Nicolaï J, De Bruyn T, Van Veldhoven P P, Keemink J, Augustijns P, Annaert P
Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, O&N2, Leuven, Belgium.
Laboratory of Lipid Biochemistry and Protein Interactions, KU Leuven Department of Cellular and Molecular Medicine, O&N1, Leuven, Belgium.
Biopharm Drug Dispos. 2015 Oct;36(7):462-80. doi: 10.1002/bdd.1959. Epub 2015 Jun 11.
The current study was designed to cross-validate rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and the isolated perfused rat liver (IPRL) model against in vivo pharmacokinetic data, using verapamil as a model drug. Michaelis-Menten constants (Km), for the metabolic disappearance kinetics of verapamil in RLM and SRH (freshly isolated and cryopreserved), were determined and corrected for non-specific binding. The 'unbound' Km determined with RLM (2.8 µM) was divided by the 'unbound' Km determined with fresh and cryopreserved SRH (3.9 µM and 2.1 µM, respectively) to calculate the ratio of intracellular to extracellular unbound concentration (Kpu,u). Kpu,u was significantly different between freshly isolated (0.71) and cryopreserved (1.31) SRH, but intracellular capacity for verapamil metabolism was maintained after cryopreservation (200 vs. 191 µl/min/million cells). Direct comparison of intrinsic clearance values (Clint) in RLM versus SRH, yielded an activity-based scaling factor (SF) of 0.28-0.30 mg microsomal protein/million cells (MPPMC). Merging the IPRL-derived Clint with the MPPMC and SRH data, resulted in scaling factors for MPPGL (80 and 43 mg microsomal protein/g liver) and HPGL (269 and 153 million cells/g liver), respectively. Likewise, the hepatic blood flow (61 ml/min/kg b.wt) was calculated using IPRL Clint and the in vivo Cl. The scaling factors determined here are consistent with previously reported CYP450-content based scaling factors. Overall, the results show that integrated interpretation of data obtained with multiple preclinical tools (i.e. RLM, SRH, IPRL) can contribute to more reliable estimates for scaling factors and ultimately to improved in vivo clearance predictions based on in vitro experimentation.
本研究旨在以维拉帕米为模型药物，针对体内药代动力学数据对大鼠肝微粒体（RLM）、悬浮大鼠肝细胞（SRH）和离体灌注大鼠肝脏（IPRL）模型进行交叉验证。测定了维拉帕米在RLM和SRH（新鲜分离和冷冻保存）中代谢消失动力学的米氏常数（Km），并对非特异性结合进行了校正。用RLM测定的“游离”Km（2.8 μM）除以用新鲜和冷冻保存的SRH测定的“游离”Km（分别为3.9 μM和2.1 μM），以计算细胞内与细胞外游离浓度之比（Kpu,u）。新鲜分离的SRH（0.71）和冷冻保存的SRH（1.31）之间的Kpu,u有显著差异，但冷冻保存后维拉帕米的细胞内代谢能力得以维持（200对191 μl/min/百万细胞）。RLM与SRH的内在清除率值（Clint）直接比较，得出基于活性的比例因子（SF）为0.28 - 0.30 mg微粒体蛋白/百万细胞（MPPMC）。将IPRL衍生的Clint与MPPMC和SRH数据合并，分别得出MPPGL（80和43 mg微粒体蛋白/g肝脏）和HPGL（269和153百万细胞/g肝脏）的比例因子。同样，使用IPRL Clint和体内Cl计算肝血流量（61 ml/min/kg体重）。此处确定的比例因子与先前报道的基于CYP450含量的比例因子一致。总体而言，结果表明，对多种临床前工具（即RLM、SRH、IPRL）获得的数据进行综合解释，有助于更可靠地估计比例因子，并最终基于体外实验改进体内清除率预测。
