169

11 METHODS FOR ASSESSING BLOOD – BRAIN BARRIER PENETRATION IN DRUG DISCOVERY

Li Di and Edward H. Kerns

11.1 INTRODUCTION

The blood – brain barrier ( BBB ) is the membrane sur-rounding the microblood capillary vessels in the brain. It plays a critical role in maintaining the homeostasis in the brain, where a fi ne balance is critical for all species. The BBB is made of endothelial cells with very tight junc-tions. There are about 400 mi of the blood capillaries in the brain with strong P - glycoprotein ( Pgp ) effl ux activity at the apical membrane of the BBB cells. Pgp plays a very important role in preventing toxic compounds from entering into the brain. The characteristics of the BBB, such as tight intercellular junctions, absence of fenestra-tions, and Pgp effl ux transport, make it diffi cult for drug molecules to enter the brain and interact with central nervous system (CNS) targets. It has been reported that only 2% of drug discovery compounds can cross the BBB and potentially become successful CNS agents [1] . The BBB is one of the major challenges for CNS therapy, leading to a low success rate in the clinic [2] .

Besides the BBB, there is a blood cerebrospinal fl uid barrier ( BCSFB ) interface with the brain. It is in a sepa-rate compartment. Delivery of drug molecules to the brain via cerebrospinal fl uid ( CSF ) at the BCSFB is diffi cult because the fl ow of interstitial fl uid ( ISF ) in the brain to the CSF is very fast. The surface area of BCSFB is also 5000 times smaller than the BBB. Taken together, the BBB is the most important delivery route and barrier for drugs targeting CNS diseases [3, 4] .

There are many mechanisms for brain penetration (Figure 11.1 ) [5, 6] . Most drugs enter the brain by tran-scellular passive diffusion through the lipid membrane into the brain. There is very limited paracellular trans-port because of the tight junctions between the endo-thelial cells of the BBB. Compounds that are substrates for uptake transporters (e.g., LAT1, PEPT1) can enter the brain at a higher rate than passive diffusion alone. Effl ux transporters, such as Pgp, oppose entry of drug molecules into the brain and can reduce compound con-centration in the brain through effl ux mechanisms. Plasma protein binding and brain tissue binding can affect distribution of drugs into the brain. Metabolism in the liver and brain can also contribute to the amount of drug present in the brain.

The multiple mechanisms and bioprocesses of the brain make it challenging to develop a simple assay to address the complicated system. There are many ways to evaluate brain penetration. There are currently two con-cepts evolving in the pharmaceutical industry and aca-demia to describe brain penetration. They are rate and extend [5, 7 – 9] . Rate is a measure of the initial slope of brain penetration, which is important for drugs that require rapid onset, such as anesthesia. Extent measures the amount of drug in the brain at steady state, which is critical for indications requiring sustained drug effects during chronic dosing. Rate is not the same as extent, but they also have common features related to molecular properties of compounds, such as lipophilicity, hydrogen

ADME-Enabling Technologies in Drug Design and Development, First Edition. Edited by Donglu Zhang and Sekhar Surapaneni.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

170 METHODS FOR ASSESSING BLOOD–BRAIN BARRIER PENETRATION IN DRUG DISCOVERY

11.3 METHODS FOR DETERMINATION OF FREE DRUG CONCENTRATION IN THE BRAIN

If the question of a project is how to predict whether a compound will be effi cacious in vivo based on in vitroactivity data, it is important to determine the free drug concentration in the brain [5, 9, 16] . Determination of free drug concentration in the biophase (brain in this case) is critical to predicting in vivo effi cacy, developing pharmacokinetic (PK)/pharmacodynamic (PD) rela-tionships and selecting dose and dosing frequency [10] . Total drug concentration in the brain is usually not rel-evant to in vivo activity [11, 16] . Drug molecules bound nonspecifi cally to lipids and proteins in the brain are nonproductive and not available to interact with the therapeutic target to produce effi cacy.

