Roche Pharmaceutical Research and Early Development, DMPK/PD project leader (H.F.), Comparative Pharmacology (C.S.), Investigative Safety, Pharmaceutical Sciences (M.U., C.C.), and Immunology, Infectious Disease and Ophthalmology (F.S.), Roche Innovation Center, Basel, Switzerland; and Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Translational and Clinical Research Center, Inc., and LIYU Pharmaceutical Consulting LCC, New Jersey, USA (L.Y.)
Roche Pharmaceutical Research and Early Development, DMPK/PD project leader (H.F.), Comparative Pharmacology (C.S.), Investigative Safety, Pharmaceutical Sciences (M.U., C.C.), and Immunology, Infectious Disease and Ophthalmology (F.S.), Roche Innovation Center, Basel, Switzerland; and Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Translational and Clinical Research Center, Inc., and LIYU Pharmaceutical Consulting LCC, New Jersey, USA (L.Y.).
J Pharmacol Exp Ther. 2021 Mar;376(3):322-329. doi: 10.1124/jpet.120.000158. Epub 2020 Dec 7.
P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) efflux transporter. In vitro approaches, including bidirectional efflux ratio (ER), are used to measure P-gp-mediated transport, but findings can be inconsistent across models. We propose a novel, more physiologically relevant, in vitro model: unidirectional apical efflux ratio (AP-ER)-a ratio of permeability rates at the apical side of the BBB with and without P-gp inhibitor. To test our approach, ER and AP-ER were calculated for 3227 structurally diverse compounds in porcine kidney epithelial cells (LLC-PK1) overexpressing human or mouse P-gp and classified based on their passive transcellular P-gp permeability or charged properties. In vivo rat infusion studies were performed for selected compounds with high ER but low AP-ER. One-third of the 3227 compounds had bidirectional ER that was much higher than AP-ER; very few had AP-ER higher than ER. Compounds with a large difference between AP-ER and ER were typically basic compounds with low-to-medium passive permeability and high lipophilicity and/or amphiphilicity, leading to strong membrane binding. Outcomes in the human model were similar to those in mice, suggesting AP-ER/ER ratios may be conserved for at least two species. AP-ER predicted measured cerebrospinal fluid (CSF) concentration better than ER for the five compounds tested in our in vivo rat infusion studies. We report superior estimations of the CSF concentrations of the compounds when based on less resource-intensive AP-ER versus classic ER. Better understanding of the properties leading to high P-gp-mediated efflux in vivo could support more efficient brain-penetrant compound screening and optimization. SIGNIFICANCE STATEMENT: To address inconsistencies associated with the historical, bidirectional efflux ratio (ER) calculation of P-glycoprotein-mediated transport, we propose to use the novel, more physiologically relevant, unidirectional apical efflux ratio (AP-ER) model. In vitro experiments suggested that compounds with strong membrane binding showed the largest difference between AP-ER and ER, and in vivo infusion studies showed that AP-ER predicted cerebrospinal fluid concentrations of compounds better than ER; outcomes in the human model were similar to those in mice.
P-糖蛋白（P-gp）是血脑屏障（BBB）的主要外排转运体。体外方法，包括双向外排比（ER），用于测量 P-gp 介导的转运，但在不同模型中发现的结果可能不一致。我们提出了一种新颖的、更符合生理的体外模型：单向顶端外排比（AP-ER）-在有和没有 P-gp 抑制剂的情况下，BBB 顶端侧的渗透速率比。为了验证我们的方法，我们在过表达人或小鼠 P-gp 的猪肾上皮细胞（LLC-PK1）中计算了 3227 种结构多样的化合物的 ER 和 AP-ER，并根据它们的被动跨细胞 P-gp 通透性或带电性质进行分类。对于具有高 ER 但低 AP-ER 的选定化合物，进行了体内大鼠输注研究。在 3227 种化合物中，有三分之一的化合物的双向 ER 远高于 AP-ER；极少数化合物的 AP-ER 高于 ER。AP-ER 与 ER 之间差异较大的化合物通常是碱性化合物，具有低到中等的被动通透性、高亲脂性和/或两亲性，导致强烈的膜结合。在人类模型中的结果与在小鼠中的结果相似，这表明至少在两种物种中，AP-ER/ER 比值可能是保守的。对于我们在体内大鼠输注研究中测试的五种化合物，AP-ER 对测量的脑脊液（CSF）浓度的预测优于 ER。与经典的 ER 相比，基于资源消耗较少的 AP-ER 可以更好地预测化合物的 CSF 浓度。更好地了解导致体内 P-gp 介导外排的性质，可以支持更有效的穿透血脑屏障的化合物筛选和优化。意义声明：为了解决与历史上 P-糖蛋白介导转运的双向外排比（ER）计算相关的不一致性，我们建议使用新颖的、更符合生理的单向顶端外排比（AP-ER）模型。体外实验表明，具有强膜结合的化合物在 AP-ER 和 ER 之间表现出最大的差异，体内输注研究表明，AP-ER 对化合物的脑脊液浓度的预测优于 ER；在人类模型中的结果与在小鼠中的结果相似。
