The brain slice method for studying drug distribution in the CNS

Fluids and Barriers of the CNS

Margareta Hammarlund-Udenaes²  Markus Fridén¹  Irena Loryan² 

[1] AstraZeneca Research and Development, Respiratory and Inflammatory Innovative Medicines, Department of Drug Metabolism and Pharmacokinetics, Mölndal, Sweden;Department of Pharmaceutical Biosciences, Translational PKPD Research Group, Uppsala University, Associate member of SciLife Lab, Box 591, Uppsala, SE-75124, Sweden

关键词: High-throughput screening;    Drug discovery;    Neuropharmacokinetics;    Unbound volume of distribution in the brain;    Brain slice method;   

Others  :  806988
DOI  :  10.1186/2045-8118-10-6

 received in 2012-11-07, accepted in 2013-01-02,  发布年份 2013

【 摘 要 】

The high-throughput brain slice method is a precise and robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (V_u,brain; ml·g brain^-1). V_u,brain describes the relationship between the total drug concentration in the brain and the concentration of unbound drug in the brain interstitial fluid, regardless of blood–brain barrier function. The brain slice method is more physiologically based than the brain homogenate method with respect to the assessment of drug distribution in the brain because the cell-cell interactions, pH gradients and active transport systems are all conserved. The method provides information that is directly relevant to issues such as nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells. For these reasons, the brain slice method is recommended for estimation of target-site pharmacokinetics in the early drug discovery process and fundamental pharmacological studies. This article provides a detailed protocol for the rat and mouse brain slice methods, with the aim of enabling simple, cost-effective profiling of compounds with diverse physicochemical properties. The procedure for assessing the viability of the brain slices after the 5 h incubation period is also described. The results are interpreted for a set of compounds covering a wide range of physicochemical properties and various pharmacological targets. Application of the method for evaluating the unbound intracellular-to-extracellular concentration ratio (K_p,uu,cell) and the unbound brain-to-plasma concentration ratio (K_p,uu,brain) is discussed.

【 授权许可】

   
2013 Loryan et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Hammarlund-Udenaes M, Friden M, Syvanen S, Gupta A: On the rate and extent of drug delivery to the brain. Pharm Res 2008, 25:1737-1750.
• [2]Hammarlund-Udenaes M: Active-site concentrations of chemicals - are they a better predictor of effect than plasma/organ/tissue concentrations? Basic Clin Pharmacol Toxicol 2010, 106:215-220.
• [3]Watson J, Wright S, Lucas A, Clarke KL, Viggers J, Cheetham S, Jeffrey P, Porter R, Read KD: Receptor occupancy and brain free fraction. Drug Metab Dispos 2009, 37:753-760.
• [4]Hammarlund-Udenaes M, Bredberg U, Friden M: Methodologies to assess brain drug delivery in lead optimization. Curr Top Med Chem 2009, 9:148-162.
• [5]Kalvass JC, Maurer TS: Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 2002, 23:327-338.
• [6]Friden M, Bergstrom F, Wan H, Rehngren M, Ahlin G, Hammarlund-Udenaes M, Bredberg U: Measurement of unbound drug exposure in brain: modeling of pH partitioning explains diverging results between the brain slice and brain homogenate methods. Drug Metab Dispos 2011, 39:353-362.
• [7]McIlwain H: Metabolic response in vitro to electrical stimulation of sections of mammalian brain. Biochem J 1951., 48
• [8]McIlwain H, Buchel L, Cheshire JD: The inorganic phosphate and phosphocreatine of Brain especially during metabolism in vitro. Biochem J 1951, 48:12-20.
• [9]Li CL, McIlwain H: Maintenance of resting membrane potentials in slices of mammalian cerebral cortex and other tissues in vitro. J Physiol 1957, 139:178-190.
• [10]Patlak CS, Hospod FE, Trowbridge SD, Newman GC: Diffusion of radiotracers in normal and ischemic brain slices. J Cereb Blood Flow Metab 1998, 18:776-802.
• [11]Becker S, Liu X: Evaluation of the utility of brain slice methods to study brain penetration. Drug Metab Dispos 2006, 34:855-861.
• [12]Kakee A, Terasaki T, Sugiyama Y: Brain efflux index as a novel method of analyzing efflux transport at the blood–brain barrier. J Pharmacol Exp Ther 1996, 277:1550-1559.
• [13]Gredell JA, Turnquist PA, Maciver MB, Pearce RA: Determination of diffusion and partition coefficients of propofol in rat brain tissue: implications for studies of drug action in vitro. Br J Anaesth 2004, 93:810-817.
• [14]Blasberg R, Levi G, Lajtha A: A comparison of inhibition of steady state, new transport, and exchange fluxes of amino acids in brain slices. Biochim Biophys Acta 1970, 203:464-483.
• [15]Newman GC, Hospod FE, Wu P: Glucose utilization of ischemic hippocampal slices. J Neurosci Methods 1989, 28:23-34.
• [16]Benkwitz C, Liao M, Laster MJ, Sonner JM, Eger EI: 2nd, Pearce RA: Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies. Anesthesiology 2007, 106:114-123.
• [17]Friden M, Gupta A, Antonsson M, Bredberg U, Hammarlund-Udenaes M: In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos 2007, 35:1711-1719.
• [18]Friden M, Ducrozet F, Middleton B, Antonsson M, Bredberg U, Hammarlund-Udenaes M: Development of a high-throughput brain slice method for studying drug distribution in the central nervous system. Drug Metab Dispos 2009, 37:1226-1233.
• [19]Stokes CE, Murphy D, Paton JF, Kasparov S: Dynamics of a transgene expression in acute rat brain slices transfected with adenoviral vectors. Exp Physiol 2003, 88:459-466.
• [20]Wellmann H, Kaltschmidt B, Kaltschmidt C: Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J Neurosci Methods 1999, 92:55-64.
• [21]Rice ME: Use of ascorbate in the preparation and maintenance of brain slices. Methods 1999, 18:144-149.
• [22]MacIntyre AC, Cutler DJ: The potential role of lysosomes in tissue distribution of weak bases. Biopharm Drug Dispos 1988, 9:513-526.
• [23]Potter SM, DeMarse TB: A new approach to neural cell culture for long-term studies. J Neurosci Methods 2001, 110:17-24.
• [24]Dos-Anjos S, Martinez-Villayandre B, Montori S, Salas A, Perez-Garcia CC, Fernandez-Lopez A: Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media. Anal Biochem 2008, 378:99-101.
• [25]Di L, Umland JP, Chang G, Huang Y, Lin Z, Scott DO, Troutman MD, Liston TE: Species independence in brain tissue binding using brain homogenates. Drug Metab Dispos 2011, 39:1270-1277.
• [26]Friden M, Winiwarter S, Jerndal G, Bengtsson O, Wan H, Bredberg U, Hammarlund-Udenaes M, Antonsson M: Structure-brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids. J Med Chem 2009, 52:6233-6243.