The gold standard method for measuring free drug concentration in the brain is in vivo microdialysis [17 – 20] . However, the assay is very low throughput, labor

bonding, and polar surface area (PSA) [5] . Ultimately, it is the free drug concentration at the site of action that exerts the pharmacological activity [10, 11] .

Many methods for assessing BBB penetration have been developed and are well documented [12 – 15] . It has been proposed that three variables are most rele-vant to predict CNS drug action: rate, extent, and brain tissue binding [10] . This chapter will cover the methods commonly used in drug discovery in assessing BBB penetration.

11.2 COMMON METHODS FOR ASSESSING BBB PENETRATION

Many methods have been developed to measure and predict the brain penetration potential of drug candi-dates [12 – 15] (Table 11.1 ). Because of the multiple mechanisms of brain penetration for small molecules [6] , the methods are designed to address specifi c aspects of BBB penetration, such as BBB membrane permea-bility, distribution of drugs between brain and blood, free drug concentration in the brain, effects of trans-porters on the BBB, and others. Which BBB method should be used for a particular drug discovery program is really dependent on what questions that project team is trying to answer. There is really no one - method - fi ts - all approach for the BBB. Methods are specifi c to particu-lar mechanisms that are most relevant to a project. Here we discuss the common methods applied in drug discov-ery to address specifi c questions of project teams with regard to BBB penetration.

FIGURE 11.1. Mechanisms for brain penetration (modifi ed from References 5, 6 ) .

Blood

Brain

TranscellularPassive

Diffusion Influx Transport (aa, peptide)

EffluxTransport

(Pgp)

MetabolismMetabolism

BrainEndothelial

Cells

Limited Pinocytosis or Paracellular Route

Protein Binding

Brain Binding Metabolism

TABLE 11.1. Commonly Used BBB Methods in Drug Discovery

Interest Methods

Free drug concentration in brain

Brain PK & brain tissue binding CSF concentration

BBB permeability In situ brain perfusion PAMPA - BBB LogD

Pgp effl ux MDR1 - MDCK Caco - 2

METHODS FOR DETERMINATION OF FREE DRUG CONCENTRATION IN THE BRAIN 171

donor and acceptor wells, respectively, for dialysis. Once equilibrium is reached (typically about 5 h), the com-pound is extracted from the matrix material with org-anic solvent and analyzed using LC - MS. The fraction unbound is the concentration in the acceptor divided by the concentration in the donor and corrected by the dilution factor. Fraction unbound, measured using brain homogenates, gives good correlation to in vivo data [25] . It is a rapid, high - throughput, and low - cost approach to obtaining free drug concentration in the brain when combined with brain PK studies. Reports suggest that there is little difference in brain binding among species [9] ; therefore, one can estimate free drug concentration using fraction unbound from a single species early in drug discovery. Brain tissue binding studies using brain slice have also been reported and a high - throughput method has been developed [28 – 30] . It has been sug-gested that data from brain slices is slightly more reli-able in predicting free drug concentration in vivo than using brain homogenates [30] , owing to unmasking of cellular material during homogenization. The protocol using brain slices to study binding is still quite involved and laborious [29] . Brain tissue binding study with brain homogenate is the most common and cost - effective approach in drug discovery to measure fraction unbound. Cassette dosing further improves the throughput [27] .

The free drug concentration in the brain for an orally administered drug is independent of brain tissue binding [7, 11, 31] . Optimization of fraction unbound is nonpro-ductive [11] .

11.3.2 Use of CSF Drug Concentration as a Surrogate for Free Drug Concentration in the Brain

CSF concentration is frequently used as a surrogate measure for in vivo free drug concentration in the brain [32 – 34] . CSF drug concentration has been shown to have good prediction of brain free drug concentration and is better than using unbound plasma concentration [24, 35] . Human and rat have similar rank ordering based on CSF information, which makes rat CSF con-centration a viable tool in drug discovery. Any differ-ence between rat and human CSF data is presumably owing to human data that was obtained under human disease states [10, 34] .

