Transport of small molecules through the blood-brain barrier: biology and methodology

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of small molecules through the blood-brain biology and methodology

Transport

barrier:

William M. Pardridge* Department

of Medicine,

Brain Research Institute, UCLA (Received

28 February

School of Medicine, Los Angeles,

1995; accepted

30 March

CA 90024. USA

1995)

Abstract

The development of small molecules as effective neuropharmaceuticals requires that these compounds undergo significant transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. While it is often assumed that any small molecule undergoes passive diffusion through the BBB, the evidence in the literature suggests only small molecules that (a) are lipid-soluble and (b) have a molecular weight <: 400-600 Da threshold, are transported through the BBB via lipid-mediation in pharmacologically significant amounts. Exceptions to this rule are lipid-soluble molecules with a molecular weight of < 400 Da that are actively bound by plasma proteins in vivo; and small molecules that are transported through the BBB via carrier-mediated transport. This paper reviews the biology of small molecule transport through the BBB, and also reviews the various methodologies available for assessing whether small molecules undergo significant transport through the BBB in vivo. Keywords: Endothelium; Drug delivery; Liposome; Hydrogen cokinetics; Endocytosis; Transcytosis; Cerebrospinal fluid

bond; Peptide transport;

Tissue culture; Pharma-

Contents I. Introduction......................................................................... 2. Biology of small molecule transport through the blood-brain barrier 2.1. Intraventricular drug infusion 2.1.1. Two barriers in brain: the blood-brain barrier and the blood-CSF barrier 2.1.2. Azidothymidine (AZT) transport exemplifies the two barriers in brain 2.1.3. Multivesicular liposome transport in CSF 2.1.4. Equivalence of intra-ventricular and intravenous injections 2.2. Lipid-mediated blood-brain barrier transport . 2.2.1. The role of hydrogen bonding in limiting blood-brain barrier drug transport 2.2.2. Molecular weight threshold of blood-brain barrier transport of small molecules 2.2.3. Plasma protein binding of small molecules 2.3. Carrier-mediated transport of nutrients or drugs through the blood-brain barrier

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(310) 8258858;

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................................................................................... 2.3.1. Nutrienttransport 2.3.1.(a) Neutral amino acid drugs .................................................................... 2.3.2. Blood-brain barrier transport of amines and monocarboxyhc acids ........................................ ............................................................. 2.3.3. Enzymatic blood-brain harrier mechanisms ....................................................................... 2.3.4. Enzymatic barrier to adenosine ............................... 2.3~5. Is P-glycoprotein an active efflux system at the brain capillary endothelium’! 3. Pharmacologically based strategies for blood-brain barrier delivery of small molecule drugs ........................... 3.1. Overview of blood-brain barrier drug delivery strategies ...................................................... 3.2. Lipidization strategies..................................................................................... ........................................................................ 3.2.1. O-methylation or O-acetylation ............................................................... 3.2.2. Amantadine and adamantane derivatives 3.2.3. Fatty acid or cholesterol esters ......................................................................... ...................................................................... 3.2.4. Dihydrotrigonellinatc derivatives ................................................................................... 3.2.S. Reverselipidization 3.3. Liposomes ................................................................................................. ...................................... 4. Methodologies for quantifymg blood-brain barrier transport of small molecules ................................................................................... 4.1. Thepharmacokineticrule 4.2. In vivo measurements of blood-brain barrier PS product ....................................................... ................................................. .................................... 4.2.1. Introduction ............................................................ 4.2.2. Carotid artery single injection technique .................................................. .......... 4.2.3. Internal carotid artery infusion technique Intravenous injection technique ...................................................................... ........................... Measurement of BBB PS product in humans with positron emission tomography Measurement of V, under conditions of unidirectional influx ............................................. Measurements of solute transcytosis through the blood-brain barrier ...................................... ................................................................. 4.2.7.(a) Capillary depletion technique ....................................................................... 4.2.7.(b) Dialysis fiber technique 4.3. In vitro measurements of blood-brain barrier PS products ...................................................... .................................................... 4.3.1. ‘In vitro’ blood-brain barrier models in tissue culture .................................................................... 4.3.1 .(a) In vivoiin vitro correlation .................................................... 4.4. Experimental artifacts of BBB permeability determinations 4.4.1. Artifact 1: A compound is said to undergo BBB transport because the brain V,, > V,, (secondary .............................................................................. endothelialendocytosis)

I’) 20 20 20 21 21 21 21 21 22 23 24 24 24 24 25 2.5 25 26 26

4.2.4. 4.2.5. 4.2.6. 4.2.7.

to

4.4.2. Artifact 7: A compound is said to undergo BBB transport because the brain V,, > V,, (secondary to .......................................................... non-specific binding to the brain cndothelium) 4.4.3. Artifact 3: A compound is said to undergo BBB transport because the brain V,, > V,, (secondary to metabolism artifacts) ........................................................................... 4.4.4. Artifact 4: The blood-brain barrier is said to bc permeable to a drug because the drug readily distributes into .......... ....................................... cerebrospinal fluid ............................ 4.45. Artifact 5: Blood-brain barrier permeability is said to hc hormonally increased based on measurements of increased %ID/g without measuring plasma AIJC ...................................................... 4.4.6. Artifact 6: Blood-brain harrier pcrmeahility is said to be hormonally increased based on measurements of %ID/g without measuring brain blood volume (V,,) .................................................... 4.4.7. Artifact 7: A pathophysiologic modulation is said to increase hlood-brain barrier permeability, as reflected by an increased brain uptake index. but cerebral blood flow is not measured .................................. 5. Conclusions ........................................................................................... ........................................................................................ Acknowledgements ................................................................................................ References

1. Introduction Many central nervous system discovery programs are designed volume receptor-based screening

(CNS) drug to use high programs to

27 2x 2X 29 20 2Y 30 30 30 30

discover small molecule receptor ligands. It is often assumed that such ‘small molecules’ will enjoy free passage through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. It is further anticipated

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that the discovery of small molecules will obviate the need for BBB drug delivery systems. However, only small molecules that are both (a) lipidsoluble and (b) have a molecular weight less than a threshold of 400-600 Da diffuse through the BBB in proportion to the lipid solubility of the molecule. Small molecules that are either watersoluble or have a molecular weight in excess of the 400-600 Dalton threshold are transported through the BBB poorly, and may require brain drug delivery systems to achieve in vivo CNS pharmacologic effects following systemic administration. In the absence of a suitable BBB drug delivery strategy, small molecules that are not both lipid-soluble and have a molecular weight less than 400-600 Da may not become effective neuropharmaceuticals, and may not proceed further along the pathway of drug development (Fig. 1). The purpose of this review is threefold. First, the biology of small molecule transport through the BBB is reviewed (section 2). Second, strategies for increasing BBB transport of small molecules are discussed (section 3). Third, the methods for quantitating the degree to which a small

7

molecule undergoes transport are reviewed (section 4).

2. Biology of small molecule the blood-brain

through the BBB

transport

through

barrier

2.1. Intraventricular drug infusion 2.1.1. Two barriers in brain: the blood-brain barrier and the blood-CSF

barrier

The BBB is situated at the brain capillary endothelial wall, which segregates blood from brain interstitial fluid (ISF) (Fig. 2). The bloodcerebrospinal fluid (CSF) barrier separates blood from CSF, and is situated at the choroid plexus epithelial barrier (Fig. 2). The capillaries perfusing the choroid plexus and other circumventricular organs (CVOs) have porous endothelial walls and circulating molecules readily distribute into the perivascular space of CVOs [l]. However, further transport into CSF is prevented by the tight junctions at the apical membrane of the choroid plexus epithelium. As depicted in Fig. 2,

SMALL MOLECULE BRAIN DRUG DISCOVERY 7 WATER-SOLUBLE MW > 600

LIPID-SOLUBLE MW < 600

TERMINATION 4 BRAIN DRUG DELIVERY TECHNOLOGY CLINICAL I_

DRUG DEVELOPMENT

J

Fig. 1. The evolution of small molecule drug discovery into clinical drug development may proceed along one of two pathways depending on the lipid solubility and the molecular weight (MW) of the drug. If the drug is lipid-soluble and has an MW <4OC-600 Da, then the drug may be transported through the BBB in pharmacologically significant amounts and may proceed directly from drug discovery to drug development. Alternatively, if the drug is water soluble or has a MW in excess of the 400-600 Dalton threshold. then the drug may not be transported through the BBB in pharmacologically significant amounts. In this situation, the drug development is either terminated, or undergoes an assimilation into a BBB drug delivery system. Exceptions include small, lipid-soluble molecules that do not penetrate the BBB because of avid plasma protein binding in viva, or water soluble molecules that penetrate the BBB via carrier-mediated transport.

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TWO BARRIERS IN BRAIN

10

t

AZT readily crosses chorold plexus and enters CSF

ARACHNOID

Fig. 2. (A) The two major extracellular compartments of bram arc the cercbrospinal fluid (CSF) and the brain interstitial fluid (ISF) and these two fluid compartments are segregated from blood by the choroid plexus or blood-CSF barrier and by the brain capillary or blood-brain barrier (BBB). respectively. Reproduced with permission from [ 1511.Although there is no anatomical barrier between CSF and ISF. there ix a functional barrier which arises out of the continuous bulk flow of CSF from the choroid plexus formation sites to the arachnoid villi absorption sites. The absorption of CSF back into systemic blood occurs completely in human brain every 3-5 h or 4-S times per day. Conversely. a small molecule with a diffusion coefficient of 6 x IO ” cm’is will diffuse S mm in It h. assuming no enzymatic metabolism or tissue sequestration of the molecule. Thus, bulk flow rates arc much faster than diffusion rates, which minimizes molecule penetration into the brain from the CSF flow tracks. (B) Rapid distribution of azidothymidine (AZT) into human CSF following oral administration. From [8]. These results arc consistent with rapid similar to that of the parent compound thymidinc [9), (C) transport of AZT through the choroid plexus in a manner Autoradiography in the rat showing no measurable transport of AZT through the BBB in viva. Reproduced with permission from [ 121. The parent compound. thymidine. is also not mcasurahly transported through the BBB [ 101.

there is no anatomical barrier separating CSF and ISF. However, there is a functional barrier that arises from the continuous bulk flow of CSF through the CSF flow tracks. CSF is produced at the choroid plexi of the lateral, third, and fourth ventricles and is absorbed into the superior sagittal sinus across the arachnoid villi [2]. CSF in the human brain is produced at a rate of approximately 21 ml/h [3]. Since the CSF volume in the human brain is approximately 100 ml. the entire CSF volume is turned over completely every 4-5 h or 4-5 times per day. The functional barrier limiting the diffusion of

small molecules from the CSF into brain parenchyma arises from the relatively slow rates of diffusion in comparison to the rapid rates of convection or bulk flow of CSF out of the brain and back to the systemic circulation (Fig. 2). For example, a small molecule such as glucose has a diffusion coefficient of 6 X 10mh cm’/s [4]. The effectiveness of diffusion decreases with the square of the distance, and it takes 11.7 h for a small molecule with this diffusion coefficient to diffuse 5 mm [4]. Additional factors including cellular uptake and cellular metabolism further limit the effective radius of diffusion of small

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molecules from the CSF to brain parenchyma. The uptake of small molecules by brain cells is a function of the lipid-solubility of the molecule, CSF protein binding of the drug, or brain cell binding of the drug. The limited diffusion of small molecules into brain parenchyma following intraventricular infusion is shown for the rhesus monkey in Fig. 3 [5]. The concentration of small molecule drugs in brain tissue is only l-2% of the CSF concentration in areas of brain parenchyma removed only 2-3 mm from the ependymal surface (Fig. 3). While intraventricular injection of small molecules may be a poor mode of drug delivery to brain parenchyma, this approach does allow for adequate drug distribution to the surface of the brain. Therefore, diseases with a predilection for the brain surface are amenable to treatment by intraventricular drug administration (Table 1). In

Table 1 Diseases of the surface tricular drug therapy

of the brain

amenable

to intraven-

Disease

Drug

Meningeal leukemia Bacterial meningitis Viral meningitis Chronic pain Spinal spasticity

Methotrexate Penicillin Immunoglobulin Morphine Baclofen

Taken

from

[4].

the case of treatment of chronic pain with intraspinal morphine (Table l), this approach is efficacious owing to the high concentration of opioid receptors on the surface of the spinal cord [6]. Similarly, GABA type B receptors, which are activated by baclofen for the treatment of spinal spasticity (Table l), are also abundant on the surface of the spinal cord [7].

