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U.S. Department of Health and Human Services • Public Health Service • Alcohol. Drug Abuse. and Mental Health Administration
Bioavailability of Drugs

to the Brain and the

Blood-Brain Barrier

Editors:

Jerry Frankenheim, Ph.D.

Roger M. Brown, Ph.D.

Research Monograph 120

1992

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers and discussions from a technical

review on “Drug Bioavailability and the Blood-Brain Barriers” held on September

13-14, 1990, in Rockville, MD. The review meeting was sponsored by the

National Institute on Drug Abuse (NIDA).

COPYRIGHT STATUS

NIDA has obtained permission from the copyright holders to reproduce certain

previously published material as noted in the text. Further reproduction of

this copyrighted material is permitted only as part of a reprinting of the entire

publication or chapter. For any other use, the copyright holder’s permission

is required. All other material in this volume except quoted passages from

copyrighted sources is in the public domain and may be used or reproduced

without permission from the Institute or the authors. Citation of the source is

appreciated.

Opinions expressed in this volume are those of the authors and do not

necessarily reflect the opinions or official policy of NIDA or any other part

of the U.S. Department of Health and Human Services.

The U.S. Government does not endorse or favor any specific commercial

product or company. Trade, proprietary, or company names appearing in this

publication are used only because they are considered essential in the context

of the studies reported herein.

NIDA Research Monographs are indexed in the ‘Index Medicus.” They are

selectively included in the coverage of ‘American Statistics Index,” “BioSciences

Information Service,” “Chemical Abstracts,” ‘Current Contents,” “Psychological

Abstracts,” and “Psychopharmacology Abstracts.”

DHHS publication number (ADM)92-1910

Printed 1992

For sale by the U.S Government Printing Office

Superintendent of Documents, Mail Stop: SSOP. Washington. DC 20402-9328

ISBN 0-16-037914-8

ii
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Jerry Frankenheim

The Basic Neurobiology of Addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Roger M. Brown

Some Relationships Between Addiction and

Drug Delivery to the Brain......................................................................... 13

William H. Oldendorf

Neuroactive Peptides and Amino Acids at the

Blood-Brain Barrier: Possible Implications

for Drug Abuse ................................................................................ 26

Berislav V. Zlokovic, J. Gordon McComb,

Lynn Perlmutter, Martin H. Weiss, and Hugh Davson

MPTP Toxicity and the “Biochemical”

Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

Sami I. Harik

An Overview of the Multiple Functions

of the Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

A. Lorris Betz

Formation and Differentiation of Brain Capillaries. . . . . . . . . . . . . . . . . . . . . . . ......... 73

John Laterra, Johannes E.A. Wolff,

Christopher Guerin, and Gary W. Goldstein

iii
Nonpermeable and Permeable Vessels of the Brain................................... 87

Milton W. Brightman and Yoshiaki Kadota

The Blood-Brain Barrier Is Not a “Barrier” for Many Drugs ......................108

Joseph D. Fenstermacher

Drug Delivery to the Brain: Barrier Modification

and Drug Modification Methodologies ................................................... 121

Stanley I. Rapoport

Cultured Brain Microvessel Endothelial Cells

as In Vitro Models of the Blood-Brain Barrier............................................. 138

Yoshinobu Takakura, Kenneth L. Audus,

and Ronald T. Borchardt

Opioid Peptide Drug Development: Transport

of Opioid Chimeric Peptides Through the

Blood-Brain Barrier ................................................................. 153

William M. Pardridge

Redox Approaches to Drug Delivery to

the Central Nervous System................................................................169

Marcus E. Brewster and Nicholas Bodor

Drug Delivery to the Brain Using an

Anti-Transferrin Receptor Antibody........................................................... 202

Phillip M. Friden, Lee Walus, Marjorie Taylor,

Gary F. Musso, Susan A. Abelleira, Bernard Malfroy,

Fariba Tehrani, Joseph B. Eckman III. Anne R. Morrow,

and Ruth M. Starzyk

Permeation of the Blood-Brain Barrier for Drug

Delivery to the Brain.................................................. 218

Heidi C. Horner, Kari Barbu, Frederique Bard,

Deborah Hall, Mary Janatpour, Katy Manning,

Janine Morales, Seth Porter, Paulette E. Seller,

Laura Tanner, and Lee R. Rubin

Pathways Into, Through, and Around

the Fluid-Brain Barriers ................................................ 230

Richard D. Broadwell

List of NIDA Research Monographs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

i v
Introduction

Jerry Frankenheim

In the past decade, understanding of the multifaceted importance of the bloodbrain barrier (BBB) has mushroomed. One reflection of this is the recent

proliferation of popular and scientific publications regarding the BBB:

“The Sciences” (Pardridge 1987)

1989 Grass Instrument calendar (Cserr 1989)

Two Scientific American” articles (Spector and Johanson 1989; Goldstein

and Betz 1986)

“Discover” (Angier 1990)

“Trends in Neurosciences” (Risau and Wolburg 1990)

There also have been detailed reviews in “Annual Review of Pharmacology

and Toxicology” (Pardridge 1988) “Acta Neuropathologica” (Broadwell 1989),

“Handbook of Neurochemistry” (Betz and Goldstein 1984), and “Basic

Neurochemistry” (Betz et al. 1989) as well as several excellent books. The BBB

is even part of an attraction called “Body Wars,” at Epcot Center in Florida.

Without the BBB scientists who participated in this technical review and

authored this monograph, these books and articles would not have been

written. Each of these scientists has his or her name on at least one brick in

the wall, one piece of the puzzle. It was a great privilege to welcome them to

the National Institute on Drug Abuse. Their discoveries are improving the

therapy of movement disorders (e.g., Parkinson’s disease), epilepsy, strokes,

and central nervous system (CNS) infection and tumors. Perhaps advances in

the treatment of drug abuse will be next. This will be a very difficult task, since

abused drugs, like almost all things that can make people feel good, are

(unfortunately) remarkably soluble in fat, and they penetrate the BBB with

extreme ease. Areas of possible interest include (but are certainly not limited

to):

Changes (including deficits) in BBB function caused by acute and chronic

abuse of such drugs as cocaine, amphetamine, phencyclidine (PCP), and

heroin

1
Improved understanding of the reinforcing properties of abused drugs in

terms of their entry into the brain and their fate at the BBB and in brain

interstitium

Other roles of the BBB (e.g., the role of its enzymes with regard to the

toxicity of abused substances)

Rational strategies for targeting potential treatment drugs (including

peptides, neurotransmitter precursors, and nutrients) to the brain

The main reason for planning the technical review was to determine the

importance of BBB research findings to drug abuse research. In planning the

review, it was confirmed that drug abuse researchers must be constantly aware

of findings about the BBB. The chapters herein demonstrate the importance of

the BBB in understanding drug abuse and affirm that the various roles of the

BBB have to be understood to fully comprehend the mechanisms of CNS

effects of drugs, including drugs of abuse, and the treatment of drug abuse.

The central effects of drugs are not isolated from their effects on the BBB nor

from the effects of the BBB on the drugs.

REFERENCES

Angier, N. Storming the wall. Discover 11(5):67-72, 1990.

Betz, A.L., and Goldstein, G.W. Brain capillaries: Structure and function. In:

Lajtha, A., ed. Handbook of Neurochemistry. Vol. 7. New York: Plenum

Press, 1984. pp. 465-484.

Betz, A.L.; Goldstein, G.W.; and Katzman, R. Blood-brain-cerebrospinal fluid

barriers. In: Siegel, G.J.; Agranoff, B.; Albers, R.W.; and Molinoff, P., eds.

Basic Neurochemistry. New York: Raven Press, 1989. pp. 591-606.

Broadwell, R.D. Transcytosis of macromolecules through the blood-brain

barrier: A cell biological perspective and critical appraisal. Acta Neuropathol

79:117-128, 1989.

Cserr, H. Biology of the Blood-Brain Barrier. Quincy, MA: Grass Instrument

Company Calendar, 1989.

Goldstein, G.W., and Betz, A.L. The blood-brain barrier. Sci Am 255:74-83,

1986.

Pardridge, W.M. The gatekeeper. The Sciences 27:50-55, 1987.

Pardridge, W.M. Recent advances in blood-brain barrier transport. Annu Rev

Pharmacol Toxicol 28:25-39, 1988.

Risau, W., and Wolburg, H. Development of the blood-brain barrier. Trends

Neurosci 13:174-178, 1990.

Spector, R., and Johanson, C.E. The mammalian choroid plexus. Sci Am

261:68-74, 1989.

2
AUTHOR

Jerry Frankenheim, Ph.D.

Pharmacologist

Neuroscience Research Branch

Division of Preclinical Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-31

5600 Fishers Lane

Rockville, MD 20857

3
The Basic Neurobiology of Addiction

Roger M. Brown

INTRODUCTION

A thorough understanding of the relationship between the brain mechanisms

that underlie compulsive drug seeking and how these processes are changed

by exposure to drugs is essential to understand phenomena such as drug

abuse, addiction, and physical dependence and also to develop a rational

biomedical treatment for the changes in central nervous system (CNS)

homeostasis that are the direct result of chemical insult.

SUBSTRATE FOR ADDICTION

The discovery of endogenous opioid peptides in the brain has explained how

a material from a poppy plant can induce narcosis in a human. Differences in

the distribution of the endogenous opioids within the brain and spinal cord imply

that they subserve specific functions. These substances have been shown to

be involved with the modulation of pain by drugs, in physiological events such

as trauma and acupuncture, as well as in the physical dependence Induced

by opiates. The opiate narcotics, as well as barbiturates and alcohol, are

well known to have the ability to induce tolerance and physical dependence,

the hallmark of addiction. Drug dependence has long been associated

with an adaptation of the CNS to the presence of the drug so that normal

functioning is only possible in the presence of the addictive substance.

When this occurs, drug seeking becomes oriented toward an avoidance of

the painful consequences of drug abstinence. Neuroscientists are examining

the changes in presynaptic and postsynaptic receptor function that occur during

drug dependence and withdrawal to intercede and “reset” the neuron to a

normative state.

Brain Reward Circuitry—Basis for Drug Abuse

Although there has never been complete agreement on what constitutes drug

addiction, cocaine certainly does not fit the older definition of an addictive drug.

Tolerance to its effects does not occur to any appreciable extent, and it does

4
not induce a profound physical withdrawal syndrome. Yet, the abuse of this

“nonaddictive” drug has reached epidemic proportions, and treatment centers

are filled to capacity in attempts to help users rid themselves of it.

Over the past decade, neuropsychopharmacologists have been examining

the biological basis of drug-seeking behavior and have developed a model to

explain why taking certain drugs is so attractive. According to the hypothesis,

drugs are sought because they directly activate the brain’s reward system,

a neural network responsible for the subjective experience of pleasure.

The foundation for the hypothesis began in 1954, when Olds and Milner

(1954) reported that animals would work for electrical stimulation of certain

neuroanatomical pathways in the brain. This finding gave rise to the concept

that the brain contains a system that is responsible for the experience of

pleasure. Presumably, this pathway is activated physiologically by any

event, activity, or stimulus that is highly reinforcing. Stated another way,

these events are rewarding because this “reward” pathway is activated.

As a result of the discovery of reward centers, neuroscientists began to map

regions of the brain that were positive for brain self-stimulation. Two findings

resulted from this line of investigation. First, the dopaminergic projections of

the ventral tegmental area (VTA), the mesolimbic and mesocortical dopamine

(DA) projections, were found to be a critical link in the reward system. Second,

it was found that animals would self-administer drugs directly into the reward

circuit. Many types of abused drugs, including morphine, heroin, cocaine,

amphetamine, and possibly nicotine, activate this system, albeit at different

loci. The involvement of DA synapses is consistent with the findings that

(1) neuroleptics block intracranial self-stimulation; (2) neuroleptics block the

systemic self-administration of a variety of drug classes: opiates, amphetamine,

cocaine, and barbiturates; and (3) systemic administration of DA blockers

prevents the self-administration of drugs directly into certain regions of brain

tissue (Wise 1983; Nakajima and McKenzie 1986; Goeders and Smith 1983;

Koob et al. 1987).

The DA synapse is not the only link in the chain. Neuroleptics block the

electrical self-stimulation of reward sites in certain regions of the cerebellum,

yet there are no DA neurons there. Therefore, the DA synapse, which is

critical to brain reward, may be several synapses removed from the activating

source. Many drugs of abuse, including amphetamine, opiates, MDMA (3,4-

methylenedioxymethamphetamine or “ecstasy”), phencyclidine (PCP or ‘angel

dust”), pentazocine and tripelennamine (a combination known as “T’s and

Blues”), and nicotine, are among the drugs found to lower the threshold for

electrical self-stimulation from the VTA and other rewarding sites such as

the medial forebrain bundle (Bain and Kornetsky 1987; Hubner et al. 1988;

5
Kornetsky 1985; Untenvald and Kornetsky 1984). The facilitation of

intracranial self-stimulation by these drugs is blocked by opiate antagonists

such as naloxone.

Thus, the reward circuitry involves at least a dopaminergic synapse and an

opioidergic synapse. Probably several others are involved as well. The cell

bodies of the VTA contain opiate and nicotine receptors, and the postsynaptic

receptors of the terminals are dopaminergic. Whether there is a single brain

reward circuit or several is a matter of investigation in contemporary research

laboratories (Koob and Bloom 1988; Wise 1983).

COCAINE: A POTENT REINFORCER

Researchers working to elucidate the chemical neuroanatomy of this reward

system began to realize that drugs are sought out initially because they induce

pleasure and not because of anything directly related to the development of

physical dependence. Indeed, physical dependence and brain reward can

be dissociated. As with cocaine, animals will self-administer opiates into their

VTA. They will not, however, self-administer drugs into the periaqueductal gray

(PAG) region, an anatomical relay station that is important in narcotic-induced

modulation of pain transmission and that, like the VTA, is populated with opiate

receptors. This demonstrates that the VTA is responsible for mediating opiateseeking behavior. On the other hand, when opiates are continuously infused

into the PAG over a 24- to 48-hour period, an abstinence syndrome is observed

when naloxone is administered. In contrast, infusions of opiate into the VTA do

not result in a precipitated abstinence syndrome following administration of

narcotic antagonist. Thus, the PAG is responsible for physical dependence,

and the VTA mediates drug self-administration (Bozarth and Wise 1984).

Abuse of a drug must be related to some positive aspect of drug action;

therefore, it is believed that different classes of drugs are abused because

they activate the brain’s reward mechanism. For example, cocaine is

potentially dangerous to health because it is rewarding (and continually used)

despite its lack of physical dependence liability, whereas heroin and other

opiates are dangerous because they are rewarding in addition to their ability

to induce physical dependence. The positive subjective effect of several drug

classes reinforces drug-seeking behavior in spite of simultaneously occurring

harmful physiological effects that may not be apparent to the user.

The strength of the rewarding impact of cocaine is illustrated by the findings of

many animal drug self-administration studies (see Johanson 1984). Cocaine is

inherently reinforcing; that is, no priming is necessary to induce animals to selfadminister the drug. Morphine, on the other hand, usually requires the

6
production of physical dependence before self-administration occurs. Also,

animals do not initially self-administer barbiturates; they have to be trained on

another drug first and then switched to barbiturates. In fact, cocaine is often

used as the training drug when shaping animals to self-administer. Cocaine is

not unique in this respect, however. Heroin, too, is inherently reinforcing and

requires no manipulations to set the condition for drug self-administration.

Another way to measure the rewarding value of a drug is to conduct a “breaking

point” study that asks how many lever presses an animal is willing to emit for

a single infusion of a test drug. This kind of abuse liability testing has made

possible the characterization (and comparison) of drugs in terms of relative

potency (i.e., the amount of drug required to maintain equivalent break points

relative to another drug) and/or reinforcing efficacy (i.e., the maximum break

point for any dose of the test drug). Studies to date indicate that the reinforcing

efficacy of cocaine far exceeds that of most other drugs. For example, among

drugs tested, cocaine’s breaking point exceeds that of methylphenidate,

fenfluramine, amphetamine, methamphetamine, nicotine, and local anesthetics.

Animals have been reported to press a lever more than 12,000 times for a

single cocaine dose of 0.5 mg/kg (Yanagita 1973).

Further evidence for cocaine’s potential danger to health comes from the finding

that, when given unlimited access, animals will self-administer the drug to the

point of toxicity and death. This is in contrast to opiate self-administration,

which is self-limiting. Opiates and barbiturates are examples of drugs that are

not self-administered to the point of toxicity. Again, however, cocaine is not

unique in this respect. Alcohol and amphetamine also are self-administered to

the point of toxicity. Thus, cocaine’s abuse potential and danger to health

surpasses that of morphine in two ways. First, cocaine is inherently reinforcing,

whereas other drugs such as morphine are less so; second, cocaine is selfadministered to the point of toxicity, whereas this is less so for opiates (Bozarth

and Wise 1985).

DEFINITION OF ADDICTION BASED ON DRUG SEEKING

Concern over today’s epidemic of cocaine abuse is probably the single most

important factor in focusing on the neural basis for drug seeking. As pointed

out above, cocaine does not appear to conform to the older concept of an

addictive drug, yet its abuse liability ranks along with that of the “hard drugs”

such as heroin. Because of inadequacies in the definition of addiction, Cohen

(1985) pointed out the need to define addiction in behavioral terms and

suggested that addiction be defined as “the loss of control over the intake of a

drug that leads to its compulsive use despite harmful effects in some area of

the person’s functioning.” Cocaine certainly falls into this category, along with

7
other abused substances, such as nicotine, that do not induce any appreciable

tolerance and physical dependence.

IMPLICATIONS OF THE BRAIN REWARD MODEL OF ADDICTION

The brain reward model explains the “why” of drug abuse and brings the

concept of psychological dependence into the realm of neurobiological

events. The model also may provide the rationale for developing effective

pharmacotherapeutic treatment strategies. According to the model, drugs are

sought because they pharmacologically activate the brain reward system to

a degree that surpasses that from natural rewards. The ventral tegmental

DA projection is critical in the circuitry, and a variety of drug classes activate

components of the neural system. The animal models used to test and

elaborate the model all have shown brain dopaminergic neurons to be

critical in mediating drug self-administration. In the human cocaine abuser,

however, greater success has been obtained with the use of DA agonists.

The discrepancy between animal and human treatments probably exists

because in the animal model the physiological reward process is the principal

determinant of the drug-seeking behavior, whereas in the clinic the therapist

may be dealing with drug reward, drug craving, or both.

Because bromocriptine, a DA agonist, has been reported effective in treating

cocaine addiction, speculations have been made concerning cocaine-induced

DA depletions (Dakis and Gold 1985). Although there is little, if any, evidence

for DA depletion following cocaine use, there may be a “functional” shortage

of neurotransmitter that is responsible for purported success with treatment

aimed at DA agonism. The “functional” DA depletion could result from

adaptive regulatory mechanisms responding to cocaine-induced excess of

neurotransmitter in the synaptic cleft, the end result being an inadequate

impulse-induced release of DA. It is not yet clear whether the most effective

way to terminate cocaine abuse should be aimed at cocaine-induced reward

(DA antagonists to block reward and possibly craving) or drug-induced

transmitter deficiencies (DA agonists to correct dopaminergic receptor downregulation). Both possibilities are being examined.