CSF contains very little protein and the sampling is much more amenable in drug discovery compared with in vivo microdialysis. Catheters are inserted into the cisterna magna or lumber intrathecal space for serial CSF sampling, and drug concentrations in CSF are determined using LC - MS. For large animals, studies can be repeated with the same catheterized animals to mini-mize variations from individual animals. CSF concentra-tion is currently the only source of information on brain

intensive, expensive, and technically challenging for lipophilic compounds due to nonspecifi c binding of test compounds to the dialysis probe membranes/equipment, which leads to low recovery and unreliable data. As a result, in vivo microdialysis has not been routinely applied in early drug discovery to determine free drug concentration in the brain, where higher throughput and rapid turnaround time is needed to support large number of compounds and programs. It is more com-monly used for late - stage drug discovery and develop-ment, when a limited number of compounds are studied in depth. Microdialysis is also widely used to determine neurotransmitters, such as dopamine, glutamate, and γ - aminobutyric acid (GABA) [21] .

Free drug concentration in the brain is a complicated variable affected by many different processes in vivo , such as absorption, metabolism, and distribution. There-fore, optimization of free drug concentration involves multiple parameters. For orally administered drugs, the strategy is to optimize solubility and permeability, to optimize fraction absorbed, and metabolic stability to optimized blood concentrations. Optimization of fraction unbound, through structural modifi cation, is counterproductive and should be avoided in drug dis-covery [11] .

11.3.1 In vivo Brain PK in Combination with invitro Brain Homogenate Binding Studies

In vivo brain PK experiments are typically used to determine total brain exposure of drug candidates (bound plus unbound). Brain PK studies are usually performed by dosing animals of selected species (typi-cally rat or mouse) with test compounds at a certain dose via a desirable route (e.g., intravenous, intraperito-neally, or orally) [12] . At different time points after dosing, blood samples and whole brains are taken. The compounds are extracted from plasma and brain tissue homogenates and analyzed with light chromatography - mass spectrometry (LC - MS). PK data, such as area under the curve (AUC) and Cmax in plasma and brain, and brain exposure/plasma exposure (B/P) ratio, can be derived from the study using statistical software. The exposure (e.g., Cmax ) obtained from brain PK studies is the total drug concentration. To obtain the free drug concentration in the brain, the fraction unbound in the brain ( fu, brain ) is determined by using in the following calculation:

C C fmax,u max u brain*= ,

Fraction unbound in the brain is often determined invitro using equilibrium dialysis with brain homogenates in drug discovery [22 – 27] . Typically, brain homogenates with test compounds and buffers are added into the

172 METHODS FOR ASSESSING BLOOD–BRAIN BARRIER PENETRATION IN DRUG DISCOVERY

Low - permeability compounds have a low rate of crossing the BBB, and thus equilibrium is usually not fully established between the blood and the brain within common experimental conditions. In this case, the free drug concentration is usually higher in the blood than in the brain.

Highly permeable compounds have a high rate of crossing the BBB. Equilibrium is fully established in a short amount of time and the free drug concentration is the same on the blood and the brain side of the BBB. Substrates for uptake or effl ux transporters have a pro-portionately higher or lower free drug concentration on the brain side, respectively, than if BBB permeability was only via passive diffusion.

BBB permeability is a pure measure of a compound ’ s characteristics and is not confounded by other mecha-nisms, such as plasma protein or brain tissue binding and metabolism. Characteristics such as B/P, which in the past was commonly used in CNS research, result from multiple mechanisms. Since passive BBB per-meability is a single variable parameter of a com-pound, medicinal chemists can develop structural – BBB permeability relationships and optimize BBB penetra-tion potential.