2.1.2. Azidothymidine

(AZT) transport exemplifies the two barriers in brain

t

.OOll

t

I

_I

.2

CENTIMETERS

FROM

I

.3 THE

t

1

.4

.5

EPENDYMAL

I

SURFACE

Fig 3. Semi-log plot of the brain/CSF concentration gradient relative to the distance (mm) removed from the ependymal surface following the intraventricular infusion of four different drugs into the rhesus monkey ventricle at an infusion rate of 0.2 ml/min for a 60 min period. These studies show a steep concentration gradient whereby the drug concentration falls log orders in magnitude with each mm moved from the ependymal surface. Reproduced with permission from [5].

The differential permeability of the BBB and the blood-CSF barrier to small molecules is exemplified in the case of AZT transport in brain. AZT is a reverse transcriptase inhibitor and is used in treatment of acquired immune deficiency syndrome (AIDS). Since the brain is a sanctuary for the human immunodeficiency virus (HIV), it is desirable to deliver AZT to brain for the treatment of cerebral AIDS. Initial studies showed that AZT rapidly distributes into human CSF (Fig. 2). The rapid transport of AZT into CSF parallels the rapid CSF uptake of circulating thymidine, the parent pyrimidine nucleoside, owing to the presence of a thymidine transport system within the choroid plexus barrier [9]. However, thymidine is not transported through the BBB owing to the absence of a pyrimidine nucleoside carrier in the brain capillary endothelial membrane [lo]. Similarly, AZT is not transported through the BBB, as demonstrated with either the carotid injection technique [ll] or with autoradiography (Fig. 2). More recent studies are consistent with the slow transport of AZT across the BBB in proportion to its lipid solubility, followed by rapid efflux from brain ISF to blood owing to the presence within the

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BBB of an AZT efflux system [13]. These findings illustrate the general point that transport systems present within the choroid plexus may or may not be present within the BBB. That is, measurements of small molecule distribution in CSF reflect transport across the choroid plexus and not transport across the BBB. Small molecules, such as AZT, may readily distribute into CSF, but not cross the BBB and enter brain parenchyma, if there is differential expression within the BBB and the choroid plexus of particular transport systems. 2.1.3. Multivesicular liposome transport in CSF Dideoxycytidine (DDC) is another inhibitor of retroviral reverse transcriptase and is a potential treatment of cerebral AIDS, should this molecule enter into brain parenchyma from blood. Recent studies have shown that when DDC is injected into the ventricular compartment in the rat, this small molecule is removed from the CSF compartment with a half-time of 1 h [14], which approximates the half-time of CSF turnover in the rat [3]. Conversely, when DDC is packaged within 40 p multivesicular liposomes (MVL), the t; of elimination of DDC/MVL from CSF is increased to 23 h [14]. However, CSF elimination proceeds via bulk flow and is independent of molecular size [2]. Therefore, the prolongation of the CSF elimination t; of DDC when the molecule is packaged within the MVL suggests that the MVL is not freely removed via bulk flow across the arachnoid villi. CSF is transported across the arachnoid membrane via giant vacuolar transport and this one-way transport to blood is a form of macropinocytosis [2]. However, the 40 p MVL may be too large to be imbibed by this macropinocytosis process and this may explain the prolongation of the CSF elimination t 1 of DDC when packaged within the MVL. The effect of MVLs on CSF flow rateg, CSF volumes, and pressure has apparently not been measured. The prolongation of the CSF elimination t; of the DDCIMVL complex has not been shown to enhance DDC penetration into brain. Indeed, packaging the DDC within the MVL may reduce the effective diffusion coefficient of the molecule and lead to sequestration of the small molecule

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within the CSF flow track, perhaps in the neighborhood of the arachnoid villi exit pathway. 21.4. Equivalence of intra-ventricular and intravenous injections Lipid-soluble barbiturates have been infused into the cisterna magna of the dog ventricular compartment for up to 24 h and the dose for maintenance of anesthesia has been determined [15]. In the case of sodium amobarbital or sodium pentobarbital, the anesthesia maintenance dose is essentially identical when the barbiturate is administered by constant CSF infusion or by constant intravenous infusion. In this case, the barbiturate small molecules in the CSF compartment undergo absorption across the arachnoid villi and enter the systemic circulation, recirculate through the cerebral circulation and cross the BBB, and enter brain parenchyma to induce the pharmacologic effect. These studies reinforce the observations made by Fishman and Christy in 1965 that “an intrathecal injection of drug is equivalent to a slow intravenous injection” [16]. One of the misconceptions underlying small molecule drug administration via intraventricular injection is that drug is distributed only to brain and not to the systemic circulation. In fact, the drug is only distributed to the surface of the brain and is transported rapidly across the arachnoid villi and distributed into the systemic blood circulation [4]. 2.2. Lipid-mediated blood-brain barrier transport 2.2.1. The role of hydrogen bonding in limiting blood-brain barrier drug transport

The simplest way to make a preliminary determination as to whether a given small molecule drug is likely to undergo transport through the BBB is to inspect the molecular structure of the drug and calculate the total number of hydrogen bonds (N) formed between the functional groups on the drug molecule and solvent water. The rules for hydrogen bonding were reviewed by Stein [17] and are given in Table 2. The permeability of plant cell membranes for a variety of small molecule drugs may be accurately predicted from determination of the total hydrogen

W.M. Pardridge Table 2 Hvdrogen

bonding

Functional

OH

hydroxyl carboxylic acid primary amine primary amide secondary amide

group

Hydrogen 2 2 2 2 1

carboxylic amide aldehydes esters ethers nitriles

1 1 1 l/2 0 1

bonds

water. Since a hydroxyl group forms two hydrogen bonds with solvent water (Table 2) the addition of a single hydroxyl group to the parent compound reduces plant cell membrane permeability by approximately one log order of magnitude. Similar inverse linear relationships between membrane permeability and hydrogen bond numbers have been demonstrated for gall bladder epithelial cells [18], as well as for the BBB (Fig. 4). The data in Fig. 4 show that the permeability of the rat BBB in vivo is reduced approximately one log order of magnitude with the addition of each pair of hydrogen bonds added to a steroid hormone nucleus [19]. Virtually identical patterns have recently been demonstrated for peptide diffusion across the cell membrane of Caco-2 cells in tissue culture [20].

(N)

I

R -co-

-o-C=N From

acid

Stein [17].

bond number [17]. For example, a small molecule such as erythritol has a N = 8, whereas a small molecule such as butanol has a N = 2. The cell membrane permeability for erythritol is approximately three log orders of magnitude reduced in comparison to the cell membrane permeability for butanol. A general rule for small molecule transport across plant cell membranes is that cell membrane permeability is reduced one log order of magnitude with each pair of hydrogen bonds formed with solvent

2.2.2. Molecular weight threshold of blood-brain barrier transport of small molecules

In addition to calculating the hydrogen bond number (N) for a given small molecule, the lipid solubility of the molecule may be determined with a variety of polar lipids, such as 1-octanol [21]. The octanol/water or octanol/saline partition coefficient (P) is frequently used to predict the BBB transport of a small molecule. However,

B

‘q=O

OH

OH

A

-‘-

0@

-2T

?

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rules

Structure

-NH, -NH-N-

I Advanced

.EZ

d

-3-

O&HO&

Progesterone

(N =2 1

Testosterone

(N=3)

Estrodiol

(N-4)

.B -z

c~,Oti 0

_o

c~,Ot-i

c~,Oti

-4r=090 \. -5

o+@

oe@o

,HO&+H

-

0

2

4

N

6

6

Corticosterone(~=6)

Aldosterone

(N=7)

Cortisol

(N:8)

Fig. 4. (A) The log PM; for the steroid hormones shown in (B) is plotted versus the hydrogen bond number (N). PM! is the product of the BBB permeability coefficient (P, cm/s) X X’M for each steroid hormone. where M = molecular weight. The N value, or hydrogen bond number, is assigned according to the rules listed in Table 2. Reproduced from [19] by copyright permission of The Society for Clinical Investigation. P = progesterone; T = testosterone, Ez = estradiol; B = corticosterone; A = aldosterone; F = cortisol. (B) Molecular structures of steroid hormones emphasizing the polar functional groups (hydroxyl and carbonyl groups) that form hydrogen bonds with solvent water. The total number of hydrogen bonds (N) formed with solvent water are given in the parentheses. Reproduced with permission from [152].