A recent pharmacotherapy investigation examined the role of DA receptor

subtypes in cocaine action. The two types of DA receptors on neurons in

the CNS, D1 and D2 DA receptors, are distinguished from each other by

their localization in brain areas, their ligand affinities, and their behavioral

characteristics. Recently, the development of drugs that are specific agonists

or antagonists for D1 and D2 receptors has made it possible to separate DA

neurotransmission by receptor type. One of the roles of the D1 receptor is to

enable D2 functioning (i.e., the behavioral events following D2 stimulation

8
require D1

 receptor stimulation for their expression). D1 antagonists have been

shown by several investigators (Clark and White 1987; Koob and Hubner 1989;

Koob et al 1987; Nakajima and McKenzie 1986) to block behavioral effects of

amphetamine and cocaine. The advantage of testing D1 receptor antagonists

to counteract cocaine self-administration is that these drugs show a wide doseresponse curve. This is in contrast to D2

 antagonists, which show very narrow,

practically all-or-none, dose-effect curves. Although these studies are still in

experimental stages, the advantage of using D1 receptor antagonists is that it

may be possible to titrate the patient so that the action against cocaine craving

is maximized while the distressing neuroleptic action is minimized.

While the involvement of DA receptor subtypes in drug reward is being

analyzed, other investigators are beginning to examine the processes

underlying craving and/or relapse. The model, as presented, may explain

why clinical treatment with dopammergic antagonists is not as useful as initially

speculated. First, the circuitry of the brain reward system is responsible for the

pleasurable effects of drugs, and this is what initially drives drug seeking. Once

the drug is experienced, its presence causes adaptive changes in the neuron

(e.g., up- or down-regulatory alterations in postsynaptic receptors, changes in

autoreceptor sensitivity, alterations in impulse flow). The perturbations in

homeostasis need to be corrected before the abuser can be drug-free. What

role DA neurons play in drug craving or relapse has yet to be evaluated,

although some evidence suggests that neuroleptics can prevent relapse in

some psychomotor stimulants (Ettenberg 1990).

Finally, much interest has been directed recently toward a methadone-like drug

for cocaine. In treating opiate addiction, methadone, a weak opiate agonist, is

given orally, and its presence in the body serves to prevent the development of

aversive withdrawal symptoms. As long as methadone is taken, physiological

processes are functional. The oral methadone is primarily a negative reinforcer,

in that it prevents the occurrence of abstinence symptoms. On the other hand,

quite a different situation exists in treating cocaine addiction because cocaine

does not induce physical dependence but acts directly on the reward substrate.

As aptly pointed out by Wise (1988), cocaine is a positive reinforcer, and any

drug that partially substitutes for cocaine not only would satisfy cocaine cravings

but also would simultaneously activate the brain reward mechanism and

perhaps even “prime” cocaine-seeking behavior. Clearly, a cocaine substitute

would not appear suitable as a treatment drug. On the other hand, if long-term

cocaine use leads to down-regulation of DA receptors, how does one go about

up-regulating the system without producing a “high”?

9
SUMMARY

This overview on cocaine’s addiction liability is presented in this monograph on

the blood-brain barrier (BBB) because the National Institute on Drug Abuse has

been given the task of finding pharmacotherapies to treat addiction. Knowledge

about the BBB might help researchers design better drugs or approaches to

keep the “good” drugs inside and/or the “bad” drugs outside. It is to be hoped

that the BBB community can be convinced that drug abuse is an exciting area

and that work on biological barriers has something to offer.

REFERENCES

Bain, G.T., and Kornetsky, C. Naloxone attenuation of the effect of cocaine on

rewarding brain stimulation. Life Sci 40:1119-1125, 1987.

Bozarth, M.A., and Wise, R.A. Anatomically distinct receptor fields mediate

reward and physical dependence. Science 224:516-517, 1984.

Bozarth, M.A., and Wise, R.A. Toxicity associated with long-term intravenous

heroin and cocaine self-administration in the rat. JAMA 254(1):16-18, 1985.

Clark, D., and White, F.J. D, dopamine receptor-the search for a function: A

critical evaluation of the D1/D2 dopamine receptor classification and its

functional implications. Synapse 1:347-388, 1987.

Cohen, S. Cocaine: The Bottom Line. Rockville, MD: American Council for

Drug Education, 1985.

Dakis, C.A., and Gold, MS. Bromocriptine as a treatment of cocaine abuse.

Lancet 8438(May 18):1151, 1985.

Ettenberg, A. Haloperidol prevents the reinstatement of amphetaminerewarded runway responding in rats. Pharmacol Biochem Behav 36:

635-638, 1990.

Goeders, N., and Smith, J. Cortical dopaminergic involvement in cocaine

reinforcement. Science 221:773-775, 1983.

Hubner, C.B.; Bird, M.; Rassnick, S.; and Kornetsky, C. The threshold

lowering effects of MDMA (ecstasy) on brain-stimulation reward.

Psychopharmacology 95:49-51, 1988.

Johanson, C.E. Assessment of the dependence potential of cocaine in animals.

In: Grabowski, J., ed. Cocaine: Pharmacology, Effects, and Treatment of

Abuse. National Institute on Drug Abuse Research Monograph 50. DHHS

Pub. No. (ADM)87-1326. Washington, DC: Supt. of Docs., U.S. Govt. Print.

off., 1984. pp. 54-71.

Koob, G.F., and Bloom, F.E. Cellular and molecular mechanisms of drug

dependence. Science 242:715-723, 1988.

Koob, G.F., and Hubner, C.B. Reinforcement pathways for cocaine. In:

Clouet, D.; Asghar, K.; and Brown, R., eds. Mechanisms of Cocaine Abuse

and Toxicity. National Institute on Drug Abuse Research Monograph 88.

10
DHHS Pub. No. (ADM)89-1585. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1988. pp. 137-159.

Koob, G.F.; Le, H.T.; and Creese, I. D, receptor antagonist SCH 23390

increases cocaine self-administration in the rat. Neurosci Lett 79:315-321,

1987.

Kornetsky, C. Brain-stimulation reward: A model for the neuronal basis for

drug-induced euphoria. In: Brown, R.M.; Friedman, D.P.; and Nimit, Y., eds.

Neuroscience Methods in Drug Abuse Research. National Institute on Drug

Abuse Research Monograph 62. DHHS Pub. No. (ADM)85-1415.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1985. pp. 30-50.

Nakajima, S., and McKenzie, G.M. Reduction of the rewarding effect of brain

stimulation by blockade of D-1 receptors with SCH-23390. Pharmacol

Biochem Behav 24:919-923, 1986.

Olds, J., and Milner, P. Positive reinforcement produced by electrical

stimulation of the septal area and other regions of the rat brain. J Comp

Physiol Psychol 47:419,1954.

Unterwald, E.M., and Kornetsky, C. Effects of concomitant pentazocine and

tripelennamine on brain-stimulation reward. Pharmacol Biochem Behav

21:961-964, 1984.

Wise, R.A. Brain neuronal systems mediating reward processes. In: Smith,

J.E., and Lane, J.D., eds. The Neurobiology of Opiate Reward Process.

New York: Elsevier, 1983. pp. 405-437.

Wise, R.A. The neurobiology of craving: Implications for the understanding and

treatment of addiction. J Abnorm Psychol 97(2):118-132, 1988.

Yanagita, T. An experimental framework for evaluation of dependence liability

of various types of drugs in monkeys. Bull Narc 25(4):57-64, 1973.

SUGGESTED READING

Brown, R.M. Pharmacology of cocaine abuse. In: Redda, K.K.; Walker, CA.;

and Barnett, G., eds. Cocaine, Marijuana, Designer Drugs: Chemistry,

Pharmacology, and Behavior. Boca Raton, FL: CRC Press, 1989.

pp. 39-51.

Clouet, D.; Asghar, K.; and Brown, R., eds. Mechanisms of Cocaine Abuse and

Toxicity. National Institute on Drug Abuse Research Monograph 88. DHHS

Pub. No. (ADM)89-1585. Washington, DC: Supt. of Docs. U.S. Govt. Print.

off., 1988.

Kleber. H.D., and Gawin, F.H. Cocaine abuse: A review of current and

experimental treatments. In: Grabowski, J.. ed. Cocaine: Pharmacology,

Effects, and Treatment of Abuse. National Institute on Drug Abuse Research

Monograph Number 50. DHHS Pub. No. (ADM)87-1326. Washington, DC:

Supt. of Docs., U.S. Govt. Print. Off., 1984. pp. 111-129.

11
Kuhar, M.J.; Ritz, M.C.; and Boja, J.W. The dopamine hypothesis of the

reinforcing properties of cocaine. Trends Neurosci 14(7):299-301, 1991.

Stewart, J. Conditioned and unconditioned drug effects in relapse to opiate and

stimulant drug self-administration. Prog Neuropsychopharmacol Biol

Psychiatry 7:591-597, 1983.

AUTHOR

Roger M. Brown, Ph.D.

Chief

Neuroscience Branch

Division of Preclinical Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-31

5600 Fishers Lane

Rockville, MD 20857

12
Some Relationships Between

Addiction and Drug Delivery to

the Brain

William H. Oldendorf

INTRODUCTION

The rapidity and abruptness of drug delivery to the brain using various routes

of administration are factors in drug addiction. The shorter the interval between

intake and perceived effect of a drug, the greater the propensity toward a more

severe addiction. For example, severity of cigarette nicotine addiction is

probably related to the approximately 3 seconds elapsing between the act of

gasping smoke accumulated in the mouth and the perceived mental response.

This chapter offers some documentation in support of the following hypothesis:

Some of the steps involved in taking an addicting substance are (1) the act of

taking the substance into the body, (2) the period of time elapsing during which

there is no subjective effect, and (3) the time at which a drug effect is perceived.

The more immediate the effect after intake, the more addicting the substance is

likely to be. The latter has been widely discussed in the literature and is not

presented here as novel; rather, some hemodynamic data obtained from human

studies related to this hypothesis are presented. Most of these studies were

radioisotopic tracer studies of human brain blood flow following carotid or

intravenous (IV) injection (Oldendorf 1962; Oldendorf and Kitano 1965a,

1965b).

It is assumed that substances reported in these hemodynamic studies,

131I-iodohippurate (hydrophilic) and 131I-iodoantipyrine (lipophilic), have no

psychotropic effects in the small tracer doses used. Their blood-brain barrier

(BBB) permeabilities closely resemble common drugs of abuse; it also is

assumed that their early distributions in the body are representative of some

drugs of addiction (i.e., nicotine, cocaine, heroin, ethanol, and morphine).

The human brain content of chromium-labeled red blood cells (51Cr-RBC)

and separately labeled plasma, radioiodinated serum albumin (131RISA),

13
after a carotid injection is shown in figure 1. Both these labeled substances

remain confined to blood and rapidly leave the brain by about 10 seconds after

their abrupt carotid injection (Oldendorf et al. 1965a). These curves show that

the labeled red blood cells pass through the brain more rapidly than does

labeled plasma. This is a well-recognized phenomenon and is included here

because it provides an indication of how rapidly blood cells and plasma pass

out of the human brain after carotid injection. Cranial counts were done by

external gamma counting. Only the cranial portion of the head is measured

in these studies.

In animal studies, a 14C test substance (such as a radiolabeled drug of abuse

mixed with 3HOH) has been extensively applied by rapidly injecting the mixture

into the common carotid artery of a barbiturate-anesthetized rat (Oldendorf

1971). At 5 seconds, the rat is decapitated and the side of the brain injected is

digested and subjected to liquid scintillation spectrometry. By 5 seconds after

carotid injection, any of the test substance that did not penetrate the BBB and

remained in brain was carried out of the head by continuing blood circulation.

The 3HOH was an internal standard that was almost completely cleared by

brain. Between 0 and 100 percent of the test substance is cleared by brain

during these 5 seconds. Comparison of the tritium-to-carbon ratio in the brain

with the same ratio in an aliquot of the injectate permits determination of the

fractional single pass clearance of the test substance, relative to the internal

standard (Oldendorf 1971). For some substances, the internal standard used

was 14C-butanol. Many classes of substances have been studied this way:

amino acids, amines, hexoses, inert polar substances, organic acids, drugs,

nucleic acid precursors, and peptides (Oldendorf 1971, 1974,198l; Oldendorf

et al. 1972).

Most of the common street drugs shown in table 1 are almost completely

cleared by rat brain during a single capillary passage. As a result of these

animal studies, it was clear that the equilibration of many drugs with brain was

essentially instantaneous through the BBB; the rapid increase in brain drug

concentration was determined largely by the rate at which drug concentration

rose in arterial blood following drug intake into the body.

DYNAMICS OF DRUG DELIVERY

It is of interest to consider several routes of administration and drug delivery

to brain: (1) A hypothetical injection into the ascending aorta, (2) inhalation,

(3) IV, (4) intranasal, and (5) ingestion. These are listed in, approximately,

a progressive lengthening of time of arrival in brain and, consequently, a

corresponding decrease in abruptness of delivery to brain. Other factors in

drug addiction (Goldstein and Kalant 1990) as well as other routes of drug

administration (Langer 1990) have been reviewed recently.

14
Common Carotid injection

5 1 Cr-RBC

- - - - 

131 RISA

Percent

Morphine

Equivalent

Seconds

FIGURE 1. Human brain content of 51Cr-RBC (solid line) and 131RISA

(dashed line) after common carotid injection (80 μc and 10 μc,

respectively). These remain intravascular and hemodynamically

would resemble morphine. In this presentation, “drug equivalent”

means that each hemodynamic curve would be similar to what

would be expected if the extensive data were transferred from

animal studies of single pass clearance by rat brain after abrupt

carotid arterial injection.

SOURCE: Oldendorf et al. 1965a, copyright 1965, American Heart Association,

Inc.

15
TABLE 1. Single pass clearance of drug (percent of 3HOH)

Drug Percent of 3HOH

Nicotine*

Ethanol*

Cocaine (percent butanol)*

Heroin*

L-methadone†

Codeine*

Morphine*

*Oldendorf 1981

†oldendorf 1974

131±7

104±4

99.5±3.4

90±3

68±6

26±2

2.6±0.2

Injection Into the Ascending Aorta

A hypothetical injection into the ascending aorta should result in the most

abrupt delivery to an entire brain.

lnhalation

It is likely that the gasp of cigarette smoke drawn into the lungs of the severely

addicted smoker closely approaches the abruptness of the hypothetical aortic

injection. The lung-to-brain arterial-capillary circulation time should be about 3

seconds, based on the arrival in human brain of radiographic contrast agents

injected into the aorta.

The serious smoker draws smoke into the mouth with the soft palate and

epiglottis closed, creating a high concentration of smoke in the mouth.

The mouth is then opened and a gasp of perhaps one-half to one liter of air

rapidly flushes this high concentration of smoke into the lungs. The fraction

of nicotine that is taken up by lung blood has a correspondingly sharp front

in its concentration curve. Three seconds after the intake gasp, about 20

percent of the nicotine front circulates to brain and all of it is lost to brain in

the first microcirculatory passage.

This inhalation method probably achieves the most abrupt front of drug

delivery to brain with the shortest transit time between act of intake and

perceived effects. The effect is essentially immediate following the act of

intake, and this is likely related to the severity and Intractability of nicotine

smoking. The immediate effect reinforces both the intake act and the central

16
response. Accordingly, smoking cigarettes is more addicting than is taking

a similar amount of nicotine by chewing gum. An extensive report of smoking

and addiction has been published recently (Surgeon General 1988).

Based on animal studies (Oldendorf 1974; Bradbury et al. 1975; Oldendorf et al.

1979), all nicotine delivered to brain after carotid injection remains there, and no

measurable nicotine leaves the brain during the first minute.

Such an immediate effect in brain also may enter into the inhalation of

freebase cocaine, where the inhalation is followed in 2 to 3 seconds by the

central stimulation sensation. This intake interval is much shorter than would

be obtained by nasal “snorting” or by IV injection; it may play a part in the

rapid and severe addiction experienced by freebase cocaine users.

IV lnjection

Figure 2 shows a common carotid arterial concentration of a commonly

used dye, cardio-green, after its abrupt IV injection (Oldendorf 1962;

Oldendorf et al. 1965b). Concentration was continually measured

calorimetrically. This shows the sharpness of the arrival in the human

brain after the initially abrupt bolus has been spread by cardiopulmonary

passage. In figure 3, an IV injection of radiolabeled 131I-iodohippurate

(hydrophilic tracer) is seen (Oldendorf 1962; Oldendorf and Kitano 1963)

measured by external gamma counting. lodohippurate brain clearance

resembles that of morphine, which has a single-pass clearance of about

1 percent, based on animal studies.

While working extensively with external gamma counting of human brain, a

method was developed that was more accurate if the bolus was delivered into

the right heart in as compact a bolus as possible. The bolus inevitably is spread

longitudinally by heart and lung passage. Following a routine IV injection, the

count rate from the human head normally began to rise in 8 to 10 seconds,

reached a peak in about 14 seconds, and was completely gone from the head

in 20 seconds.

To improve the precision of measurement, a procedure was developed for

inflating the arm veins with a blood pressure cuff placed around the upper arm

and inflated to diastolic pressure for about 1 minute (Oldendorf et al. 1965b).

The distal venous pressure in the arm rose to this drastolic pressure, creating

considerable peripheral venous distention. A small needle was placed in an

antecubital vein and the blood pressure cuff raised above systolic pressure.

This created a static inflated distal venous pool in the arm. The isotope was

injected into the vein and then the cuff was abruptly released, allowing collapse

17
FIGURE 2. Concentration of cardio-green sampled from the human common

carotid artery (c. carotid art.) after IV injection. This would be

expected to be hemodynamically equivalent to morphine.

SOURCE: Oldendorf 1962, copyright 1962, The Society of Nuclear Medicine

of the distal venous pool and driving the bolus into the right heart. This created

a measurably earlier and more dependable abrupt delivery of isotope into the

brain (Oldendorf et al. 1965b). This procedure is now in common use in nuclear

medicine laboratories to deliver an IV bolus to the right heart as abruptly as

possible.

Several drugs are almost completely cleared during a single capillary passage;

the amount delivered to any particular region depends on the fraction of cardiac

output distributing to that region. Because skeletal muscle and skin blood flow

can be altered considerably as a function of recent muscular exertion and air

temperature, it should be possible to substantially alter the fraction of highly

cleared drugs delivered to brain (e.g., cocaine or heroin). Thoroughly relaxed

muscle may differ in blood flow by a factor of about 20 from fully active muscle

blood flow.

In a wasteful scenario, the addict would, immediately after obtaining his or

her drug, hurry home, run upstairs to the highest and warmest part of the

house, apply a tourniquet while standing, insert the needle in a vein, and inject

the drug. In a less wasteful scenario, the addict would walk home, climb as

few stairs as possible, and lie down in a cool room for 5 to 10 minutes. The

tourniquet then would be applied, venipuncture accomplished, and the injection

made while sitting or lying prone.

18
40-

30-

Counts Per

Minute

(X 1000)

20-

10-

Seconds

FIGURE 3. This curve shows human brain content after IV injection of labeled

131I-iodohippurate (400 pc), which is assumed to be

hemodynamically similar to morphine.

