Regional differences in capillary density, perfusion rate, and P-glycoprotein activity: A quantitative analysis of regional drug exposure in the brain
Graphical abstract
The rate of regional perfusion flow (diazepam as marker), as well as P-gp-mediated colchicine efflux activity, was directly proportional to local capillary density (inulin as marker) in murine brain.
Introduction
The blood–brain barrier (BBB) is the primary interface between the brain and peripheral circulation. The BBB is composed of brain capillary endothelial cells, which are characterized by highly developed tight junctions and a paucity of fenestrae and pinocytotic vesicles [1]. P-gp, a member of the ATP-binding cassette (ABC) superfamily of transport proteins, is a 170-kDa membrane protein encoded by multidrug resistance gene (ABCB1 in human, Abcb1a and Abcb1b in rodents). P-gp is expressed at the luminal side of the brain capillary endothelium and mediates the flux of various endogenous compounds and xenobiotics in the brain-to-blood direction, thereby serving as an important barrier to the entry of P-gp substrates into brain [2], [3], [4], [5].
The mammalian brain is a highly specialized organ from both the structural and functional perspective [6], [7], [8]. Most of the targets for treatment of brain disorders, e.g., neurodegenative disorders (dopamine receptors), epilepsy (e.g., GABA receptors) and neuropathic pain (μ-opioid receptors) are not homogeneously distributed in the brain [8]. The rate of local cerebral blood flow under physiologic conditions varies about 18-fold among brain areas in the rat [9]. In addition, clinical practice has shown variations in sensitivity to the therapeutic effect or CNS side effects of drugs associated with different brain regions [10], [11], [12]. Previous studies in our laboratory demonstrated that P-glycoprotein (P-gp)-mediated efflux at the BBB influences the distribution of 3H-verapamil within the brain following intranasal administration to P-gp-competent and -deficient mice [13]. Pharmacokinetic modeling of these data revealed that the apparent P-gp effect on verapamil exposure was negligible in some regions, and produced nearly a 100-fold attenuation of exposure in others [13]. The regional cerebral vascular volume and blood flow rate, as well as regional P-gp-mediated efflux activity at the BBB differ between brain regions; these differences may result in regional variations in drug exposure. Thus knowledge of the regional drug exposure is of particular interest with regard to targeted treatment for cerebral diseases.
In situ brain perfusion was developed to evaluate BBB permeability in mice [14], [15]. This technique allows examination of single-pass permeability of molecules at the BBB without systemic contamination, with complete control of the composition and flow of perfusion buffer, and without other pharmacokinetic events (e.g., hepatic metabolism) confounding the experimental results. In the present study, the in situ brain perfusion technique was utilized to study the impact of local cerebral perfusion flow rate, vascular volume, and P-gp efflux activity on regional drug exposure. 14C-diazepam and 3H-inulin, selected as markers of functional perfusion flow rate and vascular volume, respectively, were perfused simultaneously, followed by microdissection of the brain to assess regional flow rate and vascular space. Subsequent experiments were directed towards examining regional brain exposure of the P-gp substrates colchicine, quinidine, loperamide and verapamil during perfusion in P-gp-competent and -deficient mice, allowing assessment of regional P-gp-mediated efflux activity in P-gp-expressing animals. The in situ perfusion experiments were complemented by an in vivo evaluation of P-gp-mediated efflux activity over 12 h following subcutaneous administration of loperamide to assess the potential time-dependency of efflux activity estimates.
Section snippets
Animals and reagents
Adult CF-1 [mdr1a(+/+) and mdr1a(−/−)] mice (6–8 weeks of age) were obtained from Charles River Laboratories (Wilmington, MA). All mice were maintained on a 12-h light/dark cycle with access to water and food ad libitum. All experimental procedures were performed under full anesthesia induced with ketamine/xylazine (100/10 mg/kg, i.p.). All procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill and were conducted in
Regional cerebral vascular volume and flow rate
During in situ mouse brain perfusion, the regional vascular volume and perfusion flow rate in mdr1a(+/+) and mdr1a(−/−) mice were comparable (one-way ANOVA, p > 0.05 for each brain region), so the data were pooled for each structure, as summarized in Table 1 (n = 6, mean ± SD). The regional vascular volume ranged from 0.62 mL/100 g in pons and medulla to 2.21 mL/100 g in colliculi. The perfusion flow rate ranged from 38.1 mL/min/100 g in medulla to 285 mL/min/100 g in colliculi. An orthogonal linear
Discussion
The current study demonstrated that regional drug exposure in the brain is a function of many drug- and region-specific factors, including physicochemical characteristics (lipophilicity), drug concentration in blood/perfusate, local cerebral perfusion flow rate, brain capillary volume, regional capillary transit time, and the flux of drugs across the BBB that may include both passive and active (transport-mediated) components. Generally, brain structures such as colliculi, thalamus and parietal
Acknowledgements
This study was supported by NIH GM61191.
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Presently at Roche R&D Center (China) Ltd., Shanghai 201203, China.