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the use of the log P as a reliable index of BBB transport of lipid-soluble small molecules has been shown to be valid only when the molecular weight is less than a threshold of approximately 400-600 Da [22]. Initially, the BBB transport of a lipid-soluble peptide, cyclosporin, was found to be several log orders of magnitude lower than that predicted from the 1-octanol log P for this compound, which was approximately 3 [23]. Cyclosporin has a molecular weight of approximately 1200 Da. Subsequently, the BBB permeability for vinca alkaloids, vincristine or vinblastine, which have a molecular weight of approximately 800 Da was found to be disproportionately low in relation to the log P of these compounds [24]. These results support the earlier work of Levin [22], which showed that the BBB permeability of lipid-soluble small molecules is proportional to the log P of the compound providing the molecular weight of the molecule is less than a threshold of 400-600 Da (Fig. 5). The molecular basis for the molecular weight threshold of BBB lipid-mediated transport of small molecules is provided for in the model of Trauble (Fig. 5). The movement of lipid-soluble molecules through biological membranes is hypothesized to be limited by thermal fluctuations in the hydrocarbon chain of the membrane phospholipids [25]. The thermal motion of the phospholipid fatty acyl chains results in the formation of ‘kinks’, which create a mobile free volume within the hydrocarbon phase of the membrane. Constant motion of the fatty acyl ‘kinks’ allows for molecular ‘hitchhiking’ through the phospholipid bilayer. The kinks increase with the membrane content of unsaturated free fatty acid, and decrease with the membrane concentration of cholesterol [25]. Thus, lipid-mediated small molecule transport through the BBB may be a function of the phospholipid and cholesterol concentrations. The presence of the kinks or ‘holes’ in the phospholipid chain segments creates a kind of pore through which small molecules may traverse. The size of the pore may define the molecular weight threshold [4]. The presence of a molecular weight threshold for lipid-soluble small molecule transport through the BBB (Fig. 5) is an important factor

A

I#

lo-’

lOCTANOL/BUFFER

Id* PARTITION

100

102

COEFFICIENT1

104 hlW“‘a

Fig 5. (A) The BBB permeability coefficient (v-axis) is plotted versus the octanol/buffer partition coefficient, normalized for ~/molecular weight (MW) on the x-axis for 27 different drugs. Drugs l-23 have molecular weights <400 Dalton threshold. The molecular weights for drugs 24 (adriamycin. 543 Da), 25 (epipodophylotoxin, 657 Da), 26 (vincristine, 825 Da) and 27 (bleomycin, 1400 Da) exceed the 400 Da threshold and the BBB permeability coefficient for these drugs is l-2 log orders less than that predicted from the lipid solubility of the compound. Reproduced from Levin [22] by permission (Copyright American Chemical Society, 1980). (B) Kinks in membrane phospholipid bilayer are formed by rotations about the carbon-carbon bond. These kinks form pores in the membrane phospholipid bilayer. The formation of a pore of finite size within the BBB membrane may explain the molecular weight threshold demonstrated in (A). That is. molecules with molecular weights above the 400-600 Da threshold may have molecular sizes in excess of the diameter of the ‘pore‘ in the BBB lipid membrane. Model (with permission) from Trauble [25].

that is not widely recognized in CNS drug discovery programs. Many CNS drug discovery programs are predicated on the discovery of ‘small molecules’ that will undergo transport through the BBB without the use of brain drug delivery systems. However, in order for such small molecules to actually undergo significant transport

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through the BBB, the molecules must be both (a) lipid-soluble, and (b) have a molecular weight less than the threshold of 400-600 Da. Indeed, virtually all of the CNS drugs that are currently in clinical practice fulfill these two criteria. CNS drug discovery programs that generate small molecule lead compounds with molecular weights of 400-600 Da or higher may not undergo transport through the BBB in pharmacologically significant amounts in the absence of a brain drug delivery system.

2.2.3. Plasma protein binding of small molecules The prediction of BBB transport of small molecule drugs on the basis of either measurements of lipid-solubility or hydrogen bond calculations assumes that there is no significant plasma protein binding of the drug. Plasma protein binding is generally measured with such in vitro techniques as equilibrium dialysis or centrifugal filtration. Although it is often assumed that plasma protein-bound drug is not available for transport through the BBB, studies in the past have shown that many plasma protein-bound drugs are available for transport through the brain capillary endothelial wall [26]. Since plasma proteins, per se, undergo negligible transcapillary transport in brain, the transport of drugs through the BBB from the plasma proteinbound pool represents a mechanism of enhanced dissociation of the drug from the plasma protein binding site within the brain microcirculation in vivo [26]. That is, the dissociation constant (K,) governing the plasma protein/ligand binding reaction in vitro is much lower than the K, observed in vivo within the brain microcirculation. Differences in K, are generally ascribed to differences in dissociation rates [27], and enhanced dissociation of ligand from the plasma protein bound pool in vivo may occur subsequent to conformational changes about the plasma protein ligand binding site [26]. Such conformation changes may be induced by non-specific adsorption of plasma proteins such as albumin or globulins to surfaces such as the glycocalyx of the brain capillary endothelium [28]. Alternatively,

dialyzable small molecules released into microcirculation or local pH effects have been implicated in cell surface catalyzed conformational changes and enhanced dissociation of drugs from albumin binding sites [29,30]. A comparison of the K, in vitro versus the K, in vivo in the brain capillary for a number of albumin-bound ligands is shown in Table 3. In addition, a number of CNS active drugs are lipophilic amines and these compounds avidly bind to a plasma globulin called c~,-acid glycoprotein, which is also referred to as orosomucoid [31]. In the case of propranolol, albumin-bound drug is not available for transport through the BBB, whereas orosomucoid-bound propranolol is available for transport into brain [26]. Al-

Table 3 Comparison of plasma protein binding in vivo within the brain microvasculature Plasma

protein

Drug

K,ln

of drugs

(CLW

In vitro Bovine

albumin

hAAG

Human

albumin

in vitro and

Propanolol Bupivacaine Imipramine

290 5 30 141 k 10 221 IT 21

Propanolol Bupivacaine

3.1 t 0.1 6.5 t 0.5

L-663,581 L-364,718 Diazepam

125 2 16 8.2 t 0.8 6.3 2 0.1

In vivo 220 5 40 2112 107 1675 2 600 1924 17t4 675 -c 18 266 -c 38 157k36

hVLDL

Cyclosporin

1.9 2 0.5

1.8 k 0.4”

hLDL

Cyclosporin

0.81 t 0.08

1.6 2 0.4”

hHDL

Cyclosporin

0.45 2 0.10

0.44 -t 0.11”

HSA

Isradipine Darodipine

63 2 8 94 2 5

221 2 7 203 z 14

hAAG

Isradipine Darodipine Imipramine

6.9 2 0.9 2.5 2 0.5 4.9 5 0.3

35 + 2 55 2 7 9029

hAAG, human a, acid glycoprotein; hVLDL, human very low density lipoprotein; hLDL, human low density lipoprotein; hHDL, human high density lipoprotein; HSA, human serum albumin. a Units are g/l. From [4].

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though albumin-bound propranolol is not available for transport across the BBB, other albumin-bound ligands are available for transport into brain from the circulating albumin-bound pool (Table 3). The observation that some albumin-bound ligands undergo enhanced dissociation in vivo within the brain microcirculation, whereas other ligands such as propranolol do not, is consistent with the finding that there are at least 6 major binding domains on albumin for small molecule ligands [32]. Thus, albumin interactions with the brain capillary endothelial surface may induce conformational changes about some albumin binding sites, but not others. The extent to which albumin or globulinbound ligands are available for transport through the BBB in vivo generally cannot be predicted from in vitro measurements of plasma protein binding, such as equilibrium dialysis. Rather, in vivo techniques, such as the carotid injection method, must be used in order to determine the extent to which albumin-bound or globulinbound ligands are available for transport through the BBB. The use of the carotid injection technique to measure in vivo plasma protein binding effects has been reviewed recently [33]. A practical example of the advantage of measuring plasma protein binding effects in vivo is the case of tryptophol, which is 3-indole-ethano1 and is a compound that induces sleep and is formed in hepatic encephalopathy after disulfiram or in the case of trypanosomal sleeping sickness [34]. Tryptophol is highly lipid-soluble and has an octanol/saline partition coefficient of 30. The compound is nearly 100% extracted by brain on a single capillary passage. Like its parent compound, tryptophan, tryptophol is bound by albumin and more than 95% of the drug is albumin-bound in vitro [34]. However, albumin-bound tryptophol is freely available for transport through the BBB in vivo owing to enhanced dissociation from the albumin binding site [34]. Thus, if tryptophol was being developed as a small molecule candidate, then in vitro plasma protein binding measurements would predict negligible transport of the compound through the BBB in vivo. In fact, tryptophol is readily taken up by brain from the circulating albumin-bound pool.

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2.3. Carrier-mediated

transport of nutrients or drugs through the blood-brain barrier 23.1.

Nutrient transport

Circulating nutrients gain access to brain via carrier-mediated transport through the brain capillary endothelial lumenal and ablumenal membranes [35]. At least eight different nutrient transport systems have been identified (Fig. 6). Each of the nutrient carriers depicted in Fig. 6 transports a group of different nutrients of the same structure. For example, the glucose carrier also transports 2-deoxyglucose analogues, 3-0methyl glucose, galactose, mannose, and other glucose analogues. The carrier is inhibited by cytochalasin B or phloretin [36], although it is not clear if the carrier actually transports these

BLOOD-BRAIN BARRIER NUTRIENT TRANSPORT SYSTEMS

Fig. 6. Eight different nutrient transport systems localized on the lumenal membrane of the brain capillary endothelium. The eight different carriers each transport classes of nutrients and a representative metabolic substrate for each system is Thown in the figure. Reproduced with permission from [153].

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two compounds through the BBB. Phloretin has been used to induce brain glucopenia in the face of a normal plasma glucose via inhibition of the BBB glucose carrier [37]. The neutral amino acid carrier at the BBB is analogous to the leucine (L)-preferring system and transports approximately 15 large and small neutral amino acids and has the highest affinity for phenylalanine [38]. The basic amino acid carrier transports ornithine, arginine, and lysine [38]. The monocarboxylic acid carrier transports pyruvate, lactate [39], and the ketone bodies, P-hydroxy butyrate and acetoacetate [40]. The purine nucleoside carrier transports adenosine and other nucleosides, including uridine, a pyrimidine nucleoside [lo]. The purine base carrier transports adenine and other purine bases, but not pyrimidine bases [lo]. The choline carrier transports choline and perhaps other endogenous compounds with a quaternary ammonium group [41]. The glutamate carrier may be an active efflux system for amino acid excitatory neurotransmitters, glutamate and aspartate [42]. Neutral amino acid drugs

The neutral amino acid carrier transports neutral amino acid-type drugs called pseudonutrients, and these includge a-methyldihydroxyphenylalanine (DOPA) [43], L-DOPA [44], (Ymethylparatyrosine [45], and phenylalanine mustards, including melphalan [46] and melphalan analogues [47]. A recently approved anticonvulsant, gabapentin, is not strictly a neutral amino acid, but is structurally related to large neutral amino acids and may be transported through the BBB via this transport system [48]. L-DOPA is used to augment brain dopamine levels, since dopamine, a catecholamine, undergoes no pharmacologically significant transport through the BBB. Subsequent to its transport through the BBB, L-DOPA is decarboxylated in brain to dopamine by aromatic amino acid decarboxylase [44]. The dopamine/t_-DOPA paradigm is a model strategy for BBB delivery of small molecules. For example, a small molecule candidate may be a catecholamine-type drug that undergoes negligible transport through the BBB. Rather than using lipidization drug delivery strategies (reviewed below), it may be easier to