SOURCE: Oldendorf 1962, copyright 1962, The Society of Nuclear Medicine

In the first scenario described, muscle blood flow would be near its maximum

and might be 12 to 15 liters per minute. Blood flow to brain is constant with

exercise; thus, the greater muscle and skin blood flow would “steal” cardiac

output from brain. Cocaine or heroin, for example, in such “stolen” blood would

be lost to muscle and skin as well as all other organs. The second scenario

might deliver 2 to 3 times as much intravenously injected blood to brain,

resulting in a corresponding increase in drug deposition in brain.

One could estimate that, of a $100-per-day cocaine habit, perhaps only 2 or

3 cents worth of cocaine would find its way to human brain receptors in the

19
first scenario. However, in the less wasteful second scenario, perhaps 10 to

15 cents worth of cocaine would find its specific brain receptor, the remainder

being dispersed elsewhere in the body, where it is nonspecifically bound,

producing no central effect.

Lipophillicity of Drug

Figures 4 and 5 show the isotope content of the human brain after IV injection

of a lipophilic tracer, 131I-iodoantipyrine (Oldendorf and Kitano 1965b). The

start of the time base is the time the tourniquet-cuff was released and the bolus

began its movement centrally (figure 4). There is then a period of about 8 to

10 seconds before the head curve begins to rise. It rises to a maximum and

then remains constant.

Figure 5 shows the externally gamma counted 131I-iodoantipyrine averaged

from 17 human subjects after IV injections (Oldendorf and Kitano 1965b).

Seven of these had angiographically confirmed arteriovenous malformations;

the remaining 10 were normal controls. The averaged curve with the initial

peak represents a substantial amount of blood shunted around the local

Seconds After Arm Release

FIGURE 4. This curve shows normal human subject brain content after

IV 131I-iodoantipyrine (8 μc), which is assumed to be

hemodynamically similar to nicotine and cocaine.

SOURCE: Oldendorf and Kitano 1965b, copyright 1965, Neurology

20
Seconds

FIGURE 5. These averaged curves show human brain content after IV

131I-iodoantipyrine (8 to 20 μc), a lipophilic tracer. Time base

starts at first appearance in head. The initially peaked curve

probably would resemble incompletely cleared heroin. The

unpeaked curve probably resembles cocaine and nicotine

hemodynamically.

SOURCE: Oldendorf and Kitano 1965b, copyright 1965, Neurology

microcirculation. The radioisotope being measured in this arteriovenous

shunt rapidly leaves the brain. The remainder of the isotope passes through

a capillary bed, and no peak is seen in the average normal subjects (dashed

line).

In the rat model, iodoantipyrine, like cocaine, is cleared 100 percent during

a single brain passage; heroin is about 65 percent cleared. These curves

(figures 4 and 5) presumably closely resemble the human brain content

following IV cocaine and approximate heroin given intravenously. The

normal 131I-iodoantipyrine curve shown in figure 4, with no initial peak,

would be representative of the behavior of cocaine. The initially peaked

curve shown in figure 5 should represent the curve expected from the

incompletely cleared heroin.

21
When a drug that is almost completely cleared by brain is injected

intravenously, its local concentration in brain some 15 seconds later is

proportional to regional blood flow to that region. This regional distribution

of blood flow ordinarily is unrelated to regional distribution of specific receptors.

Radiolabeled heroin and morphine have been injected intravenously into

mature rats and then the amount of drug in total brain assessed 1 minute

later (unpublished data). Approximately 50 times as much heroin distributed

to brain relative to morphine. From this, one might expect heroin would be

about 50 times more potent, mole for mole, as an analgesic than morphine.

However, the usual relative analgesia attributed to these drugs is that heroin is

3 to 5 times as efficacious as morphine. One explanation of this unexpectedly

low ratio is that the heroin probably is delivered to various regions of brain in

proportion to blood flow, independent of receptor density.

Morphine, which is much more hydrophilic than heroin, is only about 1 percent

cleared in a single rat brain passage but accumulates slowly in various regions

in proportion to the local opiate receptor density. This may relate to morphine’s

clinically observed 10- to 15-minute delay before peak analgesia after IV

injection.

lntranasal

From informal discussion with cocaine users, the stimulation after intranasal

intake is more delayed than after IV injection. According to the hypothesis

under discussion here, this stimulation delay should make intranasal cocaine

slightly less addicting than IV cocaine. One also would expect other factors to

enter into this, such as the relative simplicity of snorting vs. the paraphernalia

required for an IV injection.

Ingestion

The major drug under consideration here is ethanol (alcohol). The partition

coefficient (PC) of ethanol is ideal for a socially usable drug. Its olive oil:water

PC is 0.04 (figure 6) (Oldendorf 1974). This means that ethanol achieves

25 times the concentration in water as in olive oil. If one assumed that olive

oil is reasonably representative of human depot fat, then distribution of ethanol

probably is predominantly to lean body mass. As such, obese drinkers can

become intoxicated with little more ethanol than those of normal habitus.

If the PC of ethanol were one or two orders of magnitude larger, ethanol

would tend to accumulate in depot fat and therefore require a much larger

dose. Assuming its rate of metabolism would be unchanged, the effects of

22
FIGURE 6. The relationship between olive oil:water PC at 37 oC, pH 7.5

(refined olive oil, Sigma, St. Louis) and single pass rat brain

clearance following carotid injection. In addition to the drug’s

substantial clearance when its PC is greater than 0.05 in this

system, there is another high uptake group having much lower

PCs that lie above the average curve. They are encircled by a

dashed line and are metabolized substances.

SOURCE: Oldendorf 1974, copyright 1974, Society for Experimental Biology

and Medicine

ethanol would be considerably prolonged. Instead, the situation that exists is

that a “social drinker” can drink during the evening, have a few hours of effect,

and be sober by the next morning. The effects of alcohol ingestion are noted

after perhaps 5 minutes. If one consumed alcohol in the evening and did not

become intoxicated until the next morning, alcohol probably would largely

cease to be a substance of abuse.

If the PC were one or two orders of magnitude lower, ethanol would equilibrate

very slowly with brain through the BBB. As shown in figure 6, there seems to

be a broad threshold at around 0.05 olive oil:water PC, above which the single

pass clearance of a substance in the rat model is substantial. A drug such as

23
ethanol has a PC just high enough to get into brain freely, yet not accumulate

excessively in depot fat.

SUMMARY

Hemodynamic radioisotope studies of brain blood flow in humans have been

correlated with the delivery of some common addictive drugs. Both lipophilic

and hydrophilic tracers were used in the hemodynamic studies. lodoantipyrine

is lipophilic and is completely cleared by brain during a single brain circulatory

passage, as are cocaine and nicotine. lodohippurate is hydrophilic, so its brain

clearance after IV injection resembles that of morphine.

The earlier studies performed in humans have been related here with recent

studies of blood brain penetration of drugs of abuse. As presented, these

separate studies are consistent with the proposed hypothesis that the interval

between drug intake and perceived effect is a significant consideration when

explaining severity of addiction. The shorter the time interval between drug

intake and its perceived effect, the more severe the addiction appears to be.

This relationship may explain differences in severity of addiction to the same

drug taken by various routes of administration.

REFERENCES

Bradbury, M.W.B.; Patlak, C.S.; and Oldendorf, W.H. Analysis of brain uptake

and loss of radiotracers after intracarotid injection. Am J Physiol

229(4):1110-1115,1975.

Goldstein, A., and Kalant, H. Drug policy: Striking the right balance. Science

249:1513-1521, 1990.

Langer, R. New methods of drug delivery. Science 249:1527-1533, 1990.

Oldendorf, W.H. Measurement of the mean transit time of cerebral circulation

by external detection of an intravenously injected radioisotope. J Nucl Med

3(5):382-398, 1962.

Oldendorf, W.H. Brain uptake of radiolabeled amino acids, amines, and

hexoses after arterial injection. Am J Physiol 221(6):1629-1639, 1971.

Oldendorf, W.H. Lipid solubility and drug penetration of the blood brain barrier.

Proc Soc Exp Biol Med 147:813-816, 1974.

Oldendorf, W.H. Clearance of radiolabeled substances by brain after arterial

injection using a diffusible internal standard. In: Marks, N., and Rodnight,

R., eds. Research Methods in Neurochemistry. Vol. V. New York: Plenum,

1981. pp. 91-112.

Oldendorf, W.H.; Braun, L.; and Cornford, E. pH dependence of blood-brain

barrier permeability to lactate and nicotine. Stroke 10(5):577-581, 1979.

24
Oldendorf, W.H.; Hyman, S.; Braun, L.; and Oldendorf, S.Z. Blood-brain barrier:

Penetration of morphine, codeine, heroin, and methadone after carotid

injection. Science 178:984-986. 1972.

Oldendorf, W.H., and Kitano, M. Clinical measurement of brain uptake of

radioisotope. Arch Neural 9:574-585, 1963.

Oldendorf, W.H., and Kitano, M. Isotope study of brain blood turnover in

vascular disease. Arch Neural 12:30-38, 1965a.

Oldendorf, W.H., and Kitano, M. The symmetry of 131I4-iodoantipyrine uptake

by brain after intravenous injection. Neurology 15( 11):994-999, 1965b.

Oldendorf, W.H.; Kitano, M.; and Shimizu, S. Evaluation of a simple technique

for abrupt intravenous injection of radioisotope. J Nucl Med 6:205-209,

1965b.

Oldendorf, W.H.; Kitano, M.; Shimizu, S.; and Oldendorf, S.Z. Hematocrit of the

human cranial blood pool. Circ Res 17:532-539, 1965a.

Surgeon General, U.S. Public Health Service. The Health Consequences of

Smoking. Nicotine Addiction. Department of Health and Human Services,

Office of Smoking and Health. DHHS Pub. No. (CDC)88-8406. Washington,

DC: Supt. of Docs., U.S. Govt. Print. Off., 1988.

AUTHOR

William H. Oldendorf, M.D.

Senior Medical Investigator

West Los Angeles VA Medical Center

B151C

Wilshire and Sawtelle Boulevards

Los Angeles, CA 90073

Professor of Neurology and Psychiatry

University of California, Los Angeles School of Medicine

Los Angeles, CA 90024

25
Neuroactive Peptides and Amino Acids

at the Blood-Brain Barrier: Possible

Implications for Drug Abuse

Berlslav V. Zlokovlc, J. Gordon McComb, Lynn Per/mutter,

Martln H. Welss, and Hugh Davson

INTRODUCTION

The blood-brain barrier (BBB) normally serves to isolate the brain, to a large

degree, from compounds dissolved in blood plasma. However, the BBB must

not be regarded as an absolute restriction to blood-borne molecules, but rather

as a multiple regulatory brain unit(s) with highly developed transport, metabolic,

and receptor-mediated functions (Oldendorf 1971; Davson 1976; Betz and

Goldstein 1986; Pardridge 1988). These functions are of vital importance for

regional homeostasis of the neural microenvironment and maintenance of

normal local cell-to-cell communications within the brain. The BBB is described

anatomically as a continuous cellular layer of the endothelial cells that are

sealed by tight junctions (Brightman 1977). Several homeostatic BBB

mechanisms are related to substances that act as neurotransmitters and/or

neuromodulators in the brain and hormones at the periphery (Segal and

Zlokovic 1990). This review is limited to the authors’ observations with the

vascular brain perfusion (VBP) model.

BBB TO NEUROACTIVE SUBSTANCES

The pioneering work of Oldendorf (1971) has shown that BBB restricts rapid

penetration of monoamines (e.g., norepinephrine, dopamine, serotonin [5-HT),

amino acid neurotransmitters (e.g., glutamate, glycine, gamma aminobutyric

acid [GABA]), and neuropeptides (e.g., enkephalins, thyrotropin-releasing

hormone FRH]) (Cornford et al. 1978), whereas some neuroactive drugs, such

as amphetamine (Pardridge and Connor 1973) as well as cocaine and nicotine

(Oldendorf 1981), can cross the BBB quite rapidly. Once in the brain, these

drugs act on various neurotransmitter and neuropeptide systems to produce

distinct psychostimulant and neurotoxic effects. A moderate BBB permeability

to synthetic analogs of naturally occurring opioid peptides has been

demonstrated by Rapoport and colleagues (1980). An enzymatic barrier with

26
a role in terminating actions of neurotransmitters (e.g., monoamines) also

has been suggested (Harik, this volume). The hypothesis that the brain

capillary receptor for peptide is a component of a BBB transport system has

been formulated by Pardridge (1966), and specialized carrier-, receptor-, or

absorption-mediated transcytosis mechanisms within the brain capillary

endothelium have been shown for large peptides and proteins (e.g., insulin,

insulin-like growth factor [IGF] I and II, transferrin, cationized albumin, and

immunoglobulin G [IgG]) either by autoradiographic analysis of the in situ

perfused brain or by capillary isolation methods (Pardridge 1988).

During recent years, the authors have developed a long-term (up to 20

minutes) VBP model in the guinea pig to investigate interactions of neuroactive

substances at the blood-brain interface (Zlokovic et al. 1986). Investigations

conducted with this technique suggested that extremely low initial brain

extractions of neuropeptides (e.g., leucine-enkephalin [leu-enk], delta sleepinducing peptide [DSIP], arginine-vasopressin [AVP]) and neurotransmitters

(e.g., glutamic acid) may have resulted from relatively slow but specific uptake

mechanisms at the luminal side of the BBB (Zlokovic 1990). The method has

proven to be sensitive to measure discrete transient changes of impaired BBB

permeability during chronic drug intoxication (Rakic et al. 1969) and in the brain

affected by an autoimmune process (Zlokovic et al. 1989a).

VBP MODEL: TECHNICAL CONSIDERATIONS

The VBP model allows for experimental control of the concentration of test

molecules and the composition of arterial Inflow. In this preparation, brain

perfusion is carried out through the righ! common carotid artery in the

guinea pig, which is cannulated by fine polyethylene tubing connected to

the extracorporeal perfusion circuit (Zlokovic et al. 1986). The contralateral

carotid artery is ligated, and both jugular veins are cut to allow drainage of

the perfusate. The perfusion medium is an artificial plasma and 20 percent

sheep red blood cells. The surgical procedure in the guinea pig is reduced

compared with the rat (Takasato et al. 1964) since the cauterization of the right

superior thyroid, ophthalmic, and pterygopalatine artery is not required. The

unique anatomy of the cerebral circulation in the guinea pig has not been found

In any mammals other than cavoids (Bugge 1974). The forebrain supply relies

largely on the external carotid artery, and the Internal carotid artery normally

does not exist. The blood flow in the internal ophthalmic artery Is reversed

under physiological conditions, due to five anastomotic new branches derived

from the proximal part of the external carotid artery and the stapedial arterial

system. The circle of Willis is complete, but communication between carotid

and vertebral circulations is poor, resulting in the retrograde postcannulation

pressure in the right common carotid artery between 10 and 15 mm Hg. The

27
functional separation between artificial and vertebral circulations has been

confirmed by isotope experiments (Zlokovic et al. 1966a). The molecules under

study are protected from hydrolysis and, therefore, are presented to the bloodbrain interface in the intact form.

During brain perfusion, jugular venous outflow can be collected for

radioimmunoassay hormone analysis. Placement of an arterial carotid loop

may be required when brain microdialysis is conducted simultaneously with

vascular brain perfusion. The physiological (e.g., perfusion and arterial blood

pressure, heart rate, respirations, electrocardiogram, electroencephalogram,

cerebral blood flow, PCO2 acid-base status), biochemical (e.g., water,

electrolyte, adenosine triphosphate, and lactate brain content) (Zlokovic et al.

1986), and morphological (e.g., brain immunogenicity, ultrastructural integrity

of the neural tissue at the BBB and non-BBB regions) (figure 1) parameters

remain normal in the perfused brain.

Biomathematical modeling of kinetic data from isotopic experiments relies on

previously published treatments (Pardridge and Mietus 1962; Patlak et al. 1963;

Smith et al. 1987). A novel computer-assisted microimaging of the perfused

brain is based on immunohistochemical analysis of blood-borne peptide/protein.

NEUROPEPTIDES

The multiple time point/graphic analysis was used to estimate the unidirectional

transfer rate constant, Kin (μL min.1 g.1), for radiolabeled neuropeptides and

inert polar molecules (figure 2). A time-dependent progressive linear brain

uptake of leu-enk and TRH during the 20 minutes was significantly higher than

for the inert polar molecules. Figure 2 shows that, during relatively short

periods of 1 to 2 minutes, it is not possible to distinguish between the extraction

for a given peptide from either those of inert polar molecules or those of other

peptides. Kin values for neuropeptides, IgG, and inert polar molecules are

given in table 1.

It has been demonstrated that Kin for the tracer estimated in the absence of

potential inhibitors and/or competitors represents its maximal permeability

surface area product (Smith et al. 1967). The cerebrovascular permeability

constants for leu-enk, AVP, TRH, DSIP, cyclosporin A, and inert polar

molecules have been correlated with their olive oil:water partition coefficients,

or reciprocal values of the square root of their molecular weight (Zlokovic et al.

1990). This analysis indicated that lipophilicity and molecular weight are not

good predictors for cerebrovascular permeability of small peptides.

28
FIGURE 1. Ultrastructural integrity of the ipsilateral supraoptic nucleus from

guinea pig brain vascularly perfused for 10 minutes with the

medium containing 20 percent sheep red blood cells suspended

in artificial plasma. 1a. The capillary and cellular elements

within the neuropil remain intact, retaining their appropriate

morphological relationships. E, endothelial cell; L, capillary

lumen; N, neuron; D, dendrite. Print magnification=X 7,000. 1b.

Two dendrites cut in cross-section are contacted by several axon

terminals (ax), and a synapse (arrowheads) is evident. Print

magnification=X 31,1 000.

2 9
Time (minutes)

FIGURE 2. Uptake of small neuropetides, D-mannitol, and polyethylene

glycol (PEG) by the parietal cortex of perfused guinea pig brain

NOTE: Kin=[CBR(T)/CPL-Vi]/T where CBR(T) and CPL are dpm per unit mass

of brain and plasma, respectively, at perfusion time T; VI is the initial

volume of distribution (ordinate intercept).

SOURCE: Values are taken from Zlokovic et al. 1987, 1988a, and Rakic et al.

1989.

30
TABLE 1, Uptake of neuropeptides, IgG, and inert polar molecules at the

luminal side of the BBB

Tracer

KIn

(μL min-1 g-1)

V I

(mL 100 g-1)

[

3H] Leu-enk 3.62±0.11 0.40±0.07

[

3H] AVP 2.78±0.04 0.27±0.04

[

3H] TRH 1.22±0.08 0.78±0.09

[

125I] DSIP 0.93±0.14 0.94±0.13

[

125I] IgG 0.58±0.05 0.29±0.03

[

3H] D-mannitol 0.24±0.04 0.35±0.07

[

3H] Dextran 0.05±0.01 0.31±0.02

NOTE: Values are means&E of 7 to 12 perfused brains. Kin, values are

significantly different by analysis of variance (ANOVA). Kin and V,

values were graphically estimated in the parietal cortex using the

equation given in figure 2. Molecular weight of dextran was 70,000.