11.4.1 In situ Brain Perfusion Assay

The in situ brain perfusion assay is the most common insitu / in vivo method for studying BBB permeability in whole animals [36, 37] . Wild - type and knockout mice can both be used in order to evaluate the roles of transport-ers in BBB transport. The assay is performed by infusing a test compound with a reference in a perfusate to the external carotid artery at 5 – 20 mL/min to keep the arte-rial pressure at 80 – 120 mm Hg. The perfusion time is very short, typically around 30 s and no more than 2 min [15, 38] . At the end of the experiment, animals are sacri-

exposure in human. Timing of the sampling is important since the concentration – time profi les may be different between CSF and brain. Cassette dosing can be applied to improve throughput [34] . When compounds have good membrane permeability, using CSF as a surrogate for ISF concentration works well because equilibrium is rapidly established between CSF and ISF in the brain. However, in some cases, CSF concentration can either overpredict or underpredict free drug concentration in the brain. CSF tends to underestimate free drug concen-tration for compounds with very high brain penetration. CSF overestimates ISF concentration for compounds with very low brain penetration [10, 34] . For compounds that are substrates of effl ux transporters, such as Pgp substrates, the CSF drug concentrations tend to be higher than free drug concentration in the brain. This is because Pgp pumps from brain side to the blood side on the BBB, but pumps from the blood side to the CSF on the BCSFB (see Figure 11.2 ). For this reason, Pgp substrates can accumulate at the CSF and lead to over-estimation of free drug concentration in the brain. On the other hand, for compounds that are substrates for uptake transporters, the free drug concentration will be higher in the brain than in the CSF. Despite the com-plexity of the brain physiology, CSF can be used a sur-rogate to predict free drug concentration in the brain when compounds are not substrates of transporters.

11.4 METHODS FOR BBB PERMEABILITY

If one is interested in the ability of a compound to cross the BBB, permeability methods should be used. Perme-ability is a rate of how fast a compound crosses the BBB and it is important for drugs requiring a rapid onset, such as anesthesia. BBB permeability can also affect the free drug concentration in the brain.

FIGURE 11.2. Direction of effl ux for Pgp on BBB and BCSFB [3, 4, 12] .

Blood

Choroid plexus Blood-brain barrier

ISF (280 mL)CSF (140 mL)

Arachnoid

villi

1 cm

Intracellullar

compartment

of the brain

Neurons

Neuroglia

Pgp

Pgp

METHODS FOR PGP EFFLUX TRANSPORT 173

gives very different selectivity [39] . As a result, the pre-diction of BBB permeability using cell - based models is usually unsatisfactory. In addition, cell - based assays are typically much more expensive and laborious than PAMPA - BBB. They involve cell cultures in transwell fi lter plates. Cell - based assays are best used as models for transporters of the BBB, rather than as BBB perme-ability models.

11.4.3 Lipophilicity (LogD 7.4 )

Lipophilicity (LogD 7.4 ) is one of the dominant factors for BBB permeability [44] . LogD has been shown to have good correlations with in situ brain perfusion data [39] . Calculated LogD can be used early in the drug discovery process to guide structural modifi cation in order to enhance BBB permeability. It is important to note that one should use LogD 7.4 rather than LogP since it is more physiologically relevant. LogP has been shown to have poor correlation with in situ brain perfusion data for BBB permeability [38] .

11.5 METHODS FOR P GP EFFLUX TRANSPORT

If the question is whether a compound is a substrate for Pgp and how to “ dial out ” the effl ux properties, a Pgp effl ux transporter assay should be applied. Even though several effl ux transporters have been identifi ed on the BBB, such as, Pgp, breast cancer resistance protein (BCRP), multi-drug resistance protein 4 (MRP4), and multi-drug resistance protein 5 (MRP5) [45] , Pgp by far is the most prevalent transporter preventing drugs from entering the brain. Most commercial CNS drugs do not have Pgp effl ux activity [46, 47] . Therefore, screening of Pgp effl ux is important for CNS drug therapy.