15

synthesize the a-amino acid analogue of the catecholamine. The latter compound may undergo transport through the BBB via carrier-mediated transport on the neutral amino acid carrier and then be subsequently decarboxylated in brain enzymatically. Neutral amino acid-type drugs compete with circulating endogenous amino acids for BBB transporter sites. Therefore, when hyper-aminoacidemia is induced, such as after a high protein meal, there is a greater saturation of BBB neutral amino acid transporter sites and less drug will be available to brain. Conversely, in hypoaminoacidemia, such as after carbohydrate-induced hyperinsulinemia, there is less saturation of BBB neutral amino acid transport sites and greater delivery to brain of neutral amino acidtype drugs [43,49]. 2.3.2. Blood-brain barrier transport of amines and monocarboxylic acids Lipophilic amine compounds, such as amphetamine, methylphenidate, propranolol, lidocaine, or diphenhydramine, have a component of BBB transport that is saturable by relatively high concentrations (5-50 mM) of drug (50-52). The amine transport is pH dependent and is inhibited by acid pH [50,51]. The saturability of BBB transport of lipophilic amines is somewhat unexpected and there is no known endogenous ligand for the putative transport system. It is conceivable that these compounds undergo lipidmediated transport through the BBB and the saturability arises from the pores that are the normal conduits for lipid-mediated transport through the endothelial membrane (Fig. 5). For example, the concentration of ‘kinks’ in the fatty acyl membrane is estimated to be on the order of lo-50 mM [25]. This concentration, in fact, approximates the apparent K,,, of lipophilic amine transport through the BBB (49-52). A variety of monocarboxylic acid drugs may have a weak affinity for the monocarboxylic acid or lactate carrier within the BBB (Fig. 6). For example, previous studies have shown that probenecid inhibits the BBB transport of lactate, butyrate, propionate, and pyruvate [53]. Other investigations have demonstrated that a nonmetabolizable p-lactam antibiotic, cefodizime,

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has a BBB permeability-surface area (PS) product of 3.1 pl/min/g and that the co-administration of high concentrations of penicillin markedly reduce the BBB PS product for this drug [54]. However, it is possible that the slow influx of penicillin-like drugs through the BBB is counterbalanced by the efflux of these drugs from brain back to blood, which may be a probenecid-sensitive acid efflux system. Evidence supporting the existence of a BBB acid-efflux system at the BBB was reported for dipropylacetate or valproate, wherein it was shown that the BBB PS product in the blood-to-brain direction was considerably reduced compared to the BBB PS product for the drug in the brain-to-blood direction [55].

2.3.3. Enzymatic blood-brain mechanisms

barrier

The brain microvasculature, like other epithelial barrier systems, contains a variety of enzymes that may either activate or inactivate drugs that traverse the capillary wall in brain [56]. Some examples of BBB enzymatic mechanisms are listed in Table 4. Microvascular enzymes may inactivate pharmacologically active substances that traverse the endothelial wall. This enzymatic inactivation prevents the entry into the brain interstitial fluid of the pharmacologically active compound. For example, microvascular monoamine oxidase (MAO)-type B inactivates catecholamine substances that traverse the BBB [56]. Similarly. brain microvascular glutamyl aminopeptidase inactivates angiotensin II by converting it to angiotensin III

Table 4 Blood-brain

barrier

enzymatic

[57]. Brain microvascular aminopeptidase M inactivates enkephalins that are locally released at the microvasculature by releasing the aminoterminal tyrosine [58]. y-glutamyl transpeptidase ( y-GTP) inactivates leukotriene (LT) C4 by converting it to LTD4 [59]. Certain BBB enzymatic mechanisms are actually localized in the microvascular pericyte, and not the brain microvascular endothelium. The pericyte shares the basement membrane with the capillary endothelium and is believed to have a contractile [60], phagocytic [61], or antigen presentation function [62]. One model of pericyte function is that this cell is a smooth muscle analogue of the microcirculation. However, recent studies show that brain microvascular pericytes, under normal conditions, lack the cyactin isoform, typical of contractile cells [63,64]. Nevertheless, pericytes are believed to have an important enzymatic function and indeed, certain enzymatic mechanisms such as glutamyl aminopeptidase [57] and aminopeptidase M [65] are localized to the pericyte. Even y-glutamyl transpeptidase is expressed both in brain microvascular endothelium [66] and in pericytes [67]. There may be species differences as to whether certain enzymatic mechanisms at the BBB are localized in pericytes versus the endothelium. For example, butyrylcholinesterase is localized in pericytes in canine brain capillaries but is an endothelial enzyme in rat capillaries [68]. brain also called pseudochButyrylcholinesterase, olinesterase, deacetylates heroin, which results in the formation of morphine. Similarly, brain microvessels contain cytochrome P450 enzymes, which 0-demethylate codeine to form morphine

mechanisms

Enzyme

Function

Aromatic amino acid decarboxylase Monoamine oxidase, type B Cytochrome P450 Pseudocholinesterase y-glutamyltranspeptidase Glutamyl aminopeptidase Aminopeptidase M

Covert I.-DOPA to dopamine Inactivate catecholamines 0.Demethylate codeine to form morphine Deacetylate heroin to form morphine Convert leukotriene C4 to leukotriene D4 Convert angiotensin II to angiotensin III Inactivate opioid peptides

From

[56-59,

681.

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[.56]. In the case of these enzymatic mechanisms, the drug is activated via passage through the BBB enzymatic mechanism. 2.3.4. Enzymatic barrier to adenosine Adenosine is a potential neuropharmaceutical that is not effectively transported to brain from blood in vivo. Adenosine is a cerebral vasodilator when applied topically to pial vessels [69], yet the intracarotid infusion of adenosine fails to increase cerebral blood flow in most species [70]. Adenosine analogues are also potential sedatives or anticonvulsants [71]. The inability to achieve pharmacologic effects in brain following the systemic administration of adenosine is somewhat anomalous since this molecule is a ligand for the BBB adenosine transporter (Fig. 6). The presence of a BBB transport system for adenosine, yet the failure to achieve CNS pharmacologic effects with systemic adenosine, suggests there is an enzymatic barrier within brain for this nutrient. This enzymatic barrier has been confirmed recently with internal carotid artery perfusion experiments showing that 90% of adenosine taken up by brain during a 15 s internal carotid artery perfusion is converted to adenosine metabolites [72]. These recent in vivo studies confirm earlier in vitro results showing that 90% of adenosine is metabolized at 37°C within 10 min in isolated bovine brain capillaries [73], yet is metabolically stable in isolated brain synaptosomes [74]. The enzymatic blood-brain barrier to circulating adenosine may be anatomically situated in either the endothelial cell, the pericyte, or the astrocyte foot process. The endothelial cell and pericyte share a common capillary basement membrane and the astrocyte foot processes invest more than 95% of this basement membrane. Adenosine deaminase, which converts adenosine to inosine, is localized to astrocyte foot processes with minimal localization to capillary endothelial cells [75]. Therefore, it is possible that adenosine is transported through the brain capillary endothelium and is then metabolized at the level of the astrocyte foot process. Such an enzymatic BBB may prevent further distribution of circulating adenosine to receptors on either cerebrovascular smooth muscle cells or neurons [72].

2.3.5. Is P-glycoprotein an active efjkx at the brain capillary endothelium ?

system

P-glycoprotein is the product of the multi-drug resistance (mdr) gene, is a 170 kDa membrane protein [76] and is detectable immunocytochemitally in human brain sections at the microvasculature [77]. P-glycoprotein is also found in isolated animal or human brain capillaries using either immunocytochemistry or Western blotting techniques [4,78]. P-glycoprotein binds a variety of anticancer drugs [79-811 including vinca-alkaloids, actinomycin D, daunomycin, taxol, prazosine, verapamil, nifedipine, progesterone, or cyclosporin [4]. The expression of P-glycoprotein is believed to confer multi-drug resistance on cells by serving as a drug active efflux system, which minimizes the cellular uptake of the drug. The expression of P-glycoprotein is markedly down-regulated in primary cultures of brain capillary endothelial cells [4]. Residual P-glycoprotein activity in cultures of brain capillary endothelium may function as a drug active efflux system in tissue culture [82,83]. However, the finding of residual P-glycoprotein activity in cultured brain capillary endothelium does not make certain that P-glycoprotein is an important active efflux system at the brain capillary endothelium in vivo. Indeed, careful examination of the immunocytochemical detection of P-glycoprotein in human brain capillaries [78] reveals a discontinuous immunostaining pattern characteristic of either a pericyte and/or astrocyte foot process origin of the brain microvascular P-glycoprotein immunoreactivity. The discontinuous immunostaining of isolated brain microvessels obtained when the microvascular antigen is of pericyte origin has been demonstrated previously [62], and is to be contrasted with the continuous immunostaining characteristic of endothelial antigens [4]. The expression of P-glycoprotein in brain cells such as astrocytes may prevent the cytoplasmic accumulation of drugs in brain. Under such conditions, an inhibition of brain P-glycoprotein would be reflected by an increased steady state brain volume of distribution (V,), but not an increase in the BBB PS product (on the brain side of the BBB). The latter would arise from an inhibition of a brain endothelial P-glycoprotein

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active efflux system. A reduction in brain V,, is shown by studies using transgenic ‘knockout’ mice lacking the mdrla isoform of P-glycoprotein [84]. In these animals, brain concentration of drugs that are substrates for P-glycoprotein are increased relative to the control animals. These brain V,, values are steady state values that are complex functions of BBB transport, brain cell metabolism, and brain cytoplasmic binding of drugs (see section 4.2.6). What is needed is a formal quantitation of the BBB PS product (in the brain-to-blood direction) for drugs that are putative substrates for P-glycoprotein in both the control and ‘knockout’ mice. Such studies would differentiate whether P-glycoprotein functions to increase drug efflux across the BBB or to reduce the brain V, of the drug via inhibition of drug access to brain cell cytoplasmic binding systems.

3. Pharmacologically brain barrier delivery

based strategies for bloodof small molecule drugs

3.1. Overview of blood-brain delivery strategies

barrier drug

Blood-brain barrier drug delivery strategies may be broadly classified as to whether these are invasive, physiologically based, or pharmacologically based strategies [4], and these are briefly reviewed in the Preface to this issue. Invasive or neurosurgical-based strategies include intraventricular drug infusion, cerebral implants, or blood-brain barrier disruption. The advantages and limitations of intraventricular drug infusion are reviewed in section 2.1. A variant of intraventricular drug infusion is the use or intracerebral implants or peri-ventricular implant of cells genetically expressed to secrete therapeutic molecules. The release of drugs from periventricular cell implants and diffusion into brain will be subject to the same factors as those limiting brain penetration of drugs administered by intraventricular infusion devices. Intracerebral implants may also alter BBB permeability and represent a local form of BBB disruption. For example, a recent study shows that the periventricular implant of an adrenal medulla graft

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results in a local increase in dopamine secondary to opening of the BBB in the region surrounding the implant [85]. BBB disruption using either osmotic or biochemical means are reviewed in the next article (K.L. Black) of this volume. Physiologically based strategies for delivery of small molecules through the BBB include the use of pseudonutrients that are substrates for BBB nutrient carrier systems and these mechanisms have been reviewed in section 2.3. Pharmacologically based strategies for BBB delivery of small molecules include lipidization approaches and liposomes.

3.2. Lipidization strategies 1.2.1. 0-methylation

or 0-acetylation

Lipidization of a small molecule involves chemical modification of the drug that substitutes one of the hydrogen bond-forming polar functional groups listed in Table 2 with a apolar functional group that forms no hydrogen bonds with solvent water. A variety of different approaches for lipidizing small molecules has been developed and some of these are listed in Table 5. The classical approach of lipidization involves either 0-methylation of morphine to form codeine or 0-acetylation of morphine to form heroin (Table 5). As reviewed in section 2.2.1.. blood-brain barrier permeability to a small molecule increases by a log order of magnitude with the removal of each pair of hydrogen bonds from the parent compound. Therefore, 0-methylation of morphine, to form codeine, results in the removal of two hydrogen bonds and increases BBB permeability tenfold [86]. The double Oacetylation of morphine. to form heroin, increases BBB permeability by approximately l(M)fold for the parent morphine compound [86]. The ideal lipidization strategy is reversible, and once in the brain, the molecule is enzymatically converted back to the parent compound. For example, codeine or heroin is converted in the brain to morphine (Table 5). The efflux of morphine from brain to blood is as slow as the influx from blood to brain. Therefore, the enzymatic conversion of the prodrug to the drug helps trap the drug in the brain.