SOURCE: Values are taken from Zlokovic et al. 1987, 1988a, 1988b, 1989a,

1989c, 1990.

The first interacting step of circulating neuropeptide at the BBB is the luminal

site contact, The kinetics of radiolabeled peptide BBB uptake were examined in

the presence of (1) fully saturated L-amino acid transporter, (2) aminopeptidase

and amidase inhibitors, (3) unlabeled peptide, (4) peptide fragments, and

(5) peptide receptor antagonists and agonists. These experiments failed to

demonstrate cross-inhibition between neuropeptides and L-amino acids,

confirming a hypothesis that peptide bond prevents utilization of amino

acid BBB transporter (Zlokovic et al. 1983). It also has been difficult to

demonstrate a carrier-mediated transport of amino acid residues released

from peptides, which may suggest that the N-terminal enzymatic hydrolysis

may not necessarily be the primary event during peptide interactions at the

luminal BBB site. These findings have been supported by a lack of significant

inhibition of leu-enk and AVP uptake in the presence of aminopeptidase

Inhibitors, bestatin, and bacitracin (Zlokovic et al. 1987, 1989b, 1990). The

authors’ earlier studies indicated that bacitracin reduced leu-enk brain

uptake index (BUI) values to the level of intravascular markers, most likely

by preventing actions of blood-borne aminopeptidases on the bolus (Zlokovic

et al. 1985a). It has been shown that aminopeptidase rapidly degrades leu-enk

in vitro (Pardridge and Mietus 1981), so the lack of this effect in the VBP model

may suggest that the enzyme is situated at the abluminal site. It has been

31
demonstrated that microvascular aminopeptidase M is a membrane-bound

protein contributing to about 1 percent of the total brain “enkephalinase” activity,

but its exact cellular location in the BBB has not been determined (Churchill et

al. 1987).

The dose-dependent self-inhibition of KIn values was demonstrated for leu-enk

and AVP, and Michaelis-Menten parameters are given in table 2. Table 2

shows that the Km for leu-enk uptake is similar to the large neutral amino acids

determined during brain perfusion in the rat (Smith et al. 1987), but the capacity

of this system is about three orders of magnitude less. It would seem that leuenk kinetic parameters compare best with the BBB transport mechanisms of

purine bases and nucleotides, whereas the AVP system has Km similar to the

carrier-mediated transport system for thyroid hormones, but its capacity was

about 35 times less (Pardridge 1988). The diffusion component, K01 for both

peptides was negligible. Leu-enk peptide fragments, Tyr-Gly, Tyr-Gly-Gly,

and Gly-Gly-Phe-Leu (Zlokovic et al. 1987, 1989b), and AVP peptide fragments,

AVP-(1-8) pressinoic acid, and [pGlu4,CytS]AVP-(4-9) (Zlokovic et al. 1990),

did not influence BBB uptake of their parent peptides. These results indicate

saturable luminal BBB uptake of intact peptides, as well as the absence of

significant saturable metabolism of the peptide fragments.

The specific a-opioid receptor antagonist ally2-Tyr-AIB-Phe-OH, μ-opioid

receptor agonist Tyr-D-Ala-Gly-Me-Phe-NH(CH,),OH, and naloxone did

not affect significantly uptake of [3H]-leu-enk (Zlokovic et al. 1989b). On

the other hand, the V1-vasopressinergic receptor antagonist, TMeAVP

TABLE 2. Kinetic parameters for leu-enk and AVP uptake at the luminal side

of the BBB

Peptide Km (μM)

Leu-enk 39±3.2

AVP 2±0.3

Vmax (pmol min-1 g-1)

160±22

5.49±0.74

NOTE: Values are means±SE for 5 to 9 Kin observations in the parietal cortex.

Michaelis-Menten parameters were estimated by fitting equation

KIn=Vmax(Km+CPL)+Kd to e rain perfusion data with weighted

nonlinear least squares. Kd values were not significantly different

from zero.

SOURCE: Values are taken from Zlokovic et al. 1989b. 1990.

32
(1-ß-mercapto-ß,ß,cyclopentamethylenepropionic acid,2-(O-methyl)tyrosinevasopressin], but not the V2

-agonist, dDAVP (1 -deamino-[8-D-argininelvasopressin), significantly inhibited AVP BBB uptake. The affinity of TMeAVP

was about two times less than for the peptide (Zlokovic et al. 1990). The AVP

BBB receptors may represent either a part of peptide BBB transport system, as

demonstrated for insulin, IGF I and II, and transferrin (Pardridge 1988), or they

can mediate hormone effects on the BBB. In the latter case, the cellular uptake

of circulating peptide would be limited primarily to its binding at the blood-brain

interface, similar to that observed for the in situ steroid hormone binding

(Pardridge 1987).

The fact that small neuropeptides may be taken up intact at the luminal side

of the BBB does not rule out the possibility that they may be subsequently

metabolized in the adjacent compartments, such as the cytosolic endothelial

space, abluminal surface of the BBB, and surrounding neuropil. This

matter was investigated in more detail with AVP. High-performance liquid

chromatography analysis of ipsilateral forebrain homogenates depleted from

capillaries indicated that about 50 percent of the AVP remained in its intact

form after 1 minute, whereas the percentage of the intact peptide progressively

fell with time; after 10 and 15 minutes, the radioactivity was eluted primarily in

the fraction corresponding to L-(3H]-phenylalanine (Zlokovic et al. 1991 a). This

suggests that there is normally a time-dependent, progressive aminopeptidase

degradation of circulating AVP once it has been transferred across the luminal

BBB side; the possibility that AVP is a precursor of more potent centrally active

fragments, such as [pGlu4,Cyte)-AVP-(4-9), has been suggested (Burbach

et al. 1983). On the other hand, thin-layer chromatography analysis of forebrain

homogenates has shown that DSIP remained predominantly in intact form in the

brain during the lo-minute period of perfusion, which is consistent with

previously demonstrated resistance of this peptide to hydrolysis after crossing

biological barriers (Banks et al. 1983).

Permeability of the special regions also has been examined by the VBP

model and in the in situ isolated perfused choroid plexus of the sheep. Kinetic

analysis revealed saturable AVP (Zlokovic et al. 1991 b) and DSIP (Zlokovic

et al. 1988b) uptake at the basolateral face of the choroid plexus with respective

Km values of about 30 and 5 nM. It is suggested that specific AVP uptake

mechanisms in the choroid plexus can detect circulating hormone primarily

by V1-receptors. Saturable cellular uptake in the choroid plexus also has been

found for leu-enk (Zlokovic et al. 1988c), but not for TRH (Zlokovic et al. 1985b).

Selective permeability to blood-borne AVP has been demonstrated by

immunohistochemical studies in the paraventricular nucleus (PVN) of the

hypothalamus. In these experiments, brains were perfused with unlabeled

AVP, and immunoreaction product was found to lightly stain only the

33
perivascular space of the PVN, but not of the other BBB regions (B.V. Zlokovic,

L. Perlmutter, J.G. McComb, and C. Segal, unpublished data). This pattern

may be due either to selective permeability of PVN to the circulating AVP or to

locally induced hormone release. Specific uptake mechanisms for AVP also

have been described in the hypothalamo-pituitary axis and pineal gland

(Zlokovic et al. 1991 c), and it Is suggested that AVP uptake in these regions

represents hormone binding in situ primarily to the V1-type receptors.

CIRCULATING NEUROACTIVE AMINO ACIDS

The BUI method indicated the presence of a low-capacity independent

carrier system at the BBB, transporting two neurotransmitters, L-glutamic

and L-aspartic acid (Oldendorf and Szabo 1976). Those studies were

extended to determine Michaelis-Menten parameters for L-glutamic acid

using the VBP model (Davson et al. 1990). Kinetic experiments revealed

regional Michaelis constant between 3 and 5 μM and capacity that was three

orders of magnitude lower than for neutral and basic amino acids. Regional

unidirectional saturable influx of L-glutamic acid was inhibited by 72 to 94

percent in the presence of 0.5 mM L-aspartic acid. These results indicate that

the carrier-mediated system for L-glutamic acid located at the luminal BBB side

may be fully saturated at normal plasma concentrations of this amino acid.

The physiological importance of this system and its relationship with N-methylD-aspartate (NMDA) receptors are under investigation. Unidirectional transfer

rate constants for amino acid neurotransmitters and 5-HT are given in table 3.

It can be seen that studied neurotransmitters exhibit significantly higher

cerebrovascular permeability than N-methyl-a-aminoisobutyric acid (MeAIB),

a model substrate for the A amino acid transport system (Zlokovic et al. 1986).

IMPLICATIONS FOR DRUG ABUSE

It has been shown that tricyclic antidepressants (e.g., amitriptyline, imipramine),

which act as indirect adrenergic agonists, increase the diffusibility of water

across the BBB (Prescorn et al. 1980). On the basis of these experiments,

it has been suggested that there is a neuronal system acting in a humoral

fashion to regulate the movement of substances across the BBB. The

authors have explored this hypothesis by testing BBB permeability to inert

polar molecules, leu-enk, AVP, glutamic acid, and GABA using the chronic

amphetamine intoxication animal model. The VBP technique was employed to

ensure control over peripheral factors (e.g., central hypertension) during tests

of BBB functions. The behavioral syndrom—consisting of increased locomotor

activity, stereotypy (continuous gnawing and sniffing of the cage floor and side

bars), absence of normal exploratory and grooming behavior, and loss of

weight-was developed in all animals treated with amphetamine for at least 14

days (Rakic et al. 1989). The effect of withdrawal was studied in animals that

34
TABLE 3. Uptake of amino acid neurotransmitters, 5-HT, and MeAlB at the

luminal side of the BBB

Neurotransmitter KIn (μL min-1 g-1)

L-[3H] Glutamic acid

[

3H] Glycine

[

3H] GABA

5-[3H] HT

[

14C] MeAlB

6.73±0.20

4.62±O.49

1.09±0.20

1.02±0.35

0.75±0.07

NOTE: Values are means±SE of 5 to 24 perfused brains. KIn, values are

significantly different by ANOVA, except for GABA and 5-HT. KIn

values were graphically estimated using the equation given in figure 2

SOURCE: Values for glutamic acid and MeAlB are taken from Davson et al.

1990 and Zlokovic et al. 1986.

had been treated with amphetamine for 20 days. The behavioral syndrome in

these animals progressively subsided and was absent by 7 days. Figure 3

illustrates the changes in the ratio of cerebrovascular permeability constants for

D-mannitol and PEG 4,000 simultaneously perfused in the guinea pig forebrain

during the course of the induced amphetamine intoxication and recovery. It can

be seen that ratio falls to about 1 after 14 days of treatment and stays about the

same after the extension of treatment to 20 days. A spontaneous recovery of

BBB to metabolically inert molecules was established 28 days after cessation of

treatment. Opening of the BBB also was confirmed histologically by penetration

of the fluorescent dye Lucifer yellow in animals treated with amphetamine for 14

days. A marked amphetamine-induced increase in leu-enk and AVP BBB

uptake was shown after 20 days of amphetamine treatment. KIn values for

both peptides exhibited similar time-dependent changes, suggesting an initial

fourfold to fivefold increase in BBB uptake (figure 4). KIn vs. time curves may

indicate no change in affinity but a significant increase of capacity of both

peptide systems. It is noteworthy that only a modest increase in BBB

permeability to TRH was measured. It also has been demonstrated that

amphetamine significantly increases BBB uptake of glutamic acid and GABA

(table 4).

Metaphit, an isothiocyanate analog of phencylidine (PCP) has been shown to

induce audiogenic seizures (Debler et al. 1989). The authors have examined

BBB permeability to glutamic acid In the guinea pig with metaphit-induced

audiogenic seizures. A significant reduction of glutamic acid brain uptake was

found in comparison with control animals. This finding is in contrast with an

35
days

treatment withdrawal

FIGURE 3. Cerebrovascular permeability constant, P, mannitol/PEG ratio

for vascularly perfused guinea pig forebrain during the course

of chronic amphetamine intoxication after 14 and 20 days of

treatment with D-amphetamine sulphate (i.p., 5 mg/kg daily), and

7 and 28 days after cessation of 20 days chronic treatment with

amphetamine. Interrupted line represents Pmonnitor/PPEG ratio

obtained in control, nontreated animals; dashed line represents

mannitol/PEG ratio of reciprocal values of the square root of

molecular weight. Olive oiL:water partition coefficient ratio of

mannitol/PEG is 1:30. Values are means of 5 to 12 animals.

SOURCE: Rakic et al. 1989, copyright 1989, Elsevier Science Publishers

increased cerebrovascular permeability to horseradish peroxidase observed

in electrically induced seizures (Westergaard et al. 1978) and may suggest

inhibition of specific uptake mechanism for the neurotransmitter. It has been

proposed that metaphit may bind irreversibly to a site allosterically linked to

NMDA receptor (Debler et al. 1989), and the authors’ preliminary experiments

demonstrated cross-inhibition between glutamic acid and NMDA BBB uptake.

36
FIGURE 4. Time-dependent changes of KIn leu-enk values in the forebrain of

guinea pigs treated with amphetamine for 20 days as explained in

figure 3. Curve is computed on the basis of 10 animals that were

perfused at different times up to 20 minutes; interrupted lines are

confidence bands.

It has been demonstrated that tobacco smoking results in increased plasma

AVP levels, and it has been suggested that some reinforcing and withdrawal

effects of nicotine may depend on AVP psychoactive actions (Fuxe et al.

1989). The authors have shown that chronic 14-day treatment with nicotine

administered to guinea pig by osmotic minipumps alters BBB permeability to

AVP and hormone binding to non-BBB regions. An increased uptake was

found in the caudate nucleus, as well as in capillary-depleted brain tissue and

cerebral microvessels (M. Lipovac, B.V. Zlokovic, L. Perlmutter, and E. Barrone,

37
TABLE 4. Uptake of L-glutamic acid and GABA by the parietal cortex in the

guinea pig treated with amphetamine for 20 days

Treatment [

3H] Glutamic Acid [

3H] GABA

Control

Amphetamine

0.105±0.006 (9) 0.021±0.001 (5)

0.151±0.008 (6)* 0.079±0.004 (4)*

NOTE: Values are means±SE (n); *p<0.001. CBR/CPL is the ratio of

neurotransmitter radioactivity per unit mass of the brain and plasma

measured during the 10-minute period of brain perfusion. Treatment

with amphetamine is explained in figure 3.

unpublished data). Parallel preliminary ultrastructural examination of

neurohypophysis has revealed fewer dense core vesicles than in control

animals. As many of these vesicles contain AVP, this decrease is consistent

with the reported increase in plasma AVP levels that accompanies chronic

nicotine exposure. In addition, neurohypophyseal capillaries from nicotinetreated animals had prominent finger-like projections into the lumen, and the

enclosure of axon terminals by pituicyte cytoplasm, a well-documented normal

configuration of the region in times of low hormone demand (Hatton et al. 1984)

was seen only in the control animals.

CONCLUSION

Cerebrovascular permeability to slowly penetrating neuropeptides (leu-enk,

AVP, DSIP, TRH) and neurotransmitters (glutamic acid, glycine, GABA, 5-HT)

is 5 to 120 times higher than for metabolically inert polar molecules. Olive

oil:water partition coefficients and molecular weights are not good predictors

of BBB permeability to small peptides. Neuropeptides do not use BBB amino

acid transporters, and enzymatic hydrolysis may not necessarily be the primary

event at the BBB. Specific uptake systems for leu-enk, AVP, DSIP, and

glutamic acid have been demonstrated at the BBB and basolateral face of the

choroid epithelium. There is no saturable brain uptake of peptide metabolites.

Specific peptidergic receptors can mediate slow BBB uptake of circulating

peptide and binding to non-BBB regions and the anterior pituitary. BBB

postluminal step may result in transformation of peptide to its more potent

neuroactive fragments. BBB restrictive properties, specific uptake mechanisms,

and receptor sensitivity to neuroactive peptides and amino acids are influenced

by behaviorally active drugs, such as tricyclic antidepressants, amphetamine,

metaphit, and nicotine. The psychotropic drugs should no longer be assumed

38
to cause their effects solely by altering neuron interactions. Therefore,

therapeutic approaches In the treatment of addiction should complement

restorative neurotransmitter and BBB therapy.

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ACKNOWLEDGMENTS

This work was supported by the Division of Neurosurgery, Children’s Hospital of

Los Angeles: the Wellcome Trust: the British Council Senior Fellowship (BVZ);

and by funds provided by the Cigarette and Tobacco Surtax Fund of the State

of California through the Tobacco-Related Disease Research Program of the

University of California, grant 2RT 0071.

AUTHORS

Berislav V. Zlokovic, M.D., Ph.D.

Associate Professor of Neurosurgery, Physiology, and Biophysics

J. Gordon McComb, M.D.

Professor of Neurosurgery

Lynn Perlmutter, Ph.D.

Assistant Professor of Neurology

Martin H. Weiss, M.D.

Professor and Chairman of Neurosurgery

University of Southern California School of Medicine

RMR 407

2025 Zonal Avenue

Los Angeles, CA 90033

Hugh Davson, D.Sci.

Professor of Physiology

Sherrington School of Physiology

UMDS of Guy’s & St. Thomas’s Hospitals

London SE1 7EH

UNITED KINGDOM

42
MPTP Toxicity and the “Biochemical”

Blood-Brain Barrier

Sami I. Harik

The discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes

parkinsonism in humans (Davis et al. 1979; Langston et al. 1983) caused a

major revolution in scientific research on the etiology, treatment, and prevention

of Parkinson’s disease (Snyder and D’Amato 1986; Singer et al. 1987; Kopin

and Markey 1988). MPTP is now used to induce in nonhuman primates a

clinical, pathological, and neurochemical condition, which is probably the best

available experimental model of Parkinson’s disease (Burns et al. 1983;

Bankiewicz et al. 1986).

Although the mechanisms that underlie MPTP neurotoxicity are not entirely

clear, investigations during the past few years have revealed several important

points. First, MPTP is neurotoxic to humans and other primates when given

parenterally in small doses, but chemists have synthesized MPTP in large

quantities for years without taking unusually strict precautions and, with one

exception (Langston and Ballard 1983) without evidence of neurotoxicity.

Thus, MPTP neurotoxicity might not have been discovered had the unfortunate

drug users not injected MPTP intravenously. It is likely that MPTP is detoxified

by other organs when taken by other routes. Second, MPTP is not the ultimate

neurotoxin. MPTP oxidation, mainly by the enzyme monoamine oxidase B

(MAO-B), appears to be a necessary step for neurotoxicity since pretreatment

of experimental animals with MAO-B inhibitors prevents MPTP neurotoxicity

(Heikkila et al. 1984; Langston et al. 1984; Markey et al. 1984). MPTP oxidation

to the intermediate metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium

(MPDP+), and eventually to the neurotoxic culprit, 1-methyl-4-phenylpyridinium

(MPP+), is presumed to take place in the brain, primarily in glia (Uhl et al. 1985;

Snyder and D’Amato 1986). The reason for considering glia as the main site

for MPTP oxidation is that most of the MAO-B activity of the brain is thought to

be in glia (Levitt et al. 1982; Westlund et al. 1985; Konradi et al. 1988). Third,

MPDP+ and MPP+ are then exuded via an unknown mechanism into the

extracellular space where they are selectively taken up by dopaminergic

neurons that are equipped with the high-affinity dopamine uptake pump (Javitch

et al. 1985). Concentration of MPP+ by dopaminergic neurons may explain their

4 3
selective vulnerability. This view is supported by the finding that specific

inhibitors of high-affinity dopamine uptake decrease MPTP neurotoxicity

(Ricaurte et al. 1985). Fourth, MPP+ is further concentrated in mitochondria of

dopaminergic neurons by an energy-dependent uptake process (Ramsay and

Singer 1986), eventually causing metabolic failure by interfering with cellular

respiration at Complex I (Nicklas et al. 1985; Ramsay et al. 1986, 1987).

