• Abstract
• Table of Contents
• Figures
• Literature Cited

Abstract

The blood?brain barrier (BBB) is a physical and metabolic entity that isolates the brain from the systemic circulation. The barrier consists of tight junctions between endothelial cells that contain egress transporters and catabolic enzymes. To cross the BBB, a drug must possess the appropriate physicochemical properties to achieve a sufficient time?concentration profile in brain interstitial fluid (ISF). In this overview, we review techniques to measure BBB permeation, which is evidenced by the free concentration of compound in brain ISF over time. We consider a number of measurement techniques, including in vivo microdialysis and brain receptor occupancy following perfusion. Consideration is also given to the endothelial and nonendothelial cell systems used to assess both the BBB permeation of a test compound and its interactions with egress transporters, and computer models employed for predicting passive permeation and the probability of interactions with BBB transporters. Curr. Protoc. Pharmacol . 62:7.15.1?7.15.30. © 2013 by John Wiley & Sons, Inc.

Keywords: blood?brain barrier; brain disorders; drug discovery; neuroscience; in vivo microdialysis; MDCK cells; receptor occupancy; brain medicines; brain disorders

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Table of Contents

• Introduction
• Structure and Function of the BBB
• The BBB and Immune System Function
• Assessing BBB Permeability
• Physicochemical Determinants of Transcellular BBB Transport
• Assessing Efflux Transport
• Brain Pharmacokinetics, Pharmacodynamics, and their Relationship
• Conclusions
• Literature Cited
• Figures
• Tables

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Materials

GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures

• Figure 7.15.1 The blood‐brain barrier. Endothelial cells are joined by cell adhesion molecules to form a permeability barrier (the blood‐brain barrier) between blood and brain interstitial fluid. Other components of the blood–brain barrier include pericytes, glial cells (astrocyte endfeet), and the basal membrane. These cells form an integrated (neurovascular) unit that controls critical BBB functions.

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• Figure 7.15.2 Localization of the major compound efflux transporters on brain capillary endothelial cells of the blood‐brain barrier (BBB). They include P‐glycoprotein (P‐gp), multidrug‐resistant protein (MRP)‐1 to MRP6 and breast cancer resistance protein (BCRP). These ATP‐binding cassette proteins transport a variety of substrates, including amino acids, sugars, drugs, toxins, lipids, sterols, endogenous metabolites, nucleotides, and ions. In addition to these ABC transporters, several members of the organic anion‐transporting‐polypeptide (OATP) family and the organic anion transporter (OAT) family are expressed in brain endothelial cells. In the OAT family, there is evidence for OAT3 expression in human brain capillary endothelial cells with predominant abluminal localization.

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• Figure 7.15.3 A schematic diagram showing the various routes of transport across the blood‐brain barrier (BBB). (A ) Under normal circumstances, the tight junctions severely restrict penetration of hydrophilic compounds. (B ) Lipophilic compounds with a molecular weight <450 Da can diffuse across the BBB; this is the route by which most CNS drugs enter the brain. (C ) Both hydrophilic and lipophilic compounds can be transported out of the brain by a variety of egress transporters, principally P‐glycoprotein and BCRP. (D ) The BBB contains transport proteins (carriers) for glucose, amino acids, nucleosides, and other substances. (E ) Certain proteins, such as insulin and transferrin, are transported by specific receptor‐mediated endocytosis and transcytosis. (F ) Plasma proteins, such as albumin, are poorly transported, but cationization can increase their uptake by adsorptive‐mediated endocytosis and transcytosis. Modified from Begley ().

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• Figure 7.15.4 Concentration‐time profile in plasma following administration of a compound. T max = time to reach maximal concentration. T 1/2 = the period of time required for the concentration or amount of a compound to be reduced by one‐half. C AE = concentration required to cause an adverse event. C max = the maximal concentration achieved. C TE = the concentration required to evoke a therapeutic effect. AUC = ∫ Cdt , and represents a measure of total drug exposure. The therapeutic ratio = C AE / C max .

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• Figure 7.15.5 Structure of BBB tight junctions. The tight junctions contain occludin and claudins, particularly types 3, 5, and 12, which associate and bind to each other across the intercellular cleft. Both the claudins and occludin are associated with the scaffolding proteins, zonula occludens 1, 2, and 3 (ZO‐1 to ZO‐3), which are, in turn, linked (via cingulin dimers) to the actin/myosin cytoskeletal system within the cell. This system is activated by increases in the concentration of intracellular calcium. The role of the junction‐associated molecules (JAMs) is not yet clear, but like the endothelial cell‐selective adhesion molecule (ESAM), it is a member of the immunoglobulin superfamily. Other proteins that span the intercellular cleft and are bound by homophilic adhesion constitute adherens junctions. They are the platelet endothelial cell adhesion molecule (PECAM‐1), also known as cluster of differentiation 31 (CD31), and VE‐cadherin.

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• Figure 7.15.6 Use of Transwell apparatus cells to provide a measure of the BBB permeation of compounds and their ability to act as P‐gp substrates. Monolayers of cells (e.g., MDCK cells expressing the gene for human P‐gp) are cultured on a semipermeable membrane to form tight cell‐cell junctions. The inner well containing the cells is called the apical (A) compartment while the outer well is called the basolateral (B) compartment. A CNS‐active agent is added to either compartment A or to compartment B and the movement of CNS‐active agent is measured over time and P app values are determined.

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• Figure 7.15.7 A microdialysis probe of concentric design. The probe is stereotaxically placed into the CNS (usually the brain) and isotonic perfusate is pumped through the probe at a slow flow rate. CNS‐active compound in the interstitial fluid then diffuses through the semipermeable membrane at the end of the probe and the flow of perfusate moves it out of the probe, where the dialysate is collected for subsequent analysis.

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• Figure 7.15.8 Model of the disposition of a CNS‐active compound in blood, brain, and CSF. The unbound concentration of a CNS‐active agent in blood [ C u(blood) ] is in equilibrium with the bound concentration in blood [ C (blood) ] and determines its unbound concentration in brain interstitial fluid [ C u(brain ISF ] and cerebrospinal fluid [ C u(CSF) )] by equilibration across the blood–brain barrier (BBB) and the blood‐CSF barrier (BCSFB), respectively. Clearance from brain ISF occurs by bulk flow [ Cl (bulk) ] and brain metabolic clearance [ Cl (metab) ], and the unbound concentration of CNS‐active agent in CSF [ C u(CSF) ] is in equilibrium with its bound concentration in CSF [ C (CSF) ]. Movement of CNS‐active agents through both the BBB and the BCSFB can occur by passive diffusion (red arrows), active uptake (green arrows), or active efflux (blue arrows).

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