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Table 5 Lipidization strategies Drug

Lipid moiety

Reference

Enkephalin AZT AZT Enkephalin

Amantadine Adamantane Phosphatidtylation 1,4-Dihydrotrigonellinate Cholesterol ester Laurie acid Fatty acid or cholesterol ester Dihydropyridine N-acylation O-methylation O-acetylation

87 90 147 93 93 148 144 149 1.50 86 86

TRH GABA Estradiol TRH Codeine Heroin

3.2.2. Amantadine

and adamantane derivatives

of 1-amino-adamantane The conjugation (amantadine) to the carboxy-terminal of [DAla2]leucine-enkephalin has been shown to result in pharmacologic activity of the opioid peptide following systemic administration, presumably by increasing BBB transport of the peptide [87]. In this study, the [D-Ala21 analogue was used to block inactivation of the enkephalin by brain microvascular aminopeptidase M activity. The opioid peptide was not pharmacologically active when the amantadine moiety was conjugated to the amino-terminus, consistent with the need for a free amino terminus to preserve opioid peptide activity [88]. Conjugation of the amantadine moiety to the carboxy terminal of [D-Ala’lleucine-enkephalin increased the log P of this compound from -0.22 to 2.12 [87]. Amantadine, and a closely related compound, rimantadine, are primary amine derivatives of adamantane, which is tricyclodecane. The transport of amantadine and rimantadine through the BBB in vivo has been measured, and the BBB PS product for rimantadine, 5.1 ml/min/g, is 8-fold greater than the BBB PS product for amantadine [89]. Therefore, rimantadine may be a preferred lipidization vehicle as compared to amantadine. Adamantane has been used as a lipidization moiety to enhance the lipid-solubility of AZT [90]. The tricyclodecane moiety was placed at the 5’-hydroxyl position of the AZT via an ester linkage. In these experiments, a series of

19

adamantane 5’-esters of AZT were prepared and one form was found to have a slower rate of enzymatic hydrolysis of the ester bond in rat plasma in vitro. The brain delivery of the adamantane/AZT conjugate was considerably increased over the brain delivery of AZT alone; in this case, brain delivery is measured as a percent of injected dose delivered per gram brain (%ID/g). (See next section for discussion of %ID/g and pharmacokinetic parameters). The increased brain delivery of the AZT/adamantane conjugate was observed despite the much lower plasma area under the concentration curve (AUC) of the AZT/adamantane moiety, as compared to AZT alone [90]. The reduced plasma AUC is consistent with the increased lipid-solubility of the AZT/adamantane conjugate, as compared to AZT alone. Compounds of increased lipid-solubility will penetrate all organs to a greater extent and have reduced mean plasma concentrations. As reviewed in the next section on pharmacokinetics, the brain delivery (expressed as %ID/g) is a function of both the BBB PS product, which is proportional to the lipid-solubility of the compound, and the plasma AUC, which is generally inversely related to the plasma AUC. That is, lipidization increases the BBB PS product and decreases the plasma AUC and this may have offsetting effects in terms of brain delivery of a compound. This was found in the study of AZT delivery to brain [90], where the %ID/g of AZT, following release by hydrolysis in brain of the AZT/adamantane conjugate, was not much higher than the brain delivery of AZT alone. Thus, it would appear that the AZT/adamantane moiety may have effluxed from brain back to blood faster than the release of the AZT due to prodrug hydrolysis in brain.

3.2.3. Fatty acid or cholesterol esters Another lipidization strategy involves the attachment of free fatty acyl or cholesterol groups and the formation of either fatty acid or cholesterol esters. While such compounds may undergo enhanced transport through the BBB following intravenous injection, a similar increase in brain uptake may not ensue following oral administration. This is because fatty acyl or cholesterol

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esters are incorporated into lipoproteins following oral absorption. The BBB transport of lipoprotein-bound cholesterol is log orders lower than the BBB transport of free cholesterol [91]. The intravenous injection of cholesterol does not result in rapid incorporation of cholesterol into lipoproteins [92]. Indeed, in order to incorporate cholesterol into serum lipoproteins in vitro, it is necessary to disperse the cholesterol on a celite surface and incubate human serum overnight at 37°C [91]. Therefore, incorporation of orally administered free fatty acid esters or cholesterol esters into lipoproteins is an extreme case of plasma protein binding of small molecules and exemplifies the case where plasma protein (lipoprotein) binding may reduce BBB transport of the drug. 3.2.4. Dihydrotrigonellinate derivatives Cholesterol esters have also been used to render the carboxy terminus of a hexapeptide less polar [93]. The [Ala”]leucine-enkephalin peptide was lipidized by the amidation of the amino terminus with a 1,4-dihydrotrigonellinate moiety. The combination of blocking the carboxy terminus with the cholesterol ester and the conversion of the amino terminus to the 1.4dihydrotrigonellinate amide increased the lipidsolubility of the leucine-enkephalin analogue. However, the molecular weight of the lipidized enkephalin analogue (1128 Da) exceeds the molecular weight threshold of 400-600 Da for lipid-mediated transport through the BBB (Fig. 5). Therefore, on the basis of the high molecular weight (1128 Da), it is not clear if significant BBB transport of the lipidized enkephalin analogue actually takes place. Unfortunately, no quantitative measurements of BBB PS products were reported. Lipidization is expected to increase uptake by peripheral tissues, which results in a decreased plasma AUC. A reduced plasma AUC was apparently observed in the case of the lipidized enkephalin analogue, since no detectable trigonellinate was found in blood collected only 5 min after an intravenous injection, A final consideration is that in order to solubilize the trigonellinate / enkephalin / cholesterol conjugate, it was necessary to dissolve the compound in 25% dimethylsulfoxide and 50% ethanol, which

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resulted in the administration of approximately 1 g/kg of these two solvents to rats. At this dose, either DMSO or ethanol is capable of causing BBB disruption via solvent-mediated dissolution of the membrane [94,95]. Nevertheless, the use of the 1.4 dihydrotrigonellinate moiety to lipidize the drug is a novel approach to brain drug delivery that includes a strategy to sequester the drug in brain. Brain dehydrogenases oxidize the dihydrotrigonellinate moiety to quaternary ammonium group that prevents efflux of drug from brain back to blood [93]. 3.2.X Reverse lipidization Sometimes it may be desirable to inhibit the BBB transport of a compound so that CNS side effects of a drug are minimized while still maintaining the peripheral uptake of the drug. The rules for reverse-lipidization are the inverse for increasing the lipid solubility of a compound and include the following: (a) addition of free amino or carboxyl groups, (b) addition of quaternary ammonium group, and/or (c) increase in polar or hydrogen-bond forming functional groups on the molecule. One example of reverse-lipidization is the demonstration that the BBB transport of nicotine is abolished by N-methyl quaternization of the compound [96]. 3.3. Liposomes Based on the molecular weight threshold of BBB lipid-mediated transport (Fig. 5) it might be expected that liposomes, even small unilamellar vesicles (SUV) on the order of 50 nm, do not undergo significant BBB transport. Several studies confirm that liposomes, even SUVs, do not undergo transport through the BBB (97-99). One study shows that multi-lamellar vesicles (MLV) on the order of 0.3-2 ,u in diameter accumulate in brain, but this accumulation is a toxic sequelae subsequent to MVL-induced embolism in brain vessels [98]. In another study of BBB transport, 60 nm liposomes radiolabeled with “lIndium do not undergo measurable transport through the BBB of normal brain but can be induced to undergo transport following pharmacologic BBB disruption associated with the intracarotid infusion of high doses (25 mg/kg) of

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etoposide [97]. Another recent study reports that liposomes bearing negatively charged sulfatide moieties undergo transport through the BBB in mice [loo]. However, this study was not confirmed as the BBB transport of sulfatide-bearing liposomes in rats was found to be negligible [99].

4. Methodologies

for quantifying blood-brain of small molecules

barrier transport

4.1. The pharmacokinetic

rule

The brain delivery of a given drug or solute may be quantified by measuring the percent of injected dose (ID) delivered per gram brain following systemic administration and this parameter is expressed as %ID/g. The %ID/g is a direct function of two other parameters expressed as the pharmacokinetic rule: %ID/g ; = PS

x

AUC ;

0

(1)

0

where PS = the BBB permeability-surface area product and AUCI; = the plasma area under the concentration curve at a given time (t) after injection. Unless all three parameters (%ID/g, PS product, and plasma AUC) are measured, it is possible, indeed likely, that artifactual estimates are made pertaining to whether a given molecule undergoes enhanced transport through the BBB. Therefore, the following review of methodologies for quantifying BBB transport focuses on the approaches available for measuring either the BBB PS product and/or the plasma AUC. Then, a series of experimental artifacts will be reviewed in order to demonstrate the importance of measuring all three parameters for effective quantification of BBB solute transport. 4.2. In vivo measurements barrier PS product

of blood-brain

4.2.1. Introduction

There are three principle methodologies for quantifying the BBB PS product in anesthetized or conscious laboratory animals. These methods are the single carotid injection technique, the

21

internal carotid artery perfusion technique, and the intravenous injection technique [loll. The single carotid injection technique is useful when the BBB PS product of the compound exceeds 10 pllmin I g. The internal carotid artery perfusion technique is used when the BBB PS product is in excess of 0.5 pllminlg. The intravenous injection technique may be used for virtually all compounds, even those with PS products less than 0.5 pl/min/g. The principle caveat with respect to using the intravenous injection technique is that if the compound rapidly metabolized, then brain transport measurements are subject to metabolisminduced artifacts. 4.2.2. Carotid artery single injection technique The carotid artery single injection technique may either involve tissue sampling in the case of the brain uptake index (BUI) technique [102] or venous sampling in the case of the multiple indicator dilution (MID) technique [103]. In the BUI method, a diffusible indicator reference is used and in the MID technique, a non-diffusible reference indicator is used. Because tissue sampling is involved, the BUI technique is amenable to a regional analysis of BBB transport, whereas this is not possible with the MID technique. The MID technique was originally developed for a large experimental animal, such as the dog [103], but was subsequently adapted to small laboratory animals such as rats [104]. The BUI technique may be used in virtually all laboratory animals, including mice or neonatal animals [105]. In the BUI technique, as applied to adult rats, an approximate 0.2 ml bolus of buffered Ringer’s solution containing a “H-labeled test compound and a 14C-reference compound (e.g., “C-butanol) or a ‘“C-test compound and a ‘H-reference compound (e.g., “H-water or ‘H-diazepam) is rapidly injected ( < 0.5 s) into the common carotid artery of the anesthetized or conscious animal. The bolus passes through the brain within 2 s after the single injection and the animal may be decapitated 5-15 s after injection. There is minimal eftlux of the test or reference compound from brain during this short experimental time period [106]. Following decapitation, the brain is solubilized and the brain and injection solution are counted for ‘H and 14C