However, this story has some loopholes and is dependent on several factors

that are not universally accepted. For example, it is thought that MPTP

permeates cell membranes easily in view of its lipophilicity, while MPP+ cannot

cross the lipid bilayer of cell membranes because it is a polar, water-soluble

compound. Systemic administration of MPP+ is not neurotoxic (Fuller and

Hemrick-Luecke 1986; Johannessen et al. 1986) because of its inability to cross

the blood-brain barrier (BBB) (Riachi et al. 1989). However, most scenarios of

MPTP toxicity assume that MPP+ exudes easily out of astrocytes (Snyder and

D’Amato 1986). This matter needs clarification and further examination,

A most intriguing aspect of MPTP neurotoxicity is that not all animals are

equally susceptible. In fact, the most frequently used laboratory animals are

quite resistant to MPTP neurotoxicity. Wistar and Sprague-Dawley rats are

unaffected by MPTP in doses as high as 50 mg/kg/day given subcutaneously

for several weeks (Harik et al. 1988). In contrast, as little as 0.2 mg/kg/day of

MPTP given for 2 days to African Green monkeys causes dramatic clinical

and biochemical parkinsonism and often results in death (Elsworth et al.

1987). The dramatic differences among mammals in their susceptibility to

MPTP neurotoxicity drew attention to the biological bases that underlie these

differences, not only because of their theoretical and heuristic value but also

because of their possible immense practical implications in designing strategies

for the treatment or prevention of Parkinson’s disease. If MPTP toxicity has

anything to do with Parkinson’s disease, and if the ability of rats to withstand

massive doses of MPTP can be transferred to humans, this would have major

clinical significance.

Several possible explanations have been put forward for the differences

among mammalian species, and even among strains of the same species,

in their susceptibility to MPTP neurotoxicity. One explanation is related to

neuromelanin. Humans and higher primates have more melanin in the

substantia nigra than other mammals. It was hypothesized that MPTP and/or

MPP+ bind to neuromelanin (D’Amato et al. 1986). Suggestions that aged

animals and strains of mice with skin pigments are more sensitive to MPTP

neurotoxicity than younger animals and albino strains were used to support the

melanin hypothesis. The implications are that neuromelanin accumulates with

age and that mouse or rat strains with dark skin have more melanin in their

44
midbrain. There is no direct evidence to support either of these contentions.

Furthermore, the melanin hypothesis does not explain why, after MPTP

administration, the toxin and its metabolites accumulate primarily in the striatum

(Johannessen et al. 1985), where there is no melanin. The melanin theory

also ignores evidence suggesting that MPTP causes death of dopaminergic

substantia nigra neurons by retrograde degeneration after massive destruction

of their nerve endings in the striatum (Elsworth et al. 1987).

Another frequently used explanation for the differences in susceptibility to

MPTP neurotoxicity among species and strains is the variation in brain MAO-B

activity. CF1 albino mice, for example, were thought by Zimmer and Geneser

(1987) to be less sensitive to MPTP than C57BI mace because albino mice

have lower MAO-B activity in their brains, and thus, MPP+ will not be formed in

sufficient concentrations to cause neurotoxicity. However, biochemical assays

show that MAO-B activity is similar among crude mitochondrial preparations

of the brains of mice, rats, and humans, despite the major differences in their

susceptibility to MPTP neurotoxicity (Kalaria et al. 1987, 1988; Kalaria and Harik

1987),

The author and colleagues’ research group at Case Western Reserve

University first addressed the question of the rat’s resistance to systemic MPTP

neurotoxicity by microinfusing MPTP directly into the substantia nigra of Wistar

rats. One μmole of MPTP selectively destroyed the dopaminergic cell bodies

of the pars compacta and caused near total depletion of dopamine and its

metabolites in the ipsilateral striatum, without affecting nondopaminergic

neurons in the pars reticulata of the substantia nigra or glial cells (Sayre et

al. 1986; Harik et al. 1987). These experiments suggested that the unique

resistance of Wistar rats to MPTP neurotoxicity is not due to resistance by

dopaminergic neurons but is probably a reflection of insufficient MPP+

concentrations that accumulate in the rat substantia nigra.

Failure of MPTP metabolites to achieve toxic levels in the brarns of Wistar

rats can be due to detoxification of MPTP either in other organs or at the

BBB. Indeed, Riachi and colleagues (1988) found that Wistar rats have a

high capacity to detoxify and metabolize MPTP in the liver. To determine

whether brain capillaries from different mammals can metabolize MPTP,

isolated cerebral microvessels were assayed for their MAO activity. Large

variations in MAO activity were found among mammals that were inversely

related to their susceptibility to systemic MPTP neurotoxicity (Kalaria et al.

1987, 1988; Kalaria and Harik 1987). Initially this was demonstrated in

humans (who are exquisitely sensitive to MPTP), Wistar rats (which are

resistant to MPTP neurotoxicity), and albino mice (which are intermediate in

their sensitivity to MPTP neurotoxicity). MAO was assessed by irreversible

45
binding to [3H]pargyline and by oxidation of several substrates. Based on

substrate specificity, on the ability of the selective MAO inhibitors (clorgyline

and deprenyl) to block substrate oxidation, and on polyacrylamide gel

electrophoresis studies, the vast majority of rat brain capillary MAO was of the

B type (Kalaria et al. 1987; Kalaria and Harik 1987). The list of species was

extended later to include rabbits, pigs, and guinea pigs. MAO specific activity

was highest in brain microvessels of Wistar rats (Kalaria and Harik 1987). It

was surprising that the MAO content of brain microvessels of Wistar rats was

even higher than in the liver, the traditional tissue from which MAO is usually

obtained. In fact, MAO activity was higher in brain microvessels of Wistar rats

than in any other tissue that was ever investigated. This finding was even more

surprising because immunocytochemical studies have failed to identify MAO

activity in rat brain microvessels (Levitt et al. 1982). The reason(s) for this

anomaly remains unknown.

On the other hand, human cerebral microvessels are almost devoid of

MAO (Kalaria et al. 1987, 1988; Kalaria and Harik 1987). This is not

because human brain microvessels were obtained at autopsy after a

considerable postmortem delay. MAO is known to be quite resistant to

the effects of postmortem autolysis (Mackay et al. 1978). Also, rat brain

microvessels that were harvested from brains of rats that were decapitated

many hours earlier did not show diminution in their MAO content (Kalaria et al.

1987). The author and colleagues now have extended this work to monkeys.

Brain microvessels obtained from several types of monkeys were all as deficient

in MAO activity as human brain microvessels (Riachi and Harik 1990).

In addition to studying the differences among animal species, we also

investigated strains of the same species for differences in their susceptibility

to MPTP neurotoxicity. Two strains of mice, CF1 albino and C57BI, were first

studied because of the known difference in their sensitivity to MPTP (Zimmer

and Geneser 1987). The more sensitive strain had higher MAO activity in its

brain, which was thought to account for the more facile conversion of MPTP to

MPP+ (Zimmer and Geneser 1987). Riachi and Harik (1988) confirmed that the

C57BI mice are more sensitive to MPTP and also found that regional brain

levels of MPTP and its metabolites were higher in the striata of C57BI mice

but not in their cerebral cortices or cerebella. When MAO activity was assayed

in the cerebral cortices and striata of the two strains of mice, no significant

differences were found in the striata but higher MAO activity was found in the

cerebral cortices of C57BI mice. More important, significantly higher MAO

activity was found in isolated cerebral microvessels from CFl albino mice than

in cerebral microvessels of C57BI mice (Riachi and Harik 1988). This, coupled

with the finding that isolated brain microvessels from CFl albino mice also were

more capable of converting of MPTP to MPP+ in vitro than those from C57Bl

46
mice, agreed well with the hypothesis that the higher activity of MAO in brain

microvessels correlated inversely with the susceptibility of experimental animals

to MPTP.

This work has now been extended to five rat strains (Riachi et al. 1991). It was

found that not all rat strains are equally resistant to MPTP neurotoxicity. The

differences were correlated among rat strains in their susceptibility to MPTP

administered into the internal carotid artery in vivo, with MPTP oxidation by

the cerebral cortex, striatum. and brain microvessels in vitro. The in vivo work

was performed by assessing ipsilateral striatal depletion of dopamine and its

metabolites 1 week after injecting MPTP directly into the cerebral arterial

circulation. Dopamine depletion was found to be minimal in Sprague-Dawley

and Wistar rats, but there was considerable dopamine depletion in the striata

of the more sensitive rat strains that was MPTP dose-dependent. Differences

in striatal dopamine depletion among these rat strains correlated best with

differences in MAO activity in their isolated brain microvessels, but not with

differences in MAO activity in either their striata or cerebral cortices (Riachi et

al. 1991). These findings in the various rat strains are also consistent with the

hypothesis that the resistance of Wistar and Sprague-Dawley rats to MPTP

neurotoxicity is largely a property of their unique brain endothelium, which has

high MAO-B activity.

A major portion of the MPTP that is systemically administered to experimental

animals is sequestered and metabolized in peripheral organs, principally the

liver. To ascribe the rat’s resistance to systemic MPTP neurotoxicity to its

unique BBB. it had to be shown that neurotoxicity does not occur even when

MPTP is injected directly into the arterial circulation of the brain. In primates,

internal carotid arterial administration of MPTP was shown to be a most

effective means of inducing unilateral parkinsonism and nigrostriatal

degeneration, even when given in doses that are not effective when

administered systemically (Bankiewicz et al. 1986). Riachi and colleagues

(1989) assessed the ability of MPTP, MPP+, and butanol to cross the BBB in

rats by the indicator-fractionation method. As expected in view of their high

lipid solubility, both MPTP and butanol are almost completely extracted by the

brain on the first pass, but only minimal amounts of MPP+ cross from blood to

brain. However, within seconds to minutes, butanol is rapidly washed out of the

brain, while the [3PH] label of the MPTP tracer is retained for long periods. The

retention of MPTP by the brain, at least in the early periods, was not due to its

rapid metabolism by MAO because pargyline pretreatment did not affect MPTP

retention by the brain (Riachi et al. 1989). However, 30 minutes after MPTP

injection, brain retention of the [3H] tracer was reduced significantly by pargyline

treatment. Another finding of the pargyline pretreatment experiments is that a

substantial amount of the MPTP that was taken by the brain was converted to

47
MPP+

 even in the face of near total inhibition of MAO activity (Riachi et al.

1989). Other oxidative enzymes, such as the P450 enzyme system and the

flavin-containing monoxygenases, were previously suspected of having a role

in MPTP metabolism by other tissues (Weissman et al. 1985; Cashman and

Ziegler 1986), but little is known about these enzyme systems in the brain or its

microvessels. Also, pargyline pretreatment had no effect on the formation of

the lactam metabolites of MPTP (Riachi et al. 1989) that were previously

described by Arora and colleagues (1988).

Even when MPTP was administered into the internal carotid artery of Wistar

rats, up to 5 mg of MPTP had relatively minor effects on dopamine levels in

the ipsilateral striatum (Riachi et al. 1990, 1991). These minor effects were

abolished after pargyline pretreatment. Larger doses of MPTP caused death

from acute cardiorespiratory failure, and therefore larger doses of MPTP could

not be studied. Riachi and coworkers (1990) calculated that between 15 and

20 percent of the MPTP dose was taken up by the ipsilateral cerebrum, which

weighs about 0.7 g. This corresponds to more than 1mg of MPTP/g of rat

cerebrum. For comparison, injection of 2.5 mg of MPTP Into the internal carotid

artery of monkeys, which perfuses about 40 g of ipsilateral cerebrum, caused

near complete depletion of striatal dopamine and major behavioral and motor

abnormalities. Thus, the difference between rats and monkeys in their

response to internal carotid MPTP injections is obvious. These experiments

also provide crucial evidence that the resistance of Wistar rats to systemic

MPTP toxicity is not simply due to failure of MPTP to reach the brain circulation

because of sequestration and metabolism by peripheral organs but is indeed

a reflection of true resistance to blood-borne MPTP. This, coupled with the

selective degeneration of dopaminergic neurons when MPTP was infused

directly into the rat substantia nigra (Sayre et al. 1986; Harik et al. 1987),

strongly suggests that in the rat the blood-brain interface is a barrier to the

entry of MPTP to the brain extracellular space where it can act on dopaminergic

neurons.

If MPTP is taken up and retained by the rat brain but remains relatively

ineffective in causing dopaminergic neurotoxicity, where is it going and how is

it handled? The author and colleagues hypothesize that much of it is trapped

within the brain endothelium. However, a high concentration of MPTP and

MPP+ in rat brain endothelial cells may lead to their damage with consequent

BBB dysfunction. In another study, Riachi and coworkers (1990) investigated

in detail the effects on the BBB of MPTP given to rats via the internal carotid

artery. They found that 5 hours, 1 day, or 1 week after the Injection of MPTP

into the internal carotid artery of Wistar rats there were no significant differences

in the blood-to-brain transfer values for aminoisobutyric acid between the

ipsilateral and contralateral cerebral hemispheres. Also, ultrastructural studies

48
looking for evidence of BBB leakage of horseradish peroxidase after MPTP

injections were unrewarding. These results indicate that rat brain endothelial

cells are either insensitive to MPTP and its metabolites or that, if they are at all

affected, such affection does not result in acute or chronic breaks in the integrity

of the BBB. Thus, if MPTP and its metabolites are sequestered in rat brain

endothelium, they must be within subcellular organelles and not available to

brain endothelial mitochondria. It is also possible that polar metabolites of

MPTP are being more efficiently dumped back into the circulation from rat brain

endothelium. This would be consistent with the results of Johannessen and

coworkers (1985), who showed higher levels of radioactivity in monkey brain

than in rat brain at longer time intervals after [3H]MPTP administration.

CONCLUSION

The major theme of this chapter is that brain endothelial cells are not simple

“tiles” that line capillaries to prevent the extravasation of blood cells and large

plasma proteins. The author and colleagues believe that brain capillary

endothelial cells are highly sophisticated and complex and, in addition to

their uniquely tight intercellular junctions that are credited with the “physical”

attributes of the BBB, they have a variety of active enzyme systems that are

capable of metabolizing lipid-soluble substances that can easily permeate the

“physical” barrier. These enzymes, in addition to specialized subcellular

organelles, can metabolize and sequester lipid-soluble toxins and prevent

their entry into the brain. These enzyme systems constitute the basis of the

“biochemical” blood-brain barrier, a concept first introduced by Bertler and

colleagues in 1966.

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ACKNOWLEDGMENTS

The work described in this chapter was supported by U.S. Public Health Service

grant HL-35617 and by the David S. lngalls Fund.

AUTHOR

Sami I. Harik, M.D.

Professor

Departments of Neurology and Neuroscience

Case Western Reserve University

2074 Abington Road

Cleveland, OH 44106

53
An Overview of the Multiple Functions

of the Blood-Brain Barrier

A. Lorris Betz

INTRODUCTION

The existence of a permeability barrier between the blood and the brain has

been known for more than a century; however, during the past two decades

there has been a tremendous expansion in knowledge of the multiple functions

of the blood-brain barrier (BBB). Besides forming a permeability barrier that

limits the movement of some solutes between blood and brain, the BBB is now

seen as a metabolically active tissue that facilitates and controls the brain

uptake of certain solutes while helping to maintain homeostasis within the

central nervous system (CNS). Current studies are stimulated not only by an

interest in understanding the normal physiology of the barrier and the role that it

plays in disease processes, but also by a desire to be able to manipulate barrier

permeability and thereby control the delivery of pharmaceuticals to the brain.

This chapter serves as an overview of the functions of the BBB. For a more

detailed discussion, the interested reader is referred to the other chapters in

this monograph and to other sources in the literature (Betz et al. 1989; Bradbury

1979; Fenstermacher 1984; Fishman 1980; Goldstein and Betz 1986; Oldendorf

1982; Pardridge 1988; Rapoport 1976; Spector and Johanson 1989).

THE BBB AS AN IMPERMEABLE WALL

German scientist Paul Ehrlich was the first to describe the existence of

a permeability barrier between the blood and the brain (Ehrlich 1885).

Following the injection of a vital dye such as trypan blue into the bloodstream,

he observed that virtually all organs of the body stained blue except for the

brain. Whereas Ehrlich believed that the dye did not stain the brain because

the brain lacked the ability to bind the dye, his associate, Edwin Goldmann.

later disproved this hypothesis by showing that trypan blue, when injected into

the cerebrospinal fluid (CSF), did indeed stain the brain (Goldmann 1909).

This experiment also demonstrated the presence of a brain-blood barrier as

54
well as a BBB and, in addition, showed that there was no permeability barrier

between CSF and brain.

Although several investigators later hypothesized that the blood vessels in

the brain were the site of the BBB (Krogh 1946; Spatz 1933), this view was

not widely held. However, in the 1960s Reese and Karnovsky (1967) and

Brightman and Reese (1969) repeated the EhrlichIGoldmann experiments at

the ultrastructural level using electron microscopy to observe the distribution of

the protein tracer horseradish peroxidase following intravenous or intrathecal

administration. These experiments conclusively identified the brain capillary

endothelial cell as the site of the BBB as well as the brain-blood barrier. Of

primary importance in the formation of a permeability barrier by these cells is

the presence of continuous tight junctions that seal together the margins of the

endothelial cells (figure 1). Furthermore, in contrast to endothelial cells in many

other organs, brain capillary endothelial cells contain no direct transendothelial

passageways such as fenestrations or channels (Brightman and Kadota, this

volume; Broadwell, this volume). Recent ultrastructural studies suggest that

there may be an endocytotic process that is capable of moving protein tracers

from the blood to the brain through the lysosomal and/or Golgi compartments

of the brain capillary endothelial cell (Broadwell, this volume); however, the

flux mediated by this pathway is substantially less than the transvascular

permeability observed in most other vascular beds.

The brain capillary endothelial cell forms a barrier even to molecules that are

much smaller than the protein tracers typically used in ultrastructural studies.

For example, the electron-dense ion lanthanum has been shown by electron

microscopy to be excluded by the tight junctions of the brain capillary

endothelial cells (Dorovini-Zis et al. 1983). A multitude of radiotracer

experiments using small molecules such as sucrose and mannitol also

demonstrate highly restricted brain uptake (Fenstermacher, this volume).

Finally, electrical measurements across the wall of blood vessels on the

surface of the frog brain (Crone and Olesen 1982) or rat brain (Butt et al.

