀ 测定实验设计。这些发现对药物相互作用风险评估的影响在相关文章中进行了讨论（Ellens 等人，2013 年），并提供了建议。