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radioactivity lows:

and

the

BUI

I

is calculated

Advanced

Drug

as fol-

[‘Hldpm BUI =

[‘4C]dpm [“H]dpm [ 14C]dpm

=2x 100 r

(brain)

x IO0

(injectate)

(2)

where E, = the unidirectional extraction of the “H-test compound and E, = the extraction of the ‘“C-reference compound. Methods for measuring the E, have been described previously [106]. Given values for the E,, the BUI may be converted into E, values. Given measurements of cerebral blood flow (F), which have been described previously [106], E, values may be converted into BBB PS products using the KetyRenkin-Crone equation of capillary physiology (107-109):

where f = the fraction of plasma exchangeable drug, also called the bioavailable drug fraction [llO]: ,f = 1 for drugs that are not plasma protein bound and f < 1 for drugs that undergo significant plasma protein binding within the brain microcirculation in vivo (see section 2.2.3). Owing to the rapid injection, mixing of the injection bolus with circulating rat plasma is on the order of 2-5%. This mixing may be incorporated in the quantitative analysis in certain circumstances, such as related to high affinity amino acid transport [ Ill]. The advantages of the BUI technique is that it is fast and many different compounds under many different physiologic conditions can be evaluated in a relatively short period of time. The disadvantage of the technique is that brain extraction is measured over a time period as short as the single 1 s capillary transit time in brain. Therefore. it is difficult to measure BBB PS products less than approximately 10 pl/min/ g. A fully quantitative treatment of the BUl technique requires independent measurements of both E, and F. Failure to obtain these additional measurements may lead to the recording of experimental artifacts (see next section).

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4.2.3. Internal carotid artery infusion technique Extension of the BUI technique to a longer experimental time period involves internal carotid artery perfusion of brain and tissue sampling of radioactivity. Carotid artery infusion techniques have been described for both the rat [112] and guinea pig [113]. When the internal carotid artery infusion technique is applied to rats. the anesthetized animal is cannulated at the external carotid artery, and the carotid artery tributaries (the superior thyroidal artery, the occipital artery, and the pterygopalatine artery) are electrocoagulated. The ipsilateral common carotid artery is then ligated, and the internal carotid artery perfusion is initiated at a rate of approximately 3.5-4 ml/min for 0.25-l min [ 1121. A perfusion flow rate of 3.5-4 ml/min induces an arterial pressure equal to the ambient systolic pressure and this prevents mixing of the perfusate with circulating rat plasma within the cerebral circulation [ 1121. Following decapitation, the radiolabeled test compound and radiolabeled reference compound (e.g., sucrose for measurement of brain plasma volume or diazepam for measurement of brain plasma flow) is measured and the BBB PS product is quantitated as follows:

where t(min) = the length of the perfusion period, V, = [dpm/g (brain)] + [dpml,ul (perfusate)] for the test compound, and V, = V,, for a plasma volume compound, e.g. sucrose or albumin. The internal carotid artery perfusion technique is more sensitive than the BUI technique because the experimental time period is prolonged from 1 s to 15-60 s. Both techniques have the advantage that there is no systemic exposure of the test compound prior to transport through the BBB and thus, metabolism artifacts caused with either technique are restricted to metabolic events that occur within the brain microcirculation. The length of the perfusion has been increased to 10 min for certain compounds with low PS values [114]. Under these conditions, the flow is reduced to 1.25 ml/min, and the blood volume is maintained constant by withdrawal of blood via a femoral artery. However, under these conditions

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of reduced perfusate flow and pressure, there is mixing with the circulating rat plasma. This mixing can be minimized by increasing the flow rate to 23.5 ml/min. 4.2.4. I&ravenous injection technique The intravenous injection technique has the dual advantage of not requiring access to a carotid artery and the increased sensitivity of the technique whereby BBB PS products less than 0.5 pllmin/g may be easily measured. In this technique, a femoral vein is cannulated and the radiolabeled test compound is injected. At various times after injection, groups of animals are sacrificed and arterial blood is collected. Alternatively, a femoral artery may be cannulated and blood samples may be obtained at various times (e.g., 0.25, 1, 2, 5, 10, 15, 30, and 60 min) after injection in individual animals. The plasma radioactivity [A(t)] is measured as a percentage of the injected dose (ID)/ml of serum, plasma, or whole blood, and these data are fit to the following biexponential equation: A(t) = A,e mK1’+ A2emK2’

(5)

where A(t) = %ID/ml at a given time period and K,, K2, A ,, A, are the slopes and intercepts of the two exponents defining the plasma concentration curve. At the end of the experimental time period, which may range anywhere from 0.25 to 60 min or longer, the animal is sacrificed and brain radioactivity is measured for computation of the BBB PS product as follows [4]: ps =

F’,,- bIC,(V

(6)

I

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metabolism of the test compound by peripheral tissues and this may seriously confound interpretation of the brain uptake data and prevent accurate computation of the BBB PS product [115]. Even if plasma chromatographic analysis is performed, it is not possible to differentiate radiolabeled metabolites in brain from those metabolites that are produced in the periphery and taken up across the BBB versus those metabolites that are generated within brain. Thus, computation of BBB PS products with the intravenous injection technique is hazardous when there is a measurable metabolic degradation of the test compound during the experimental time period. A variant of the single intravenous injection technique is the external organ technique [116], and this approach eliminates the need for the pharmacokinetic analysis described above. In this technique, a femoral artery is cannulated and arterial blood is collected during the experimental time period either by gravity or by connecting the cannula to a syringe withdrawal pump. Following the single intravenous injection of [3H]test compound and [‘4C]sucrose (a V,, marker), blood is collected through the femoral artery external organ for a defined time period (t). At the end of the experimental time period, which may range from t = 0.25 to 10 min, the concentration of radioactive test compound in the femoral arterial serum, plasma, or whole blood is measured and this is equivalent to the integral of plasma radioactivity that is computed with the graphical method described above. The BBB PS product determined with the external organ technique is calculated as follows:

AUC I PS=,

VIJ- Y,

AUC ; = A ,(l - emK1’) + A,(1 - emK2’) 0

K,

K,

(7)

where V,, = the brain volume of distribution of the test compound, V,,= the plasma volume of distribution for a plasma volume marker such as radiolabeled rat albumin, and C,(T) = the terminal plasma concentration. The V, is computed from the ratio of dpm/g brain divided by dpmlpl plasma at the terminal time period. The disadvantage of the single intravenous injection technique is that there may be extensive

‘3H!dpm (brain)

v,=

6

(8)

3

“ Hpm ““pm vo=

(external organ) (brain)

14 ’

‘pm

(external organ)

If the BBB PS product is sufficiently high such

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that measurable brain radioactivity is achieved with a very short experimental time period, e.g.. 0.25 min, then the amount of peripheral metabolism of the radiolabeled test compound may be minimal and accurate estimates of BBB PS products may be determined with this technique. The external organ technique is probably the simplest approach to quantifying the BBB PS product since it eliminates the need for either the mathematical analysis described in Eqs. 5-7 or the cannulation of small arteries in the neck.

4.2.5. Measurement of BBB PS product in

Delivery

Reviews

IS (IELf)

5%3%

4.2.6. Measurement of V, under conditions of unidirectional influx The V,, parameter in Eqs. (4) (6) and (8) with respect to use of the internal carotid artery perfusion, intravenous injection, or external organ techniques, respectively, is a measure of unidirectional influx into brain, and can only be measured during the linear phase of brain uptake of a radiotracer molecule. During this ‘initial rate’ phase of the experiment, the brain V,, is defined by rearranging Eq. (6) PSxAUC (9)

humans with positron emission tomography

Changes in BBB permeability in humans is measurable with quantitative PET scans using tracers such as X2Rb,which has a t 1 = 1.25 min or “XGa-EDTA, which has a tl of 68 ‘min [117]. The PS product for hXGa-EDT’A in humans is very low ( < 0.07 ~llminlg) and 180 min scans are required to measure the PS product [ 1181. The BBB PS product for X2Rb in humans is substantially higher than the PS for hXGa-EDTA and is 7.8 pllmin/g [119], which is about 20-fold greater than the PS product for sucrose in laboratory animals [112]. h8Rb is a potassium analogue and may undergo slow transport through the BBB via cation transport systems. The quantification of the BBB PS product for either isotope in humans requires that care be taken to measure both the plasma AUC and the plasma volume. As shown in Eq. (6) above. both the plasma AUC and the V,, must be measured in order to accurately compute the BBB PS product. If the change in BBB permeability is measured under both control and pathophysiologic conditions, there may be changes in either the plasma AUC and/or the V,,. It is important to arrive at independent measurements of these parameters in the process of performing BBB PS measurements in humans. Computation of the plasma AUC requires serial sampling of blood radioactivity and a mathematical analysis of the plasma decay profile. Measurements of the V,, requires independent methods such as CO inhalation method [120]. Failure to measure the AUC and V,, can lead to artifacts (see section 4.4.).

The PS product is constant during the initial rate phase of the brain uptake curve. Subsequent efflux of drug from brain to blood occurs after the initial rate phase, and computation of valid PS products is no longer possible. At this phase, the brain V, is a complex function of BBB permeability, cellular metabolism, and cytoplasmic binding processes, and a change in any of these parameters may alter the brain V,,. 4.2.7. Measurements of solute transcytosis through the blood-brain barrier Capillary depletion technique

The basis of the computation of the BBB PS product is a difference between the V, of the test compound and the V, of the plasma volume as defined in Eq. (6) above. However, V, may exceed V,, without significant transport of the test compound through the BBB because the compound is specifically or non-specifically bound to the brain microvascular wall or even endocytosed into the microvascular endothelium. For example, the brain V,, for acetylated low density lipoprotein (LDL) exceeds the V,, blood volume owing to receptor-mediated endocytosis of the acetylated LDL into the brain microvascular endothelium, a process that is not followed by subsequent exocytosis into the brain interstitial fluid [114]. In order to differentiate endothelial binding/endocytosis from actual transcytosis, a capillary depletion technique was developed and has been described in detail previously [114].