1990) demonstrate a very significant barrier to the movement of ions since

the resistance of the vessel wall is at least 1,500 to 2,000 ð. cm2.

Although the vast majority of the brain’s microvasculature is endowed with

these special features that result in the formation of a BBB, there are specific

localized areas of the brain, such as the circumventricular organs, that lack a

BBB (Gross and Weindl 1987). These brain nuclei are involved in the hormonal

regulation of other organ systems and therefore must be able to respond to

changes in the concentration of peptides and other substances in the blood.

The absence of a BBB in these regions is accompanied by differences in the

ultrastructure of the endothelial cells, including open junctions and fenestrations

55
Blood Endothelial

Cell

Brain

Polar

Solute

FIGURE 1. The BBB as an impermeable wall. The tight junctions and lipid

membrane of the brain capillary endothelial cell limit the diffusion

of polar solutes in both directions between the blood and the

brain.

and increased distance between the endothelial cells and the surrounding

astrocytes (Laterra et al., this volume; Fenstermacher, this volume; Broadwell,

this volume). To help limit the brain uptake of substances through these leaky

brain regions, the ependymal cells that the the ventricular surface and normally

do not form a barrier are differentiated into barrier-forming cells in these

localized areas (Brightman et al. 1975). Although the signals that induce brain

capillary endothelial cells and special ependymal cells to form a barrier are not

known, recent studies point to the astrocyte as an important source for such an

inductive influence (Laterra et al., this volume).

THE BBB AS A SELECTIVE SIEVE

Clearly, the BBB cannot be absolute. The brain IS dependent on the blood to

deliver metabolic substrates and remove metabolic wastes; therefore, the BBB

must facilitate the exchange of selected solutes. Lipid-soluble fuels and waste

56
products, such as O, and CO,, readily cross the lipid bilayer membranes of

the endothelial cell and, thus, encounter little difficulty in quickly exchanging

between blood and brain (figure 2). Polar solutes such as glucose and amino

acids, however, must depend on other mechanisms to facilitate their exchange.

This is accomplished by the presence of specific, carrier-mediated transport

proteins in the luminal and abluminal membranes of the brain capillary

endothelial cell (figure 2).

The permeability of the 666 to lipid-soluble substances is not surprising in view

of the large surface area of endothelial cell membrane (e.g., 100 cm2/g brain in

the rat [Bradbury 19791) that separates the brain from the blood. In fact, the

Blood Endothelial

Cell

Brain

Lipid-Soluble

Solute

Transported

Solute

FIGURE 2. The BBB as a selective sieve. Lipid-soluble solutes can cross the

BBB because they are able to penetrate the lipid bilayers of the

endothelial cell. In addition, selected polar solutes can cross the

BBB because specific, carrier-mediated transport systems are

present in the endothelial cell membranes.

57
relationship between the BBB permeability and the lipid solubility of a

nontransported solute follows exactly the relationship predicted by studies

with artificial lipid bilayers (Fenstermacher, this volume; Oldendorf, this volume).

Thus, highly polar compounds such as mannitol penetrate the barrier very

slowly, whereas lipid-soluble compounds such as butanol penetrate so rapidly

that they are completely extracted from the blood by the brain during a single

pass through the cerebral circulation. Uptake of very lipid-soluble compounds

is so high that it is only limited by blood flow (Fenstermacher, this volume), and

these substances are useful both clinically and experimentally for measuring

the cerebral blood flow rate.

Seemingly small differences in lipid solubility can make a large difference in

brain uptake. For example, the olive oil:water partition coefficient of heroin

is only 10 times greater than that of morphine, yet the brain uptake of heroin is

30 times higher (Oldendorf 1982). This explains the greater speed with which

heroin exerts its CNS effects compared with morphine, and it may contribute to

its higher abuse potential (Oldendorf, this volume).

Although lipid-soluble compounds can enter the brain quite readily, most

important metabolic substances are too polar to enter brain efficiently by

simple diffusion. For these compounds, carrier-mediated transport systems

are present to facilitate their brain uptake. The existence of carrier-mediated

transport systems that operate across the BBB can be deduced on the basis

of the selectivity that results from the binding of the solute to a recognition site

on the carrier protein. Thus, carrier-mediated transport is saturable because

the number of binding sites is limited; it is stereospecific because a specific

structural arrangement of the solute is required for it to be recognized by the

carrier; it can be competitively inhibited by structurally related compounds that

compete for binding to the same carrier or noncompetitively by compounds

that bind elsewhere on the carrier protein; and the rate of transport may be

regulated by intracellular second messengers and/or by adding or removing

carrier molecules from the membrane.

Because the brain uses glucose almost exclusively as a metabolic fuel, the

BBB is richly endowed with glucose transporters, and this carrier system has

been extensively studied (Betz et al. 1976; Lund-Andersen 1979; Pardridge

1983). The BBB glucose carrier recognizes D-glucose, D-galactose, Dmannose, and certain synthetic hexoses such as 2-deoxy-D-glucose and 3-Omethyl-D-glucose, but not L-glucose. Transport is half saturated at a plasma

glucose concentration of 5 to 10 mM, which is within the normal physiologic

range. BBB glucose transport is not energy dependent; therefore, it cannot

move glucose against a concentration gradient. It is also symmetrical and

transports glucose out of as well as into the brain. Thus, it is a facilitative58
diffusion type of transporter that facilitates the equilibration of blood and brain

glucose concentrations. In contrast to glucose transport in certain other cells,

BBB glucose transport is not regulated by insulin. Recent studies of the

quantity and distribution of glucose transporters in brain indicate a very rich

endowment in brain capillary endothelial cells, consistent with their role in

providing glucose for all the cells in the brain (Dick et al. 1984; Gerhart et al.

1989).

Another prominent transport system in the BBB enhances the brain uptake of

large neutral amino acids, which are important precursors for neurotransmitter

and protein synthesis. This carrier is similar to the L-system for amino acid

transport described in other cells in that it has high affinity for large but not

small neutral amino acids and is not sodium dependent (Betz and Goldstein

1978). In contrast to the glucose transporter for which glucose is the only

naturally occurring substrate present in the blood at a significant concentration,

the L-system amino acid carrier has affinity for at least 10 different amino acids

that are present in the blood (Smith et al. 1987). Indeed, the carrier seems

to be quite insensitive to the size of the hydrophobic side chain (Rapoport,

this volume). The presence in blood of multiple substrates for the same

carrier means that they compete with each other for transport into brain.

Consequently, when the plasma concentration of one amino acid is increased,

it can reduce the uptake of other amino acids, which may play a role in the

neurologic damage that occurs in certain metabolic diseases such as

phenylketonuria (Pardridge 1986).

It is now apparent that the BBB has many transport systems in addition to

those for glucose and amino acids (table 1). Those with the greatest transport

capacity were described first (metabolite transport systems), but as more

sensitive techniques are developed to study BBB permeability, low-capacity

transporters such as those for vitamins (Spector 1989), ions (see next section),

and peptides (Zlokovic et al., this volume; Pardridge, this volume) have been

detected. The peptide transport systems may involve a process of receptormediated endocytosis (Broadwell, this volume), a mechanism that is quite

different from the carrier-mediated transport of small solutes.

THE BBB AS AN ACTIVE PUMP

Not all BBB transport systems operate equally well in both the blood-to-brain

and brain-to-blood directions. For example, potassium is transported out of

the brain by a saturable transport system that can be inhibited by ouabain

(Bradbury and Stulcova 1970), whereas the flux of potassium from blood to

brain is low (Hansen et al. 1977) and not inhibited by ouabain (Betz 1989). This

asymmetrical transport of potassium is explained by the presence of the active

59
TABLE 1. Transport systems that operate from blood to brain

Transport System Typical Substrate

Metabolites

Hexose

Large neutral amino acid

Basic amino acid

Acidic amino acid

Monocarboxylic acid

Amine

Purine

Nucleoside

Saturated fatty acid

Micronutrients

Thiamine

Pantothenic acid

Biotin

Vitamin B6

Riboflavin

Niacinamide

Carnitine

lnositol

Electrolytes

Sodium-chloride cotransport

Cation channel

Heavy metal

Hormones

Thyroid hormone

Vasopressin

Insulin

Other Peptides

Transferrin

Enkephalins

Glucose

Phenylalanine

Lysine

Glutamate

Lactate

Choline

Adenine

Adenosine

Octanoate

Thramine

Pantothenic acid

Biotin

Pyridoxal

Riboflavin

Niacinamide

Carnitine

Myo-inositol

Sodium chloride

Sodium, potassium

Lead

T3

Arginine vasopressin

Insulin

Transferrin

Leu-enkephalin

60
pump Na,K-ATPase on the abluminal but not the luminal side of the brain

capillary endothelial cell (Betz et al. 1980). A similar asymmetry in the

distribution of ion transporters is seen in fluid-transporting epithelial cells such

as those in the choroid plexus. This cellular polarity forms the basis for their

ability to actively secrete ions and water (Betz and Goldstein 1981). Other

features that the brain capillary endothelium has in common with a typical

epithelium (Betz 1985a; Crone 1986) include the presence of continuous tight

junctions, a transcellular electrical resistance of approximately 2,000 cm2

(Crone and Olesen 1982), a low hydraulic conductivity (Fenstermacher 1984)

and a high mitochondrial content (Oldendorf et al. 1977), which is believed to

be related to a high capacity for active transport. Since the brain capillary

shares so many structural features in common with epithelia, it has been

proposed that it functions like an epithelium as well (Betz 1985a; Crone 1986).

If the BBB does indeed secrete fluid, this could be the explanation for the 10 to

30 percent of CSF that is produced from extrachoroidal sources (Milhorat et al.

1971); and it could account for the bulk flow of fluid through the brain’s

interstitial space (Szentistvanyi et al. 1984).

In addition to the presence of Na,K-ATPase on one side, fluid-secreting

epithelial cells contain other sodium transporters on the opposite side that

allow sodium to enter the cell (figure 3). Using intracarotid bolus injections to

study single-pass sodium uptake, Betz (1983) observed two saturable sodium

entry systems in the BBB. One is inhibited by furosemide, suggesting the

presence of a Na-Cl cotransport system. The other is inhibited by amiloride.

but its properties are not clearly identical to either the known amiloride-sensitive

sodium channel or the Na/H exchanger. Specifically, Betz (1983) showed that

the brain capillary sodium channel is highly sensitive to inhibition by amiloride

as IS the sodium channel in tight epithelia, but it is nonselective since amiloride

also inhibits brain rubidium uptake. Recent studies confirm that brain sodium

uptake can be inhibited by amiloride (Murphy and Johanson 1989) and that

brain capillary endothelial cells contain an amiloride-sensitive cation channel

that is permeable to both sodium and potassium (Vigne et al. 1989).

Active transport of ions across the brain capillary endothelium may play a role

not only in fluid secretion but also in regulation of the potassium concentration

of the brain’s interstitial fluid (ISF). The potassium concentration of the CSF

and ISF is held remarkably constant at approximately 3 mM despite acute

or chronic changes in plasma potassium between 1.6 and 7 mM (Bradbury

and Stulcova 1970; Jones and Keep 1987). The low permeability of the BBB

to potassium in the blood-to-brain direction contributes to this potassium

homeostasis (Hansen et al. 1977). However, potassium efflux (Bradbury and

Stulcova 1970), mediated by Na,K-ATPase located on the abluminal side of the

brain capillary endothelial cell (Betz 1986; Betz et al. 1980) and the apical side

61
Blood Endothelial

Cell

Brain

Luminal Abluminal

Membrane Membrane

FIGURE 3. The BBB as an active pump. Not all transport systems are evenly

distributed between the luminal and abluminal membranes of the

brain capillary endothelial cell. An asymmetric distribution of ion

transporters and channels could provide the basis by which the

BBB secretes fluid and helps maintain potassium homeostasis

in brain.

of the choroid plexus epithelial cell (Ernst et al. 1986), also plays a major role.

Given that the total influx of potassium across the BBB is 10 times greater than

across the blood-CSF barrier, the brain capillary is probably more important

than the choroid plexus in brain potassium homeostasis (Bradbury 1979).

Astrocytic glial cells also are believed to play an important role in maintaining

potassium homeostasis in brain (Bradbury 1979; Kimelberg and Norenberg

1989). This specialized glial cell has a large potassium conductance in its

62
end-feet that allows it to quickly take up potassium from the surrounding fluid

(Newman 1986). Localized uptake in an area where potassium is high is

accompanied by the dumping of potassium in another area where its

concentration is low. This type of spatial buffering of potassium is believed

to be important for the second-to-second regulation of localized ISF potassium

concentration (Kimelberg and Norenberg 1989) but it cannot by itself account

for long-term global regulation of ISF potassium. For this, the brain capillary

endothelial cell probably plays the primary role. One reason the brain capillary

endothelial cell is so closely invested with glial foot processes may be to permit

astrocytes and endothelial cells to work together to maintain potassium

homeostasis.

The presence of other asymmetrically distributed transporters in the brain

capillary endothelial cell (table 2) can be deduced by differences in transport

rates in the blood-to-brain and brain-to-blood directions as explained above.

Another approach is to study uptake by isolated brain capillaries in vitro.

Since these structures have their abluminal membranes exposed to the

incubation medium, transporters that are present on this side of the cell

can be studied easily. Thus, those substances that are taken up by isolated

capillaries, but that do not appreciably enter the brain from the blood, probably

have transporters on the abluminal but not the luminal membrane of the

endothelial cell. This approach has been used to demonstrate an asymmetry

of A-system (Betz and Goldstein 1978) and ASC-system (Tayarani et al. 1987a)

transporters for small neutral amino acids as well as transporters for taurine

(Tayarani and Lefauconnier 1989) and monoamines (Hardebo and Owman

1980a). However, one must be aware that the isolated brain capillaries contain

other cells, such as pericytes, that also might be transport sites.

TABLE 2. Asymmetrically distributed BBB transport systems

Transport System Typical Substrate Probable Location

Na,K-ATPase

Sodium-chloride cotransport

Cation channel

Sodium-hydrogen exchanger

A-system for neutral amino adds

ASC-system for neutral amino acids

B-amino acid

Monoamine

Organic anion

Inorganic anion

Prostaglandin

Potassium

Sodium chloride

Sodium, potassium

Sodium, hydrogen

Glycine

Cysteine

Taurine

Dopamine

p-Aminohippurate

Iodide

pGF20

Abluminal

Luminal

Luminal

Abluminal

Abluminal

Abluminal

Abluminal

Abluminal

Abluminal

Abluminal

Abluminal

63
Finally, the presence of active transport out of the brain can be deduced by

using transport inhibitors. When a substance is present under steady-state

conditions at a lower concentration in the brain’s ISF than in blood and a

transport inhibitor increases the brain concentration, then the presence of an

active efflux system can be deduced (Davson 1976). This approach has been

used to demonstrate active BBB efflux systems for iodide (Davson and

Hollingsworth 1973) prostaglandins (Bito et al. 1976), and organic acids

such as p-aminohippurate (S.R. Ennis and A.L. Betz, unpublished results) and

acid neurotransmitter metabolites (Aizenstein and Korf 1979).

THE BBB AS A METABOLIC BARRIER

The foregoing discussion emphasizes that the BBB is formed by a cell layer

with different properties of the membranes on either side. These cells also

have an intracellular cytoplasm and organelles containing specific enzymes

that play an important role in BBB function (figure 4). One of the first

demonstrations of the enzymatic BBB was made by Bertler and colleagues

(1966) who observed intraendothelial histofluorescence of dopamine after

intravascular administration of its precursor, L-3,4-dihydroxyphenylalanine

(L-dopa). In later studies, they found that rat brain capillary endothelial cells

were richly endowed in enzymes involved with neurotransmitter synthesis

(e.g., aromatic amino acid decarboxylase) and degradation (e.g., monoamine

oxidase [MAO]) (Hardebo and Owman 1980b). Since neurotransmitter

precursors such as L-dopa can enter the endothelial cell from the blood via the

L-system for large neutral amino acid transport, the subsequent intraendothelial

metabolism provides an effective mechanism for preventing neurotransmitters

from moving beyond the endothelial cell and Into the brain. Neurotransmitterdegrading enzymes present in the endothelial cells also may play a role in

inactivating neurotransmitters released during neuronal activity since, as

discussed previously, transport systems for uptake of catecholamines by

the endothelial cell appear to be present on the abluminal membrane.

Brain capillary endothelial cells contain many enzymes in addition to those

involved with neurotransmitter metabolism (Mrsulja and Djuricic 1981). Besides

the enzymes involved In energy metabolism (Mrsulja and Djuricic 1981), these

cells also contain acid hydrolases typically found in lysosomes (BaranczykKuzma et al. 1989) and aminopeptidase (Baranczyk-Kuzma and Audus 1987).

Similar to other endothelial cells, they contain angiotensin-converting enzyme

(Gimbrone et al. 1979) xanthine oxidase (Betz 1985b), and enzymes that

protect cells from peroxidatrve damage such as superoxide dismutase,

catalase, and glutathione peroxidase (Tayarani et al. 1987b).

64
Blood Endothelial

Cell

Brain

FIGURE 4. The EBB as a metabolic barrier. Some molecules that enter the

brain capillary endothelial cell are rapidly metabolized. This

process not only helps control the brain uptake ofpotentially

harmful chemicals present in the blood but a/so assists the brain

in eliminating unwanted waste products. For example, the

neurotransmitter precursor L-dopa enters the endothelial cell on

the L-system amino acid transporter. It is then converted to

dopamine by the enzyme dopa decarboxylase present in the

endothelial cell. Dopamine is further degraded to 3,4-

dihydroxyphenylaceiic acid (dopac) by the enzyme monoamine

oxidase. Dopamine also may be taken up from the brain and

metabolized as a means of eliminating the neurotransmitter from

the brain’s ISF.

65
MAO present in brain capillaries may serve to provide protection of the

brain from circulating toxins (Harik, this volume; Takakura et al., this volume).

Indeed, brain capillary endothelial cells are well equipped to handle circulating

drugs and toxins since they contain drug-metabolizing enzyme systems

such as cytochrome P-450-linked monooxygenases, epoxide hydrolase,

NADPH:cytochrome P-450 reductase, and 1-napthol UDP-glucuronosyl

transferase, which are typically found in the liver (Ghersi-Egea et al. 1988);

they also contain a multidrug transport protein, P170 (Thiebaut et al. 1989).

REGULATION OF BBB FUNCTION

The availability of isolated brain capillaries as a model system for studying

properties of the BBB has led to the discovery that brain capillary endothelial

cells contain many neurotransmitter- and hormone-binding sites, as listed

below.

ß-Adrenergic

Adrenergic

Dopamine

Histamine

Adenosine

Muscarinic cholinergic

Prostaglandin

Leukotriene C4

Insulin

Transferrin

Vasoactive intestinal peptide

Parathyroid hormone

Atrial natriuretic peptide

Vasopressin

Angiotensin II

Bradykinin

These receptors have been detected either by directly studying binding of

radiolabeled ligands to the isolated capillaries or by observing changes in

intracellular second messengers following exposure to agonists (figure 5).

