Perspectives in Pharmacology Progress in Brain Penetration Evaluation in Drug Discovery and Development

This review discusses strategies to optimize brain penetration from the perspective of drug discovery and development. Brain penetration kinetics can be described by the extent and time to reach brain equilibrium. The extent is defined as the ratio of free brain concentration to free plasma concentration at steady state. For all central nervous system (CNS) drug discovery programs, optimization of the extent of brain penetration should focus on designing and selecting compounds having low efflux transport at the blood-brain barrier (BBB). The time to reach brain equilibrium is determined by both BBB permeability and brain tissue binding. Rapid brain penetration can be achieved by increasing passive permeability and reducing brain tissue binding. Although many drug transporters have been identified at the BBB, the available literature demonstrates only the in vivo functional importance of P-glycoprotein (P-gp) in limiting brain penetration of its substrates. Drug-drug interactions mediated by P-gp at the BBB are possible due to inhibition or induction of P-gp. For newly identified drug transporters at the BBB, more research is needed to reveal their in vivo significance. We propose the following strategies for addressing drug transporters at the BBB. 1) Drug discovery screens should be used to eliminate good P-gp substrates for CNS targets. Special consideration could be given to moderate P-gp substrates as potential CNS drugs based on a high unmet medical need and the presence of a large safety margin. 2) Selection of P-gp substrates as drug candidates for non-CNS targets can reduce their CNS-mediated side effects. Brain is separated from the systemic circulation by two barriers: the blood-brain barrier (BBB) and the blood-cerebrospinal-fluid barrier (BCSFB). The BBB is composed of cerebral endothelial cells that differ from those in the rest of the body by the presence of extensive tight junctions, absence of fenestrations, and sparse pinocytotic vesicular transport. The BCSFB is formed by a continuous layer of polarized epithelial cells that line the choroid plexus. The BBB and BCSFB exhibit very low paracellular permeability and express multiple drug transporters. These characteristics restrict the entry of hydrophilic compounds or efflux transport substrates into brain (Davson and Segal, 1995). In this review, we will summarize recent published data relevant to assess drug brain penetration and present the authors’ opinions on how to effectively address BBB issues in drug discovery and development. What Parameters Should Be Used to Assess Brain Penetration? In drug discovery, it is obvious that one should select compounds with “good” brain penetration as CNS drugs, but it is not so obvious what parameters should be used to define “good” brain penetration. Two parameters, the ratio of brain Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.130294. ABBREVIATIONS: BBB, blood-brain barrier; CNS, central nervous system; P-gp, P-glycoprotein; BCSFB, blood-cerebrospinal fluid barrier; CSF, cerebrospinal fluid; PS, BBB surface area permeability product; Kp, brain plasma concentration ratio; Kp,free and Kp,uu, brain plasma free concentration ratio; Cluptake, uptake transporter clearance at the BBB; Clefflux, efflux transporter clearance at the BBB; Clbulk, the clearance due to brain interstitial fluid bulk flow; Clmetabolism, brain metabolic clearance; fu,plasma, plasma unbound fraction; fu,brain, brain unbound fraction; Cplasma, plasma concentration; Cbrain, brain concentration; Cu,plasma, plasma unbound concentration; Cu,brain, brain unbound concentration; t1/2eq,in, intrinsic brain equilibrium half-life; DDI, drug-drug interaction; KO, knockout; WT, wild type; Mrp, multidrug resistance-associated protein; Bcrp, breast cancer resistance protein; Oatp, organic anion transport polypeptide; Oat, organic anion transporter; PXR, pregnane X receptor; CP-141938, N-(4-methoxy-3-[(2-phenyl-piperadin-3-amino)-methyl]-phenyl)-N-methyl-methane-sulfonamide. 0022-3565/08/3252-349–356$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 325, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 130294/3320967 JPET 325:349–356, 2008 Printed in U.S.A. 349 at A PE T Jornals on July 3, 2017 jpet.asjournals.org D ow nladed from and plasma concentration (Kp) and BBB permeability (quantified as the permeability surface area product, PS), have been used to describe brain penetration. Kp has been the most widely used parameter to evaluate and optimize brain penetration in drug discovery, but its relevance has been questioned (Pardridge, 2004). Analogous to the concept of the extent and rate of oral absorption, brain penetration can be assessed with two parameters, the extent and the time to reach brain equilibrium (Liu and Chen, 2005). The extent can be defined as the ratio of free brain and free plasma concentration at equilibrium, Kp,free, also known as Kp,uu (Syvanen et al., 2006). The time to reach brain equilibrium can be defined as