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This technique involves density centrifugal separation of the microvascular pellet and the postvascular supernatant in conjunction with the use of a radiolabeled plasma volume marker. If the test compound is specifically bound by the brain microvascular endothelium, then this radiolabeled compound does not separate from the vascular pellet and does not distribute into the postvascular supernatant to an extent any greater than that of a plasma volume marker, such as radiolabeled albumin. However, if the test compound is non-specifically adsorbed to the microvascular endothelium, as has recently been demonstrated in the case of an opioid peptide analogue, then there is artifactual distribution of the test compound into the postvascular supernatant during preparation of the brain homogenate [121]. Therefore, the capillary depletion technique should not be used when the test compound is not bound to the brain microvascular endothelium via a high affinity process. In lieu of the capillary depletion technique, a post-perfusion wash procedure coupled with the internal carotid artery perfusion technique has been used to differentiate vascular binding from transcytosis [121]. Dialysis fiber technique

An alternative method for measuring transcytosis through the BBB is the dialysis fiber technique [122]. In this method, a dialysis fiber is implanted in the brain so that the brain interstitial fluid contents may be experimentally sampled directly. There are two caveats with respect to the use of the dialysis fiber technique. First, the extraction of solute across the fiber wall as measured in a ‘beaker’ experiment in vitro is generally greater than the extraction that occurs across the fiber wall in vivo [123]. The inequality between the in vivo and in vitro recovery across the fiber wall has been attributed to the vasogenic edema and expanded extracellular space that occurs in vivo [124]. This raises the second caveat with respect to the use of dialysis fibers and that caution is that the technique induces local brain injury and disturbed neurochemistry [12.5,126]. For example, the concentration of glucose is reduced in parallel with the increased cerebral metabolic rates of glucose and de-

creased rates of cerebral blood flow in the region surrounding the fiber [127,128]. In addition, recent studies show that the BBB is chronically disrupted by the insertion of the dialysis fiber [154]. 4.3. In vitro measurements barrier PS products 4.3.1. ‘In vitro ’ blood-brain tissue culture

of blood-brain

barrier models in

It is generally regarded that the above in vivo methods for measuring BBB PS product require radiolabeled test compounds. In many situations, particularly in the pharmaceutical industry, it is desirable to measure the BBB PS product of an entire series of compounds which are not available in radiolabeled form. In fact, the in vivo methods described above could be used to measure the BBB PS product of a non-radiolabeled compound if the requisite methodologies for serum and brain extraction of the drug followed by sensitive detection were developed. However, such extraction procedures would be simplified by the availability of an ‘in vitro’ BBB test system. Accordingly, in vitro BBB model systems have been developed wherein primary cultures of brain capillary endothelial cells are grown on filters and transport of test compounds across the brain endothelial monolayer is measured [129,130]. The problem with this approach is that there is extensive de-differentiation of the brain capillary endothelial cell when grown in tissue culture, such that many BBB characteristics are lost [4]. For example, several of the nutrient transport systems are downregulated by as much as 100-fold [36,131]. Therefore, in vitro BBB model systems generally underestimate BBB PS products for those compounds that traverse the barrier via carrier-mediation. For those molecules that undergo lipid-mediated transport through the BBB, the in vitro BBB models generally result in a considerable over-estimation of the in vivo BBB PS product [131]. This overestimation of lipid-mediated transport arises from the dominant fluid phase pathway owing to the paucity of tight junctions in the in vitro BBB model. For example, the BBB PS product for sucrose in vivo is 0.34 ~llminlg [121]; given

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S = 100 cm*/g in vivo, then P = 3.5 x 10mh cm/ min in vivo. Conversely, the P value (also called P, value) for sucrose in the in vitro barrier, 5.1 X 10m3 cm/min [131] is 1400-fold higher than the P, in vivo. The endothelial tight junctions do exist in the vitro BBB model, but are greatly reduced [132]. For example, the trans-endothelial electrical resistance (TEER) of the endothelial model is generally on the order of 40-100 ohm. cm2 [129]. This is to be contrasted with the in vivo TEER, which is estimated to be 8000 ohm * cm* [133]. Treatment of endothelial cultures with cyclic AMP analogues and astrocyte-conditioned medium may result in an augmentation of the TEER to the level of 800-1000 ohm. cm* [134]. but this value is still lo-fold reduced relative to the in vivo TEER. The increase in TEER with cyclic AMP analogues is expected since this treatment is known to increase the polymerization of the actin cytoskeleton in cultured endothelial cells and to increase polymerized Factin [135]. Astrocyte-conditioned medium may have minimal effects on BBB-specific gene expression. Other studies have shown that astrocytes induce BBB-specific gene expression within brain capillary endothelial cells in tissue culture only when the endothelial cells and astrocytes are grown in a mixed cell monolayer [136]. More recent studies suggest that the maximal induction of BBB-specific gene expression in the mixed cell culture is observed only when an homologous system is used and the endothelial cells and astrocytes arise from the same species [137]. Until much more information is available regarding the molecular biology of brain-induced tissue-specific gene expression within the capillary endothelial cell, it is unlikely that the in vitro BBB model will accurately predict PS products for BBB transport in vivo.

area (S) of the endothelial exchange surface in vitro. The in vivo BBB PS product may be converted to an in vivo P, knowing the surface area (S) of the brain capillary endothelium, which is approximately 100 cm2/g of brain [138]. Fig. 7 shows the comparison between the in vivo and in vitro P, values for a variety of different compounds that vary in lipid solubility. The figure shows that the in vitro BBB model overestimates the in vivo BBB P, value by approximately 150-fold, and this discrepancy approximates the difference in in vitro and in vivo TEER values. In cases such as L-DOPA or glucose, where molecules traverse the BBB via carrier-mediated transport, the in vitro BBB model under-estimates the in vivo P, value by several log orders of magnitude [131]. This discrepancy is due to the downregulation in tissue culture of the BBB nutrient transporters [36]. Some studies have demonstrated an equivalence between the in vitro and in vivo models for measuring BBB permeability. For example, in one study, BBB permeability is measured both in vivo and in vitro for a variety of drugs and an equivalence is found [139]. However, in this study, the permeability of each drug was not measured directly, but was normalized by the permeability of the barrier for water. In this ratio analysis, the permeability of the diffusible substance, such as butanol or water, is just as high in vitro as for the other compounds. Therefore, when a ratio is determined for the P, of a test compound relative to the P, for water in the in vitro model, then the ratio may correlate with the same ratio in vivo. However, this correlation does not mean that the P, value measured in vitro even approximates the P, value found in vivo.

In vivolin vitro correlation The determination of how well the in vitro BBB model predicts in vivo BBB PS products is facilitated by making in vitro/in vivo comparisons. Subsequent to the determination of the in vitro BBB PS product, this value may be converted into PC values by knowing the surface

The following are a series of artifacts in the measurement of BBB permeability or BBB PS products. Claims are often made as to whether a given compound crosses the BBB and it is difficult to critically evaluate the validity of these claims without a thorough background in the methodologies used to measure BBB permeabili-

4.4. Experimental determinations

artifacts of BBB permeability

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- I T

-2

\H

-3

-

-4

. n?

c= 2 5 z-

-5

/

-6

2o:-et37

,2p!7!_gg5 , ,g:_*.;

-7 -8

-3

-2

-1

0

IN VITRO

I

2

!h[Pe

- ,/ir]

3

4

5

Fig. 7. (A) Spindle-shaped morphology of primary cultures of bovine brain capillary endothelium is shown. Cells were grown to confluency on a collagen/fibronectin-coated 13 mm polycarbonate filter with a 3 CLpore size (0.33 cm’ area). The transwell was then placed between horizontal side by side transport chambers (Crown Glass Co.) consisting of drug acceptor and donor chambers. The external chamber of the side by side diffusion cell was warmed by continuous flow of water maintained at 37°C. drug or solute to the donor chamber. 2-40 min after The transport experiment was initiated by addition of ‘H- or “C-labeled addition of isotope to donor chamber, aliquots were removed from the acceptor chamber for measurement of drug transport across the monolayer. The rate of clearance of drug across the monolayer, after correction for clearance of drug across the cell-free filter, was a measure of the permeability-surface area (PS) product. Knowing the surface area (S), 0.33 cm’, the in vitro PS value was converted into the in vitro PC value (cm/s). The in vitro PC values were then correlated with the in vivo P, values. The latter were computed from the in vivo PS products determined with the internal carotid artery perfusion technique and using an in vivo S of 100 cm’lg [138]. (B) The log natural of the BBB permeability coefficient (PC), normalized for v/molecular weight (M,), is plotted versus the same function obtained in the in vitro BBB model for 13 different drugs. The correlation analysis generates the slope and the intercept as shown in the figure. The slope of the regression line (2.2) indicates the P,: values in the in vitro BBB model are approximately 1%fold greater than the corresponding PC obtained in vivo. Reproduced with permission from [131]

ty. These methodologies are often complex, so it may be useful to review the advantages and disadvantages of the respective methodologies within the context of evaluating potential experimental artifacts. 4.4.1. Artifact 1: A compound is said to undergo BBB transport because the brain V, > V,, (secondary to endothelial endocytosis) The calculation of the BBB PS product with either the intravenous injection technique (see Eq. (6)) or the internal carotid artery perfusion technique (see Eq. (4)) requires first determining the brain volume of distribution of the test compound (V,,) and a plasma volume marker

(V,,). If the V,, > V,, then it is assumed there is measurable BBB permeability for the test compound, and the BBB PS product is calculated. However, there are settings where the V, > V,, yet there is no measurable transcytosis of the compound through the BBB. For example, acetylated LDL has a V, considerably in excess of V, after a 5 or 10 min internal carotid artery perfusion [114]. When the brain homogenate is subjected to a capillary depletion analysis, it is found there is no measurable transcytosis of acetylated LDL through the BBB as the protein is completely sequestered within the brain capillary endothelium. That is, receptor-mediated endocytosis of the acetylated LDL at the lumenal

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side of the BBB is not followed by exocytosis of the protein into brain interstitial fluid [114].

cedure should be used in lieu of the capillary depletion technique.

4.4.2. Artifact 2: A compound

4.4.3. Artifact 3: A compound is said to undergo BBB transport because the brain V, > V, (secondary to metabolism artifacts)

is said to undergo BBB transport because the brain V, > V, (secondary to non-specific binding to the brain endothelium)

Non-specific binding of the test compound may also lead to the situation where V, > V,,. In the case of DALDA, a metabolically stable opioidtetrapeptide, the BBB PS product computed with the internal carotid artery perfusion technique was manyfold greater than the BBB PS product for DALDA computed with the intravenous injection technique [121]. Subsequent studies showed that the capillary depletion technique could not be used to quantify BBB transport of DALDA owing to the non-specific binding of this opioid peptide to the brain microvasculature. This non-specific binding occurred during the internal carotid artery perfusion of the peptide in balanced salt solution. Conversely, non-specific binding was not observed when the molecule was injected intravenously and circulated under physiologic conditions. The minimal transport of DALDA through the BBB was demonstrated with a post-perfusion wash procedure [121]. With this method, the brain V,, of a small molecule such as adenosine was not diminished during the post-perfusion wash period. However, the brain V, of DALDA was decreased in proportion to the brain V,, of sucrose, a plasma volume marker. during the post-perfusion wash. These results indicated that the post-perfusion wash procedure could be used to document actual brain uptake of adenosine, or adenosine metabolites, and that the method could differentiate those molecules that were only non-specifically adsorbed to the lumenal surface of the brain microvasculature [121]. In summary the finding of brajn V, > V,, does not always indicate there is actual transport of the test compound through the BBB. If the molecule undergoes high affinity binding to the brain microvasculature, then the capillary depletion technique may be used. If the molecule is believed to undergo non-specific binding to the vasculature, then the post-perfusion wash pro-