Beyond the changes in second messengers, however, little is known about

the effects of hormones or neurotransmitters on endothelial cell function. The

ß-adrenergic receptors appear to be in some way involved with regulation of ion

transport since ablation of adrenergic input to the cerebral microvasculature

by lesioning the locus ceruleus leads to up-regulation of ß-adrenergic receptors

on the endothelial cells (Kalaria et al. 1989) and a down-regulation of their

Na,K-ATPase activity (Harik 1986). Atrial natriuretic peptide (ANP) receptors

also may play a role in ion transport regulation because ANP inhibits the

amiloride-sensitive cation channel (Doczi et al. 1990; lbaragi et al. 1989).

In vivo experiments have shown that corticosteroids reduce the passive

permeability of the BBB (Betz and Coester 1990; Ziylan et al. 1988) and in

vitro experiments suggest that cAMP, cGMP, and adenosine also may be

involved in regulating barrier permeability (Horner et al., this volume). Finally,

some receptors (e.g., those for insulin, transferrin, and vasopressin) may play

a role in receptor-mediated transcytosis (Pardridge, this volume; Zlokovic et al.,

this volume) rather than regulating endothelial cell function.

66
Blood Endothellal

Cell

Brain

FIGURE 5. Regulation of BBB function. The brain capillary endothelial cell

contains receptors for many hormones and neurotransmitters,

substances that regulate BBB function through the production

of various second messengers. Neurons and glia are probable

sources for some of these regulatory factors, and others may

be present in the blood.

The endothelial cells also must contain other, as yet unidentified, receptors that

allow them to respond to modulators present in brain and probably released

by the astrocytes. As discussed elsewhere in this volume (Laterra et al., this

volume; Horner et al., this volume), glial cells may be an important source

of such signals since astrocytes or astrocyte-conditioned medium induce

morphological and functional changes in brain capillary endothelial cells in

tissue culture.

67
SUMMARY

The BBB formed by brain capillary endothelial cells is now recognized as

more than an impermeable wall. It has specific transport systems that

facilitate the uptake of important nutrients and hormones and active pumps

that help to regulate the concentrations of ions and metabolites in the brain’s

ISF. Enzymes present in the endothelial cell metabolize neurotransmitters.

drugs, and toxins before they can enter the brain and disrupt its function.

Many of these properties are likely to be under regulation either by

neurotransmitters and hormones released in the brain or by those present

in the systemic circulation. As a result of this constellation of diverse functions,

the brain capillary endothelial cell IS able to efficiently supply the brain with the

metabolites that it requires while contributing to the maintenance of the

brain’s ionic homeostasls and protecting it from circulating toxins.

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ACKNOWLEDGMENT

This work was supported in part by National Institutes of Health grant

NS-23870.

AUTHOR

A. Lorris Betz, M.D., Ph.D.

Professor

Departments of Pediatrics, Surgery (Neurosurgery), and Neurology

University of Michigan

D3227 Medical Professional Building

Ann Arbor, Ml 48109-0718

72
Formation and Differentiation of

Brain Capillaries

John Laterra, Johannes E.A. Wolff, Christopher Guerin, and

Gary W. Goldstein

INTRODUCTION

The blood-brain barrier (BBB), by regulating the exchange of substances from

blood to brain, maintains an interstitial microenvironment optimal for neuronal

function (Goldstein and Betz 1986a). Barrier function and dysfunction have

important implications for brain physiology, pathophysiology, and therapy

(Goldstein and Betz 1986b). An intact barrier may exclude potentially beneficial

therapeutic agents. Barrier dysfunction associated with tumors, inflammation,

trauma, and certain toxins may result in considerable morbidity from vasogenic

brain edema and alterations in neuronal environments.

The BBB was first described by Paul Ehrlich (1885), who observed that

certain vital dyes injected intravenously were specifically excluded from brain

parenchyma. Detailed anatomic studies have since localized the BBB to

capillary endothelial cells (Brightman and Reese 1969; Reese and Karnovsky

1967). These specialized endothelial cell functions appear gradually during

development with the morphogenesis of leaky vessels to complete barrier

expression in mature vessels. Recent studies described in this chapter indicate

that both brain microvessel morphogenesis and barrier expression may be

regulated by astrocytic cells. This chapter reviews evidence from the authors’

laboratory and several other laboratories supporting this view. The hypothesis

is presented that the earliest steps in brain capillary morphogenesis and the

subsequent development of BBB properties are influenced by two distinct

astrocytic “signals.” The authors believe the predominant signal is dependent

on the degree of astrocyte differentiation or activation.

BARRIER DEVELOPMENT IN VIVO

The brain is vascularized by sprouting vessels from the superficial perineural

vascular plexus (Bar and Wolff 1972), which is primarily sinusoidal and lined by

a fenestrated endothelium characteristic of nonbarrier vessels. In contrast. the

73
vascular sprouts penetrating brain parenchyma are composed of

nonfenestrated endothelial cells. With continued development, a mature

capillary-astrocytic complex is ultimately generated, consisting of endothelia,

pericytes, and basement membrane ensheathed by astrocytic foot processes.

Endothelial cells within this complex develop an array of barrier-specific

properties, including a continuous network of complex tight junctions, specific

glucose and amino acid transport systems, reduced vesicular density, and

increased mitochondrial density (Goldstein and Betz 1986a; Betz, this volume).

The most significant feature that distinguishes these from peripheral capillaries

is their intimate association with perivascular astrocytes, and the expression of

barrier coincides temporally with astrocytic ensheathement (Evans et al. 1974;

Stewart and Hayakawa 1987). Thus, the phenotypic specialization of the brain

capillary endothelium appears to be dependent on its unique relationship with

the surrounding brain parenchyma or, more specifically, with the ensheathing

perivascular astrocytes.

Svendgaard and colleagues (1975) were the first to ask experimentally if

barrier characteristics of newly formed vessels are determined by inductive

properties of the perivascular tissue. They assessed the development of

barrier in vessels investing either iris tissue transplanted to caudal diencephalon

or brain stem transplanted to the anterior chamber of the eye in rats. Vessels

within central nervous system (CNS) tissue transplanted to eye expressed

dopa-decarboxylase activity and limited the diffusion of 6-hydroxy-dopamine,

two barrier properties. In contrast, vessels within iris transplanted to CNS

lacked both properties. It was concluded that CNS parenchyma induces

endothelial cells to express BBB features, although the researchers could not

be sure that the vessels found within the transplants were host derived.

These findings were confirmed by the classic chimeric transplant studies

of Stewart and Wiley (1981) which demonstrated that vessels formed

in similar transplants were, in fact, host derived. More recently, Risau and

colleagues (1986) demonstrated that implantation of embryonic mouse

brain onto the chick chorioallantoic membrane induces a barrier-specific,

74-kilodalton endothelial antigen. This experiment showed that the brainderived signals inducing endothelial expression of barrier are conserved

between species.

The CNS-derived tissues used in the transplant experiments described

above contained neurons, oligodendroglia, and astrocytes. Of these cellular

components, astrocytes are the most likely candidate for inducing the BBB

due to their intimate association with brain microvessels. Direct experimental

evidence for this hypothesis has been provided by Janzer and Raff (1987).

They showed that microvessels from rabbit iris or chick chorioallantoic

membrane form a barrier to the diffusion of vital dye after growing into an

74
aggregate of rat brain astrocytes. Controls using meningeal fibroblasts

instead of astrocytes lacked barrier. Guerin and colleagues (1990)

recently obtained evidence from human pathological specimens that

extends the relevance of these findings to humans. They compared

vascular permeability and endothelial expression of the brain-type glucose

transporter in a series of human brain tumors using contrast-enhanced imaging

and immunohistochemistty. Despite the absence of normal neuronal elements,

vessels deep within both low-grade and anaplastic astrocytomas frequently

stained with glucose transporter antibodies (figure 1). The expression of this

barrier property diminished with increasing tumor grade. Interestingly, vessels

in six of nine anaplastic astrocytomas were highly permeable but continued to

express glucose transporter (figure 2). The independent expression of these

two barrier properties suggests that distinct mechanisms may be responsible

for their induction or maintenance.

BARRIER DEVELOPMENT IN VITRO

The expression of BBB properties by microvascular endothelial cells isolated

from the CNS have been examined in several in vitro culture systems. Early

investigations from the authors’ laboratory established that bovine brain and

retinal microvascular endothelial cells can be reliably cultured and used as

a model for study of the BBB. Using freeze fracture analysis, Goldstein

and colleagues (1984) demonstrated that monolayer endothelial cultures

develop tight junctions (figure 3) that exclude the diffusion of intermediate-size

molecules such as albumin and HRP and partially exclude smaller molecules

such as monosaccharides (Bowman et al. 1983). Similar to their effects in

vivo, hyperosmotic agents reversibly opened the tight junctions in vitro

(Dorovini-Zis et al. 1987). Despite these findings, cultured endothelial cells

were found to rapidly cease expressing several other BBB properties such

as glutamyl-transpeptidase and barrier function to small polar molecules

and ions. Although various technical reasons might have been responsible

for these deficiencies, this also may signify that an appropriate inductive

environment is required for the expression of this specialized endothelial

phenotype.

DeBault and Cancilla (1980) examined the presence of the barrier marker

glutamyl-transpeptidase in a brain-derived endothelial cell line (ME-2),

cultured in the presence or absence of C6 glioma cells. ME-2 cells lack this

enzyme when cultured alone but were found to develop histochemical evidence

of the enzyme after 72 to 96 hours in coculture with C6 cells. The frequently

cited conclusion that C6 astroglial cells induce the expression of glutamyltranspeptidase in endothelial cells is complicated by the fact that cultured

C6, cells constitutively express high levels of glutamyl-transpeptidase and

75
FIGURE 1. Low-grade human astrocytoma stained with (A) the endothelial

vascular marker Ulex europis lectin I and (B) rabbit antihuman

glucose transporter type l. Positive staining is evidenced by the

horseradish peroxidase (HRP) reaction product that appears

black. All vessels react positively with antiglucose transporter

antibody.

SOURCE: Guerin et al. 1990, copyright 1990, Annals of Neurology

messenger RNA (unpublished observations from the authors’ laboratory).

The possibility that endothelial-glial contact results in transfer of enzyme to

endothelial cells rather than induction was not addressed. Shivers and

coworkers (1988) qualitatively described the induction of interendothelial

tight junctions by astrocyte-conditioned media. Using quantitative freeze

fracture analysis of cultured cells, Tao-Cheng and coworkers (1987)

demonstrated that astrocytes, but not fibroblasts, markedly enhanced the

number and complexity of interendothelial tight junctions. In contrast to the

findings of Shivers and coworkers (1988), astrocyte-endothelial contact

was required. Dehouck and colleagues (1990) recently described the

development of BBB properties in bovine brain microvascular endothelial

cells grown on one side of a permeable membrane containing astrocytes

on the other side. These cultures formed a monolayer with high electrical

resistance and selectively restricted the diffusion of inulin and sucrose, but

not leucine, consistent with the presence of complex interendothelial tight

junctions and a functioning large neutral amino acid transport system.

However, a receptor-mediated basis for the selective passage of leucine

across the monolayer was not specifically demonstrated. Interestingly, large

vessel bovine endothelial cells under identical conditions failed to develop

these properties, suggesting that endothelial cells from different tissues vary

in their ability to respond to astrocytic influences. Whether these differences

76
% GLUCOSE TRANSPORTE

% CONTRAST ENHANCING

R POSITIVE

FIGURE 2. Frequency of glucose transporter positivity and contrast

enhancement as a function of tumor type. A: low-grade

astrocytoma (n=9); AA: anaplastic astrocytoma (n=9); GBM:

glioblastoma multiforme (n=10). Essentially all vessels of

normal cerebrum (nonenhancing by definition) are known

to be transporter positive. Low-grade astrocytomas and

glioblastomas demonstrate an inverse relationship between

Transporter expression and contrast enhancement. The

majority of anaplastic astrocyiomas are transporter positive

despite increased vascular permeability.

SOURCE: Guerin et al. 1990, copyright 1990, Annals of Neurology

77
FIGURE 3. (a) Cultured bovine brain microvessel endothelial cells are

joined by tight junctional complexes (arrows) and contain few

cytoplasmic vesicles and thin apical cytoplasmic folds. (b)

lnterendothelial tight junctions have a typical pentalaminar

structure. Bar=0.1 microns.

SOURCE: Dorovini-Zis et al. 1987, copyright 1987, Journal of Neuropathology

and Experimental Neurology

result from in vivo events before endothelial isolation or from differences in the

response of these cells to culture conditions is not known.

VESSEL FORMATION IN VIVO

The organization of endothelial cells into microvessels is a precondition to

BBB expression in vivo. It has long been realized that capillaries form in

developing neural tissue in close association with primitive glia (Bar and

Wolff 1972). Ling and Stone (1988) demonstrated that the in growth of retinal

microvessels is temporally and spatially coordinated with the migration of

astrocytes from optic nerve into the retina. Furthermore, Schnitzer (1987)

demonstrated in both monkey and horse that capillaries are restricted to retinal

regions populated by astrocytes. These observations suggest that astrocytes

influence vessel formation prior to inducing barrier formation.

78
VESSEL FORMATION IN VITRO

The authors’ laboratory recently has focused on defining the cellular and

biochemical determinants of early events in brain capillary formation.

Microvascular endothelial cells grown as monolayers acquire cobblestoned

morphologies that more closely resemble endothelial cells lining large vessels

than those forming capillaries (figure 4). These distinct morphologies likely

result from specific patterns of endothelial-endothelial and endothelial-matrix

interaction determined, in part, by endothelial responses to environmental cues.

Experimental evidence for astrocyte induction of capillary formation thus far

has been missing. To approach this problem, Laterra and colleagues (1990)

recently developed an in vitro model of brain capillary formation in which

astrocytes isolated from newborn rats induce bovine retinal microvascular

endothelial (BRE) cells to reorganize into capillary-like structures. Cells of

the C6, line, which have astroglial properties, also induced this morphological

change (figure 5). These astroglial-induced, capillary-like structures mimic in

vivo cerebral capillaries by light microscopic criteria of size and shape, the

accumulation of the basement membrane-associated protein laminin at

endothelial-astroglial interfaces, and the “ensheathement” of the reorganized

endothelial cells by astroglia. This “angiogenic” interaction shows CNS

specificity since a fibroblastic normal rat kidney cell line did not induce BRE

cell reorganization, and bovine adrenal microvascular endothelial cells failed

to differentiate when cocultured with astroglia.

FIGURE 4. (A) Cultured bovine retinal microvessel endothelial cells form a

cobblestoned monolayer. (B) The endofhelial character of these

cells is demonstrated by staining with Dil-acyl-LDL.

79
FIGURE 5. Bovine retinal microvessel endothelial cells reorganize into

capillary-like structures when cocultured with C6 astroglial

cells. Composite phase contrast (A) and fluorescence (B)

photomicrographs were constructed from contiguous fields.

(A) Numerous endothelial cells are organized into a 1.3-mmlong structure surrounded by C6

 cells. (B) Dil-acyl-LDL staining

distinguishes endothelial cells from surrounding astroglia. This

fluorescent marker accumulates around endothelial cell nuclei,

some of which are indicated by arrows. Bar=50 microns.

SOURCE: Laterra et al. 1990, copyright 1990, Wiley-Liss

Laterra and coworkers (1990) used this in vitro model to explore the molecular

basis of astrocyte-endothelial interactions relevant to CNS microvessel

formation. BRE cells were grown separated from astroglia by a porous

membrane to distinguish between the effects of diffusible and nondiffusible

inducers. Capillary-like structures failed to develop in the absence of direct

heterologous cell-cell interactions. Likewise, neither astroglial-conditioned

media nor coculture-conditioned media induced capillary-like structure formation

in solo endothelial cell cultures. Although it is difficult to rule out a very labile

80
diffusible substance that functions over extremely short distances, the findings

are most consistent with induction by either a nondiffusible inducer or direct

astroglia-endothelial contact.

The effects of transcription and translation inhibition on these morphogenic

events were examined (table 1). Endothelial-astroglial cocultures were

exposed to inhibitors for 15 hours immediately before the expected time of

endothelial reorganization. Capillary-like structure formation was inhibited 50

to 70 percent in a concentration-dependent fashion by cycloheximide (0.01-0.1

pg/mL), puromycin (0.1-0.25 pg/mL), and actinomycin-D (0.01-.025 pg/mL).

These results demonstrated a requirement for transcriptional events but did not

identify either cell type as the necessary and sufficient site of inhibitory action.

The effects of steroid hormones that are physiologically and therapeutically

relevant agents that regulate gene transcription also were examined (table 1).

Glucocorticoids are known to increase astroglial differentiation (Freshney 1984);

to enhance BBB function, especially in diseases associated with blood vessel

proliferation (i.e., brain tumors) (Long et al. 1966); and to be antiangiogenic in

peripheral tissues (Folkman and lngber 1987). Dexamethasone at 10-8 M

inhibited astrocyte-induced, capillary-like structure formation by 70 to 100

percent. Inhibition was concentration dependent and receptor mediated.

Both astroglial and BRE cells have glucocorticoid receptors, so the target cell

for this effect was not immediately clear. The nonglucocorticoid progesterone,

at 10-8 and 10-6 M, inhrbited capillary-like structure formation by 30 and 60

percent, respectively. Astroglial, but not BRE, cells were found to have

progesterone receptors, indicating that inhibition by progesterone most likely

occurs by altering astroglial function. The nonglucocorticoid steroidscholesterol, hydroxyprogesterone, and tetrahydrocortisol—failed to inhibit

astrocyte-induced, capillary-like structure formation. These data demonstrate

TABLE 1, Inhibition of astroglial-induced, in vitro angiogenesis

Drug Concentration

Percent

Inhibition

Dexamethasone 10-8 M 80

Hydrocortisone 10-8 M 40

Progesterone 10-8M 35

Tetrahydrocortisone 10-8 M 0

17 hydroxyprogesterone 10-8 M 5

Cycloheximide 0.1 μg/mL 80

Actinomycin-D 0.025 μg/mL 70

81
that “angiogenic” astrocyte-endothelial interactions may be mediated and

manipulated by alterations in gene expression. Furthermore, as evidenced by

the site of action of progesterone, the astrocyte may be a selective target for

these molecular events.

Additional evidence that astrocytic functions are altered by these inhibitors was

obtained by studying another model of in vitro angiogenesis. Endothelial cells

reorganize into capillary-like forms in vitro in response to various well-defined

extracellular matrices (Ingber and Folkman 1989; Kubota et al. 1988). Matrigel,

extracellular matrix material derived from the Engelbreth-Holm-Swarm sarcoma,

induces BRE cells to form capillary-like structures that morphologically mimic

astrocyte-induced structures (Kubota et al. 1988). This response occurs in the

absence of astrocytes within 12 hours of plating BRE cells on Matrigel-coated

surfaces. Comparisons of BRE cell behavior in response to either Matrigel

or astrocytes under various experimental conditions allowed the authors to

distinguish between astrocyte-independent and astrocyte-dependent

events (Laterra and Goldstein, in press; Wolff et al., in press). Neither

cycloheximide, actinomycin-D, dexamethasone, nor progesterone were

found to affect Matrigel-induced, capillary-like structure formation. Therefore,

the endogenous endothelial processes associated with reorganization into

capillary-like structures (e.g., migration, generation of homologous endothelialendothelial adhesions, cytoskeletal changes, cell shape changes) may occur

without RNA or protein synthesis and are unaffected by steroids. Based on

these results, the authors propose that astrocytes produce signals that initiate

an endogenous endothelial morphogenic program resulting in endothelial

organization into capillaries. The activity of these astrocytic “angiogenic”

signals are under pretranslational control and may be modulated by steroids.

