Abstract. To define and differentiate relevant aspects of blood-brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: K p,uu (concentration ratio of unbound drug in brain to blood), CL in (permeability clearance into the brain), and V u,brain (intra-brain distribution). The permeability of the blood-brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. K p,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (K p ), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters K p,uu , CL in , and V u,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes.

New experimental methodologies were applied to measure the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) and the unbound CSF-to-plasma concentration ratio (K(p,uu,CSF)) in rats for 43 structurally diverse drugs. The relationship between chemical structure and K(p,uu,brain) was dominated by hydrogen bonding. Contrary to popular understanding based on the total brain-to-plasma concentration ratio (logBB), lipophilicity was not a determinant of unbound brain exposure. Although changing the number of hydrogen bond acceptors is a useful design strategy for optimizing K(p,uu,brain), future improvement of in silico prediction models is dependent on the accommodation of active drug transport. The structure-brain exposure relationships found in the rat also hold for humans, since the rank order of the drugs was similar for human and rat K(p,uu,CSF). This cross-species comparison was supported by K(p,uu,CSF) being within 3-fold of K(p,uu,brain) in the rat for 33 of 39 drugs. It was, however, also observed that K(p,uu,CSF) overpredicts K(p,uu,brain) for highly effluxed drugs, indicating lower efflux capacity of the blood-cerebrospinal fluid barrier compared to the blood-brain barrier.

ABSTRACT:Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unheplful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (V u,brain ). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with V u,brain microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the V u,brain of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.

ABSTRACT:Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f u,brain ), determined using the brain homogenate method, with the unbound volume of distribution in brain (V u,brain ), determined using the brain slice method. Discrepancies were frequent and were primarily related to drug pH partitioning, attributable to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V u,brain from measurements of f u,brain and drug pK a .This model allowed prediction of V u,brain from f u,brain within a 2.2-fold error range for 95% of the drugs compared with a 4.5-fold error range using the brain homogenate f u,brain method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin, and prototypic organic cation transporter substrates. It was concluded that intrabrain drug distribution is governed by several diverse mechanisms in addition to nonspecific binding and that the slice method is therefore more reliable than the homogenate method. As an alternative, predictions of V u,brain can be made from homogenate f u,brain using the pH partition model presented, although this model does not take into consideration possible active brain cell uptake.

ABSTRACT: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 casette 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 ⅐ 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.Current drug discovery programs strategically focus on achieving a mechanistic and quantitative understanding of the time course of pharmacological effects and how they are related to drug exposure and the drug-target interaction. This approach identifies key issues for lead optimization as well as facilitating predictions of human exposure to the drug. A critical step in the establishment of these pharmacokinetic-pharmacodynamic relationships is the estimation of exposure to the drug in experimental animals. The unbound drug concentration in plasma is the most relevant and convenient measure of systemic exposure. For centrally acting drugs, however, the bloodbrain barrier can regulate drug exposure in the CNS, and the unbound plasma concentration may no longer reflect the concentration at the target site. Our definition of CNS exposure is the unbound drug concentration in the brain interstitial fluid, C u,brainISF , which surrounds most central drug targets. If a drug target is intracellular, however, exposure would be best defined as the unbound drug concentration in the intracellular fluid. This concentration is regulated not only by events at the blood-brain barrier but also by drug transporters at the brain cell plasma membrane (Dallas et al., 2006). Calculation of C u,brainISF , a rational and increasingly common approach in drug discovery for assessing CNS exposure, is approached by measuring the amount of drug in brain tissue (A brain ) and estimating the unbound brain volume of dis...

A major challenge associated with the determination of the unbound brain-to-plasma concentration ratio of a drug (K p,uu,brain ), is the error associated with correction for the drug in various vascular spaces of the brain, i.e., in residual blood. The apparent brain vascular spaces of plasma water (V water , 10.3 lL/g brain), plasma proteins (V protein , 7.99 lL/g brain), and the volume of erythrocytes (V er , 2.13 lL/g brain) were determined and incorporated into a novel, drug-specific correction model that took the drug-unbound fraction in the plasma (f u,p ) into account. The correction model was successfully applied for the determination of K p,uu,brain for indomethacin, loperamide, and moxalactam, which had potential problems associated with correction. The influence on correction of the drug associated with erythrocytes was shown to be minimal. Therefore, it is proposed that correction for residual blood can be performed using an effective plasma space in the brain (V eff ), which is calculated from the measured f u,p of the particular drug as well as from the estimates of V water and V protein , which are provided in this study. Furthermore, the results highlight the value of determining K p,uu,brain with statistical precision to enable appropriate interpretation of brain exposure for drugs that appear to be restricted to the brain vascular spaces.

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Pulmonary drug disposition after inhalation is complex involving mechanisms, such as regional drug deposition, dissolution, and mucociliary clearance. This study aimed to develop a systems pharmacology approach to mechanistically describe lung disposition in rats and thereby provide an integrated understanding of the system. When drug‐ and formulation‐specific properties for the poorly soluble drug fluticasone propionate were fed into the model, it proved predictive of the pharmacokinetics and receptor occupancy after intravenous administration and nose‐only inhalation. As the model clearly distinguishes among drug‐specific, formulation‐specific, and system‐specific properties, it was possible to identify key determinants of pulmonary selectivity of receptor occupancy of inhaled drugs: slow particle dissolution and slow drug‐receptor dissociation. Hence, it enables assessment of factors for lung targeting, including molecular properties, formulation, as well as the physiology of the animal species, thereby providing a general framework for rational drug design and facilitated translation of lung targeting from animal to man.