The BBB PS product for a variety of neuropeptides is claimed to be in the range of l-5 pl/min/g or higher [140]. These PS products, also called Ki values, have generally been measured with the intravenous injection technique. In this approach, the radiolabeled peptide is injected intravenously and serum radioactivity is obtained at various times after injection in either rats or mice. The peptides are generally labelled with [“?]iodine on the tyrosine residues of the peptide. Alternatively, peptides have been labeled with tritiation of lysine residues of the peptide [115]. Many neuropeptides are rapidly cleared by peripheral tissues, particularly liver, and are rapidly degraded to form free iodotyrosine or radiolabeled lysine analogues [ 1151. These metabolites are then released to the plasma resulting in an increase in plasma radioactivity that is soluble in the presence of trichloroacetic acid (TCA) precipitation. The radiolabeled tyrosine or lysine may circulate through the cerebral microcirculation and undergo carrier-mediated transport through the BBB, and the molecules may even undergo incorporation into proteins and become TCA-insoluble in brain homogenates [141]. It is imperative to perform chromatographic analysis of plasma and brain extracts when the brain transport of a metabolically labile peptide is being measured. However, even with the chromatographic analyses, it is not possible to determine whether radiolabeled metabolites in brain represent metabolites taken up from blood or metabolites produced in brain subsequent to transport of the peptide through the BBB. The small residual fraction of the brain extract that co-migrates on the column with the authentic peptide may represent peptide contributed by the plasma volume of brain. When the BBB PS product of metabolically stable neuropeptides is measured, the recorded PS product is up to 10 times lower than the PS products for metabolically labile peptides [121,142]. This suggests the BBB permeability for

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water-soluble peptides, which are not substrates for BBB transport systems, is quite low. In the absence of chromatographic analysis of blood and brain radioactivity, it is difficult to evaluate the extent to which a peptide actually undergoes transport through the BBB, as opposed to the artifactual uptake by brain of radiolabeled amino acid metabolites. Whenever possible, the permeability of the BBB to neuropeptides should be measured with metabolically stable analogues. 4.4.4. Artifact 4: The blood-brain barrier is said to be permeable to a drug because the drug readily distributes into cerebrospinal fluid As reviewed in section 2.1.2., AZT is readily transported across the blood-CSF barrier, and distributes into CSF of humans. The rapid penetration of drugs into CSF is often taken as evidence that the BBB is permeable to the drug. However, as reviewed in section 2.1.1. of this article, drug transport into CSF is an index of choroid plexus permeability, not brain capillary endothelial permeability. The two barriers may have similar permeability properties only if comparable transport systems are expressed in both barrier systems. In the absence of using CSF measurements as an index of BBB permeability in humans, the most direct approach for measuring BBB permeability in humans is with positron emission tomography (PET). 4.4.5. Artifact 5: Blood-brain

barrier permeability is said to be hormonally increased based on measurements of increased %IDlg without measuring plasma AUC

Opioid peptides have been shown to increase the brain uptake of test compounds, wherein brain uptake is measured simply as brain VD values or %ID/g values [143]. In this setting, the V, is not normalized by independent measurements of the V, and AUC parameters in Eqs. 6 and 9. As shown in Eq. 9, brain V,, of a test compound is a function of both the BBB PS product and the plasma AUC. Thus, a setting may arise in which the V, of a compound is increased owing to an increase solely in the plasma AUC, without any change in BBB permeability and no change in the BBB PS product. This could happen if a given hormonal treatment

29

inhibits the renal clearance of a test compound that is largely cleared by renal excretion. The inhibition of renal excretion will raise the plasma AUC and increase the %ID/g, independent of a change in BBB permeability, and this situation is predicted by Eq. 1. This artifact is eliminated by computation of the plasma AUC using the pharmacokinetic approach, as shown in Eqs. 5-7. Alternatively, the plasma AUC may be determined indirectly with the external organ technique (Eq. 8), as reviewed in section 4.2.4. An attempt to eliminate direct measurement of the plasma AUC is embodied in the brain penetration index (BPI), which is the ratio of brain V,,:liver V,, of a test compound following intravenous or peripheral administration [144]. In this case, the plasma AUC is eliminated from the formal calculation and the brain V,, in the absence of any blood volume changes, will parallel differences in drug permeability in brain relative to liver. The problem with this approach is that a given drug modification may selectively alter drug uptake by liver and this will influence the brain penetration index (BPI) independent of any change in BBB permeability. Therefore, the best approach is direct measurement of the plasma AUC, or use the external organ technique. 4.4.6. Artifact 6: Blood-brain barrier permeability is said to be hormonally increased based on measurements of %IDlg without measuring brain blood volume (V,) The measurement of the BBB PS product in humans with positron emission tomography requires measurement of brain V, of the test compound, determination of the plasma AUC, and measurement of the plasma V,, as indicated by Eq. (6). However, the measurement of the plasma V, requires the use of an independent technique, such as the carbon monoxide inhalation method [120] and is more labor-intensive. The omission of this parameter, however, will be unfortunate in the case where a given hormonal manipulation causes cerebral vasodilatation and increases the brain plasma volume (V,). Without an independent measurement of the brain V, with a plasma volume marker, the hormonal

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expansion of the V,] will be interpreted as an increase in BBB permeability. In actuality, there has been no increase in BBB permeability and the artifactual increase in brain V,, is due to the expansion of the brain plasma volume. 4.4.7. Artifact 7: A pathophysiologic modulation is said to increase blood-brain barrier permeability, as reflected by an increased brain uptake index, but cerebral blood flow is not measured When the BUI of several molecules was measured with the carotid artery injection technique in conscious animals, the BUIs were found to be reduced by approximately one-third as compared to the BUI values recorded previously in pentobarbital anesthetized animals [145]. In the absence of independent measurements of E, (the extraction of the reference compound) or measurements of F (cerebral blood flow), it may be erroneously concluded that pentobarbital treatment increases BBB permeability to the small molecules, e.g., amino acids, under investigation. Actually, the pentobarbital treatment reduced cerebral blood flow [106], which caused an increase in the extraction of the test compounds, independent of any change in BBBPS products. The artifacts obtained with the BUI technique are easily eliminated by independent measurements of the E, and F parameters along with the BUI, as discussed in section 4.2.2., and predicted by Eqs. 2-3.

total hydrogen bond number is c 8, then there may be transport of the compound through the BBB in pharmacologically active amounts. Conversely, if the molecular weight of the small molecule exceeds the 400-600 Dalton threshold, and/or the total hydrogen bond number of the compound is > 8, then the BBB transport of the small molecule may be minimal, such that no significant pharmacologic effects in brain are observed following systemic administration of the compound. The BBB PS product for small molecules may be increased by various forms of lipidization (Table 5). In general, however, lipidization of a molecule will result in proportionate decreases in the plasma AUC, owing to increased uptake by peripheral tissues. Therefore, the increased BBB PS product caused by lipidization may be offset largely by the decreased plasma AUC, resulting in minimal increases in the %ID/g or brain delivery of the drug. The inter-relationships of the brain delivery (%ID/g), the BBB PS product, and the plasma AUC are given in Eq. (1). There are a number of different in vivo and in vitro techniques and methodologies available for assessing BBB transport of small molecules. Each technique has its own advantages and disadvantages. Moreover, the experimental estimation of BBB permeability can be fraught with experimental artifacts that arise from a failure to employ a fully quantitative approach to evaluating BBB permeability. Some common experimental artifacts and cures for these problems are reviewed in section 4.4.

5. Conclusions The BBB transport of a given small molecule may be predicted by inspection of its structure and an understanding of the biology of small molecule transport through the brain capillary endothelium. While detailed models predicting BBB transport based on lipid solubility measurements have been proposed [146], the simplest way of predicting BBB transport of a given small molecule is to determine the total number of hydrogen bonds (N) formed with solvent water, and to obtain the molecular weight of the compound. If the molecular weight of a compound is less than a threshold of 400-600 Da, and the

Acknowledgements

This research was supported by NIH grants ROl-AI-28760 and ROl-DA-06748. Emily Yu skilfully prepared the manuscript.

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M.W. (1977) Morphology Exp. Eye Res. 25 (Suppl.), R.A.

(1980)

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of the Nervous System, W.B. Saunders Company. Philadelphia. [3] Davson, H., Welch, K. and Segal, M.B. (1987) Secretion of the cerebrospinal fluid. In: The Physiology and Pathophysiology of the Cerebrospinal Fluid, Churchill Livingstone, London, p. 201. [4] Pardridge, W.M. (1991) Peptide Drug Delivery to the Brain. Raven Press, New York, pp. 1-357. [5] Blasberg, R.G.. Patlak, C. and Fenstermacher, J.D. (1975) Intrathecal chemotherapy: brain tissue profiles after ventriculo-cistemal perfusion. J. Pharm. Exp. Ther. 195, 73-83. [6] Otsuka. M. and Yanagisawa, M. (1988) Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat. J. Physiol. 395, 255-270. [7] Penn, R.D., Savoy, S.M., Corcos, D., Latash, M., Gottlieb, G., Parke, B. and Kroin, J.S. (1989) Intrathecal baclofen for severe spinal spasticity. N. Engl. J. Med. 320, 1517-1521. [S] Yarchoan, R. and Broder, S. (1987) Development of antiretroviral therapy for acquired immunodeficiency syndrome and related disorders. N. Engl. J. Med. 316, 557-564. (91 Spector, R. and Berlinger, W.G. (1982) Localization and mechanism of thymidine transport in the central nervous system. J. Neurochem. 39, 837-841. [lo] Comford, E.M. and Oldendorf, W.H. (1975) Independent blood-brain barrier transport systems for nucleic acid precursors. Biochim. Biophys. Acta 25, 825-829. [ll] Terasaki, T. and Pardridge, W.M. (1988) Restricted transport of AZT and dideoxynucleosides through the blood-brain barrier. J. Infect. Dis. 158, 630-632. [12] Ahmed, A.E., Jacob, S., Loh, J.-P., Samra, S.K., Nokta, M. and Pollard. R.B. (1991) Comparative disposition and whole-body autoradiographic distribution of [2“C]azidothymidine and [2-14C]thymidine in mice. J. Pharmacol. Exp. Ther. 257, 479-486. [13] Wang, S.L., van Belle, K. and Sawchuk, R.J. (1993) Distributional transport kinetics of zidovudine between plasma and brain extracellular fluidlcerebrospinal fluid in the rabbit: investigation of the inhibitory effect of probenecid utilizing microdialysis. J. Pharmacol. Exp. Ther. 264, 899-909. [14] Kim. S., Scheerer, S., Geyer, M.A. and Howell, S.B. (1990) Direct cerebrospinal fluid delivery of an antiretroviral agent using multivesicular liposomes. J. Infect. Dis. 162, 750-752. [ 151 Aird, R.B. (1984) A study of intrathecal, cerebrospinal fluid-to-brain exchange. Exp. Neurol. 86, 342-358. [161 Fishman, R.A. and Christy, N.P. (1965) Fate of adrenal cortical steroids following intrathecal injections. Neurol. 15, 1-6. [17] Stein, W.D. (1967) The molecular basis of diffusion across cell membranes. In: The Movement of Molecules Across Cell Membranes, Academic Press, New York, pp. 65-125. [lS] Diamond, J.M. and Wright, E.M. (1969) Molecular

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

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