Many different approaches are available to assay for Pgp substrates [48] . The most commonly used methods are cell - based monolayer bidirectional transport assays, such as MDR1 - MDCK, LLC - PK1, and Caco - 2. Cells are grown on fi lter membranes to confl uence. Pgp trans-porters are expressed on the apical membrane. The transport assay is performed bidirectionally, from apical to basal and from basal to apical directions. The perme-ability ratio of b - a over a - b is the effl ux ratio. For certain cells that express multiple effl ux transporters (e.g., Caco - 2), a Pgp inhibitor is added to confi rm if a com-pound is a Pgp substrate. Most organizations prefer using MDR1 - MDCK than Caco - 2 due to much shorter culture time (3 days vs. 21 days) and much stronger signal and sensitivity. Even though the exact expression level of Pgp in the cell lines has no relevance to BBB, they are great tools to diagnose Pgp issues and guide

fi ced and the brain is taken out quickly, homogenized, extracted with organic solvents, and analyzed with LC - MS. Because the perfusion is done rapidly, there is negligible metabolism or tissue binding. The assay is designed to measure permeability of compound across BBB without the impact of other mechanisms (plasma protein or tissue binding, metabolism, etc.). Some of the studies use high concentrations of test compounds (e.g., 50 μ M), owing to the low detection limit caused by the short perfusion time. The high concentrations of test compounds often saturate transporter (e.g., Pgp) activi-ties at the BBB and lead to higher BBB permeability. When transporters are saturated, this method essentially measures the passive diffusion component of BBB per-meability. The in situ brain perfusion assay is not widely used in the pharmaceutical industry because it does not provide drug concentration in the brain in an actual dosing setting. BBB permeability is often determined using in vitro and in silico methods in the industry.

11.4.2 High - throughput PAMPA - BBB

Since most drugs enter the brain through passive diffu-sion, the passive permeability of the BBB is a very im portant component for drug design. PAMPA - BBB has been developed to evaluate the passive BBB perme-ability in high throughput [6] . PAMPA - BBB data has been demonstrated to have good correlation to in situbrain perfusion data and is a valuable tool for screening BBB permeability in early drug discovery [6, 39] . PAMPA - BBB uses an artifi cial membrane made of polar brain lipids in dodecane. The low fl uidity nature of PAMPA - BBB is very similar to the lipid properties of brain endothelial cells. The assay is performed by dissolving test compounds in buffer, which is added to the donor wells. The acceptor has a porous fi lter bottom, which is coated with brain lipid in dodecane and fi lled with buffer. The acceptor plate is placed on the donor plate to form a “ sandwich. ” Test compounds diffuse from the donor well through the lipid membrane and into the acceptor well. After overnight incubation, the compound concentrations in the donor and acceptor wells are measured and the BBB permeability is determined [6] .

Many cell - based models, using cells of cerebral or noncerebral origin, have been developed to mimic the BBB and predict BBB permeability [40 – 43] . The major limitations of cell - based methods are as follows: (1) they typically have unstable transporter expression and do not give reliable prediction of transporter activity, usually over - or underexpressing transporters of the BBB; (2) certain cell - based models do not have tight - enough junctions, making them much leakier than the BBB; and (3) cells with noncerebral nature usually have very different lipid composition than the BBB, which

174 METHODS FOR ASSESSING BLOOD–BRAIN BARRIER PENETRATION IN DRUG DISCOVERY

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11.6 CONCLUSIONS

BBB penetration is still a rapidly growing fi eld with new concepts and technologies continuously evolving. New tools are becoming available to help drug discovery scientists develop better strategies to enhance the success of CNS drugs. Commonly used BBB methods in the industry include determination of free drug concen-tration in the brain using brain PK study and brain tissue binding; BBB permeability with PAMPA - BBB or LogD; and determination Pgp effl ux activity using MDR1 - MDCK or Caco - 2. New delivery technologies and methods for the BBB will continue to be developed to meet the challenges of CNS diseases.

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