Fridén Markus, Ducrozet Frederic, Middleton Brian, Antonsson Madeleine, Bredberg Ulf, Hammarlund-Udenaes Margareta
Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden.
Drug Metab Dispos. 2009 Jun;37(6):1226-33. doi: 10.1124/dmd.108.026377. Epub 2009 Mar 19.
New, more efficient methods of estimating unbound drug concentrations in the central nervous system (CNS) combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (V(u,brain)). Although the brain slice method is the most reliable in vitro method for measuring V(u,brain), it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4 to 5 h of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programs. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit of buffer volume. The use of cassette experiments for investigating V(u,brain) in a linear drug concentration range increased the throughput of the method. The V(u,brain) for the model compounds ranged from 4 to 3000 ml . g brain(-1), and the sources of variability are discussed. The optimized setup of the brain slice method allows precise, robust estimation of V(u,brain) for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.
估算中枢神经系统（CNS）中游离药物浓度的新的、更高效的方法，是将传统方法测得的全脑组织样本中的药物量与体外估算的游离脑分布容积（V(u,brain)）相结合。尽管脑片法是测量V(u,brain)最可靠的体外方法，但此前尚未针对药物研发研究的需求进行调整。本研究的目的是提高该方法的通量并优化实验条件。在4至5小时的孵育时间内，缓冲液与脑片之间的药物平衡是一项基本要求。然而，在药物研发项目中，对于许多具有广泛结合性的亲脂性化合物而言，很难满足这一要求。在本研究中，孵育容器的尺寸和搅拌方式会影响平衡时间，每单位缓冲液体积中的脑组织量也会产生同样的影响。使用盒式实验在药物浓度线性范围内研究V(u,brain)提高了该方法的通量。模型化合物的V(u,brain)范围为4至3000 ml·g脑（-1），并对变异性来源进行了讨论。脑片法的优化设置能够精确、可靠地估算具有不同性质药物的V(u,brain)，包括高度亲脂性化合物。这是在药物研发环境中进行中枢神经系统暴露相关测量的关键一步。