the half-life to reach the equilibrium of brain and plasma concentration. What Determines the Extent of Brain Penetration? According to a three-compartment model (Fig. 1), the following equation (eq. 1) can be derived at steady state. Cluptake and Clefflux are the active uptake and efflux transport clearance at the BBB, respectively. Clbulk is the clearance due to brain interstitial fluid bulk flow, and Clmetabolism is the brain metabolic clearance. According to eq. 1, to augment the extent of brain penetration, one needs to either increase PS and Cluptake or reduce Clefflux, Clbulk, and Clmetabolism. Kp,free PS Cluptake PS Clefflux Clbulk Clmetabolism (1) It is desirable to design a compound as a substrate of brain uptake transporters to enhance Cluptake. For example, large neutral amino acid transporter 1 transports L-DOPA and gabapentin across the BBB. Although L-DOPA has been available for more than 30 years, the same success in increasing brain penetration of other drugs has rarely been replicated, with the exception of its close-in analogs. Effective in vitro approaches have yet to be developed to screen brain uptake transporter substrates to deliver drugs through the endogenous transporters at the BBB. It would be more feasible to design lipophilic compounds (high PS values) without significant efflux transport (low Clefflux) than to design compounds as uptake transporter substrates. Clbulk can play an important role in decreasing Kp,free for low permeability compounds. It has been estimated that bulk flow clearance spans the range of 0.2 to 0.3 l/min/g (Cserr and Patlak, 1993). Take the example of mannitol, a compound of low permeability with a PS value of less than 1 l/min/g. Bulk flow becomes significant compared to its permeability, resulting in a low Kp,free (0.01). For a compound with moderate to high permeability, Clbulk is insignificant. This is illustrated by caffeine, a compound of moderate to high permeability with a PS value of 13 l/min/g. In this case, bulk flow clearance is much lower than the permeability and has an insignificant effect on Kp,free (1.0) (Hansen et al., 2002). Brain metabolism, Clmetabolism, could also play a significant role in reducing Kp,free. Metabolizing enzymes, such as monoamine oxidase, flavin-containing monooxygenase, cytochrome P450, and glucuronosyltransferases have been identified in brain endothelial cells and brain tissue (el-Bacha and Minn, 1999; Fang, 2000; Gervasini et al., 2004; Strazielle et al., 2004). Hence, the stability of a compound in brain tissue needs to be examined in early drug discovery. How to Use Kp to Assess Brain Penetration? Kp is the most commonly used parameter to evaluate brain penetration and has been used as the primary parameter to optimize brain drug delivery in CNS drug discovery. In a recent study, Kp was determined for a set of the 32 most prescribed CNS drugs and ranged from 0.1 to 24 in mice (Fig. 2A). Thus, a compound having a Kp value as low as 0.1, such as sulpiride, can still be a successful CNS drug, suggesting that it is difficult to assess brain penetration based upon Kp alone (Doran et al., 2005). According to the definition of Kp (Kp Cbrain/Cplasma), fu,plasma (fu,plasma Cu, plasma/Cplasma), fu,brain (fu,brain Cu,brain/Cbrain), and Kp,free (Kp,free Cu,brain/Cu,plasma), the relationship between Kp and Kp,free can be derived as follows: Kp fu,plasma fu,brain Kp,free (2) where Cplasma and Cu,plasma are the total and unbound plasma concentration, respectively; Cbrain and Cu,brain are the total and unbound brain concentration, respectively; and fu,plasma and fu,brain are the plasma and brain unbound fraction, respectively. From eq. 2, it is evident that a low Kp can be due to high nonspecific binding in plasma, low binding in brain, or low Kp,free. There was a 240-fold difference for Kp among the 32 most prescribed CNS drugs in mice but only a 34-fold difference for Kp,free (Fig. 2, A and B), indicating that nonspecific binding (e.g., fu,plasma/fu,brain) is a significant component for Kp. Gupta et al. (2006) reported that Kp for Sand R-cetirizine is 0.22 and 0.04, respectively. These Kp values apparently indicate that S-cetirizine penetrates brain tissue better than R-cetirizine. However, the Kp,free is 0.17 and 0.14 for Sand R-cetirizine, respectively, indicating that there is no stereoselective brain penetration. Further investigation revealed that the protein binding for these enantiomers was different. The fraction unbound for Sand R-cetirizine is 0.5 and 0.15, respectively. The stereoselective Kp is caused by differential binding to plasma proteins rather than transport at the BBB. Thus, when Kp is used in drug discovery to optimize brain penetration, it is very important to understand the impact of the binding in plasma and brain. There are several methods to estimate Kp,free. Brain microdialysis is a direct approach to determine free brain concentration. However, the utility of microdialysis in the drug discovery setting is limited because it requires extensive resources and is not easily applied to highly lipophilic comPlasma