CONCLUSION

The in vivo and in vitro experimental evidence presented in this chapter

implicates perivascular astrocytes in the regulation of two brain microvascular

endothelial cell functions, microvessel morphogenesis and the generation of the

BBB phenotype. These two processes appear to be mutually exclusive. For

instance, developing brain, cerebral inflammatory disease, and brain tumors

are associated with neovascularization and incomplete BBB function. These

states are characterized by the presence of proliferating, undifferentiated,

or reactive astrocytes. This is in contrast to the quiescent, differentiated

astrocytes of the normal adult brain in which angiogenesis is minimal and the

BBB is intact. Taken together, these observations suggest that proliferating/

reactive astrocytes generate “signals” that enhance vessel growth. On the

other hand, quiescent/differentiated astrocytes appear to lack these angiogenic

signals and instead may induce microvascular endothelial cells to differentiate

82
FIGURE 6. Model of angiogenic and barrier-inducing astrocyte-endothelial

interactions. Immature, poorly differentiated, and reactive

astrocytes influence endothelial cells to form new vessels.

Differentiated quiescent astrocytes influence already established

microvessels to express the BBB phenotype. Angiogenic

astrocyte-endothelial interactions require RNA and protein

synthesis. Glucocorticoids inhibit astroglial-induced angiogenesis

and enhance barrier expression by altering astrocytic functions.

83
and express barrier. Astrocyte-derived “angiogenic” signals are likely to differ

from those that enhance barrier, and agents that modify astrocytes (e.g.,

steroids) have the potential to influence which signal predominates (figure 6).

Elucidation of the nature of these signals should provide novel approaches to

the modulation of brain vessel behavior and BBB expression.

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brain endothelium-like tight junctions in cultured bovine endothelial cells:

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ACKNOWLEDGMENTS

This work was supported by National Institutes of Health research grants ES02380 and EY-03772 (GWG) and by a grant from the Juvenile Diabetes

Foundation International (JL). J. Laterra is a Clinical Investigator Development

Awardee of the National Institute of Neurological Disorders and Stroke (NS01329). C. Guerin is a National Research Service Awardee (CA-09574).

J. Wolff is funded by the Dr. Mildred Scheel Foundation for Cancer Research,

F.R.G.

AUTHORS

John Laterra, M.D., Ph.D.

Assistant Professor

Department of Neurology

The Johns Hopkins University School of Medicine

Assistant Professor

The Kennedy Institute

Johannes E.A. Wolff, M.D.

Research Fellow

The Kennedy Institute

Christopher Guerin, M.D.

Research Fellow

Departments of Neurosurgery and Oncology

The Johns Hopkins University School of Medicine

Research Fellow

The Kennedy Institute

Gary W. Goldstein, M.D.

Professor

Departments of Neurology and Pediatrics

The Johns Hopkins University School of Medicine

President

The Kennedy Institute

707 North Broadway

Baltimore. MD 21205

86
Nonpermeable and Permeable Vessels

of the Brain

Milton W. Brightman and Yoshiaki Kadofa

INTRODUCTION

The moment-to-moment regulation of interstitial fluid (IF) composition within

the central nervous system (CNS) is made possible by certain structural

peculiarities of the CNS vessels’ endothelium. This endothelium, enclosed

almost in its entirety by an astroglial sheath, comprises a continuous, sealed

cell layer that circulating, hydrophilic molecules first encounter before they

can reach the surface of any other cell within the CNS. The morphological

substrate of the selective exclusion of solutes as small as ions is the

circumferential belts of tight junctions between the endothelial cells (Reese

and Karnovsky 1967) and endocytotic vesicles that incorporate circulating

protein but, except for certain ligands, do not transfer protein across the

cells (Broadwell 1989). The consequence of this physical blood-brain barrier

(BBB) is to enable the endothelial cell, by excluding or selectively transferring

solutes, to exert control over what may be allowed to reach even its immediate

astroglial neighbor and neuronal cells.

However, there are seven small circumscribed areas at the ependymal border

around the cerebral ventricles, the capillaries of which do not constitute a

barrier but are, instead, passively permeable to hydrophilic solutes. It is the

epithelium that caps these vessels and that blocks the exchange of large

molecules between blood and cerebrospinal fluid. This chapter stresses the

similarity of the capillary structure in these circumventricular organs (CVOs)

to those of certain transplants in brain and to vessels of brain tumors. A

consideration of these permeable vessels also provides the basis for describing

the routes taken by molecules that have gained access to the IF compartment.

The aspects of brain vessels considered in this chapter are (1) the interactions

between endothelium and astroglia in determining their structural effects on the

BBB; (2) fenestrated vessels, the vessels of the brain that lie outside the barrier;

(3) whether there is neurovascular specificity in neuroendocrine systems; and

(4) the barrier in peripheral tissues grafted to the brain.

87
ENDOTHELIAL-ASTROGLIAL INTERACTIONS

Barrier Site

When the glycoprotein horseradish peroxidase (HRP) is administered

intravascularly, it is prevented from moving between the blood and the IF

of the CNS by zonular tight junctions that occlude the extracellular cleft

between adjacent endothelial cells and by the paucity of endocytotic pits

(Reese and Karnovsky 1967).

Until 1967 the site of the barrier to the passive migration of large hydrophilic

molecules across the cerebral capillary was ascribed either to the capillary

or to its enveloping astroglial sheath. The concept of the barrier was

brought into question when electron micrographs of mammalian CNS tissue

depicted an extremely high cellularity that left little extracellular room in which

tracer molecules could accumulate in sufficient amounts to become visually

detectable. There was purported to be no distinctive cellular barrier, either

endothelial or astroglial, but rather a penetrable but very small IF or Indicator

space. That the lack of an appreciable IF space did not account for an illusory

barrier was demonstrated in the tailed amphibian, the mudpuppy (Necfurus

maculosus). The cerebral capillaries of this amphibian are simple hairpin loops

surrounded by a connective tissue-containing perivascular space large enough

to accommodate HRP infused into its cerebral ventricles. However, when the

protein was injected intravascularly, it was prevented by the endothelium from

reaching the perivascular space. The failure to enter the IF was the outcome

of an endothelial barrier and not the absence of an appreciable IF space

(Bodenheimer and Brightman 1968).

The impermeable endothelium of the “barrier” blood vessels throughout

the CNS can be rendered permeable by opening its tight junctions or by

momentarily inducing the microinvagination of the endothelial cell membrane

to form a transient, vesiculo-tubular, transcellular channel. The reversible

opening of the BBB has been achieved by administering in vivo hyperosmotic

solutions of saccharides (Rapoport et al. 1972). The mechanism of breaching

the barrier is still controversial. Patent, HRP-filled endothelial junctions have

been demonstrated in mammals treated with hyperosmotic solutions (Brightman

et al. 1973), and comparable experiments in lower forms also open the BBB;

but the junctions appear to remain tightly closed, and there is a proliferation

of vesiculotubular profiles in the endothelial cytoplasm (Farrell and Shivers

1984). The interpretation of such profiles as transcellular channels in normal

endothelium (Hashimoto 1972) and in perturbed endothelium (Lossinsky et al.

1979) has been convincingly refuted (Balin et al. 1987).

88
Dissemination of Exogenous Protein Through Interstitial Fluid

Once the proteins cross the cerebral capillary, there is no structural

impediment to their flow through the IF clefts of the cerebral parenchyma

(Brightman 1977). However, the extracellular dissemination of the larger

ferritin molecule (Brightman 1965), the apoferritin shell being ~445 kD, is

not as extensive as the smaller, 40-kD HRP molecule (Brightman et al. 1970)

following the perfusion of these molecules through the cerebral ventricles.

The rapidity with which HRP is dispersed from the ventricles and subarachnoid

space throughout the perivascular channels of the brain has been convincingly

demonstrated to be the result of the pulsatile activity of the heart (Rennels et al.

1985). Because this rapid dissemination is confined to perivascular channels

and does not include the IF clefts around neurons and glial cells, it has been

suggested that the pulsatile action is a means of clearing molecules such as

HRP from the brain rather than spreading an appreciable amount of such

charged macromolecules to the surface of brain cells (Brightman 1989).

The fraction of HRP that penetrates the 20 nm-wide perineuronal and periglial

clefts at some distance from its original point of entry, the permeable vessels

of CVOs or peripheral tissue graft, may be so small that it cannot be detected

even with the sensitive tetramethylbenzidine method. A telltale sign that

the glycoprotein has infiltrated the extensive network of the comparatively

larger perivascularclefts is the uptake of the exogenous HRP by perivascular

pericytes. Unlike the endothelial cells, the farflung pericytes do not form a

continuous cell layer, but their ubiquity and labeling by HRP indicate that the

protein had traveled along the perivascular clefts for considerable distances.

The inference that pericyte labeling connotes the spread of HRP through the

perivascular clefts of the IF compartment is, of course, based on the reasonable

premise that the endothelium under the pericytes has an intact blood-IF barrier.

There is normally no, or very little, transendothelial migration of circulating

protein, but a small rupture of the endothelium could supplement the HRP

flowing along the periendothelial clefts from a CVO, a tumor, or a graft of

peripheral tissue in the brain.

Whether HRP enters the IF compartment from the blood or from the cerebral

ventricles, the astroglia does not prevent the protein from reaching the cell

processes of neurons or the abluminal face of the endothelium. The plaquelike gap junctions tethering astrocytes to each other are, unlike zonular tight

junctions between endothelial cells, discontinuous and cicumventable by

HRP (Brightman and Reese 1969). Although the perivascular astrocyte

does not prevent HRP from flowing into the IF, its ubiquity as a periendothelial

investment suggests that it could contribute to the composition of the IF and

89
may influence the function of the endothelium. However, the size of the

molecule, as well as its coulombic charge, determines how far it will move

through the IF. In contrast to HRP (40 kD), gamma globulin G (160 kD)

injected into peripheral blood does cross the permeable vessels of a graft

on the surface of the brain, but it penetrates the IF of the underlying brain

for a distance of only about 1 mm in 1-month-old grafts (Wakai et al. 1986).

Effect of Astrocytes on Endothelial Cell Membrane

The first experimental intimation that brain cells were involved with the

formation of the endothelial barrier came with the highly significant

observations that peripheral, permeable vessels become impermeable

where they invade brain tissue (Stewart and Wiley 1981). Embryonic avian

brain, before it had become vascularized and when it was placed on the

chorioallantois, was penetrated by vessels from this organ. On entering

the piece of embryonic brain, the vessels from the chorioallantois were no

longer permeable to circulating Evans blue-albumin complex. Something

within the brain tissue had converted once-permeable vessels into barrier

vessels.

The identity of the CNS component responsible for the conversion from

permeable to impermeable vessels was made by in vivo and in vitro

approaches. When astrocytes grown in cell culture were aggregated and

implanted into the iris of the rodent eye, iris vessels, normally permeable to

Evans blue-albumin, became impermeable when the vessels entered the

astrocyte aggregate (Janzer and Raff 1987). In another approach, the cell

membranes of bovine brain endothelial cells, maintained in solo cultures,

were assessed in replicas of freeze-fractured cell membranes (Tao-Cheng

et al. 1987). As in other cell types, the tight junctions of the endothelial cells

were characterized by strands or ridges within the lipid bilayer of the fractured

plasma membrane. However, instead of forming complex, interconnecting

strands several rows wide, the ridges were discontinuous, fragmentary, and

relatively few in number. Moreover, unlike tight junctions of brain barrier

vessels, many gap junctions were distributed among the tight junction strands

(Tao-Cheng et al. 1987). When the brain endothelial cells were cocultured

with primary cultures of rat astrocytes, the junctions became enhanced: The

constituent strands became longer and more continuous, there were more rows

of strands, many of the rows were connected, and there were few gap junctions

among the strands. The astrocytes had “normalized” the endothelial tight

junctions (Tao-Cheng et al. 1987). In similar cocultures, the induclion of strand

development in brain endothelium by astrocytes also has been described but

without a decrement in gap junctions (Arthur et al. 1987).

90
The inference that the barrier attributes of brain endothelium are imposed

by astrocytes has been further reinforced by in vitro experiments. A relatively

high electrical resistance (R) has been attained across a monolayer of brain

endothelium that is maintained in culture with astroglia or its conditioned

medium. The R between the lumen and perivascular fluid of anuran pial

vessels is about 1,800 cm2 in vivo (Crone and Olesen 1982). Until

recently, attempts to increase the R, beyond 50 to 100 cm2, across

brain endothelium grown in coculture with astroglia or its conditioned medium

have been unsuccessful. One successful method has been to select and

clone endothelial cells from capillaries, in contrast to venules, where the barrier

may be incomplete. If the cloned endothelial cells are grown on one side

of a collagen-coated filter and astrocytes are grown on the opposite side, a

continuous, barrier-type of endothelial layer is obtained (Meresse et al. 1989)

with an average R of 600 cm2 across the two cell layers (Dehouck et al.

1990). A second method, which also yields a comparable or higher R,

requires the continuous exposure of brain endothelial cells to astrocyteconditioned medium, stimulation of adenylyl cyclase, and the immunological

removal of pericytes from the cultures (Rubin et al., in press). The exciting

prospects offered by these in vitro preparations are not only the rapid and

simple electrical assay of agents that might affect the barrier but also new

glimpses of how monocytes, endothelial cells, and astrocytes might interact

under immunologically defined conditions.

As strong as the accumulating evidence is that the astrocyte affects the

structure of the endothelial cell and its permeability toward large, hydrophilic

molecules, there are several reasons for doubting this relationship. (1) The

cerebral endothelium of some elasmobranchs, such as the shark and dogfish,

has open junctions and is permeable to HRP, whereas its perivascular

astroglia forms the impermeable layer (Brightman et al. 1971; Bundgaard

and Cserr 1981). Either these particular perivascular astrocytes ensure that

the endothelial junctions remain open rather than closed, or the open junctional

configuration is an inherent one, independent of the adjacent astrocyte.

Similarly, the closed tight junction may be inherent to mammalian cerebral

endothelium and independent of astrocytes. (2) Dissociated, cloned brain

endothelial cells in vitro can form a continuous layer with an R of 157 to

783 cm2 in the absence of astrocytes (Rutten et al. 1987). (3) Astrocytes

of the hypothalamic median eminence and the astrocyte-like cells, the pituicytes

of the neural lobe of the pituitary gland, are neighbors of an endothelium that

is not tight but rather fenestrated and permeable to HRP. Therefore, it is still

conceivable that the in situ development of barrier characteristics is inherent

to the cerebral endothelium and independent of the astrocyte.

91
Influence of Endothelium on Astrocyte Cell Membrane

Just as astrocytes influence the membrane structure of brain endothelium, so

does the endothelium modulate the internal structure of the astrocyte cell

membrane. Viewed in freeze-fracture replicas, the plasma membrane of the

perivascular astrocyte contains numerous, randomly dispersed orthogonal

aggregates of intramembranous particles or assemblies (Dermietzel 1973;

Landis and Reese 1974; Anders and Brightman 1979).

In vitro primary cultures of brain cells enriched with astrocytes contain

assemblies that are greatly reduced in number but that, as in their in vivo

situation, are randomly dispersed (figure 1). In the company of brain

endothelial cells or fibroblasts, the assemblies aggregate, and a few become

aligned in rows (figure 2) similar to those in situ where the plasma membrane

of two astrocytes abut one another (Tao-Cheng et al. 1990). Some of the

randomness is converted into an orderly arrangement of the particles when

brain endothelial cells and fibroblasts are in vitro neighbors of astrocytes.

It is not surprising that the effect also is exerted by fibroblasts because the

fibroblast-like cells constituting the pia-arachnoid, in addition to the endothelium

of the brain, subtend astrocytic end-feet rich in assemblies, some of which are

in orderly array.

FIGURE 1. Solo primary culture of astrocytes at 18th day in vitro. Assemblies

(arrows) are randomly scattered within the lipid bilayer of the cell

membrane. X 105,000

92
FIGURE 2. Primary astroglial culture, 10 days old, was grown on a cover slip

and placed facing brain endothelial cells for 7 days. Assemblies

have aggregated and form orderly trains of particles radiating

from caveolae (arrows). X 105,000

The function of the assembly is not known. It is doubtful that the assemblies

are involved with active transport across the astrocytes’ cell membrane (Gotow

and Hashimoto 1988). It is more likely that they are associated with potassium

channels (Newman 1985). The high concentration of assemblies in the Muller

cell end-foot that faces the vitreous of the frog eye closely matches the sites

of high K+ conductance (Newman 1985). Whether potassium channels of

astrocytes, like assemblies, can aggregate and become aligned, whether this

redispersal into rows ensures a highly localized withdrawal of K+ from the IF,

and the significance of such a focal “spatial buffering” of potassium are not

known.

93
FENESTRATED BLOOD VESSELS

Circumventricular Organs

The BBB is focally circumvented at discrete sites that have a neuroendocrine

function and that are situated around the lateral, Ill, and IV cerebral ventricles

(Weindl 1973; Klara and Brizee 1975). These CVOs, except for the

subcommissural organ, do not have a BBB. They are characterized by a

connective tissue space enclosing fenestrated, permeable capillaries capped

by an impermeable epithelium (Hashimoto and Hama 1968; Brightman 1968).

The epithelial cells of one such CVO, the choroid plexus, are linked to each

other by zonular tight junctions impermeable to HRP (Brightman 1968)

although they are permeable to a smaller solute, lanthanum salt (Bouldin

and Krigman 1975). At other CVOs, such as the median eminence, the tight

epithelium consists of specialized ependymal cells, the tanycytes (Weindl

1973) that also block the passive, extracellular flow of HRP between

perivascular space and the cerebrospinal fluid of the ventricles.

The permeability of the CVOs to a small, neutral amino acid, alpha-amino

isobutyric acid, has been measured by quantitative autoradiography. The

correlation of morphometry with the resolution provided by autoradiography

has permitted the blood flow and permeability of CVO subregions to be

delineated in terms of the arrangement, structure, and density of their

microvessels (Shaver et al. 1990a, 1990b). A consistent pattern has

been emerging. For example, in the subfornical organ the rostral region is

supplied by barrier vessels, whereas the caudal region receives fenestrated

vessels highly permeable to the amino acid. The possible correlation within

the CVOs of regional permeability and blood flow with the presence of

neuroendocrine axons has led to an interesting and reasonable speculation.

The hypothesis is that the density of permeable vessels is concordant with a

localized, high metabolic activity that subserves complex neuroendocrine

functions (Shaver et al. 1990b).

NEUROVASCULAR SPECIFICITY?

The permeable, fenestrated capillaries of the CVO, unlike the barrier vessels

of the adjacent brain in most species, are surrounded by two basal laminae

with an intervening space containing extracellular matrix, which includes

collagen and basal lamina material (figures 3 through 5). In CVOs. such as

the median eminence of the hypothalamus and the neural lobe (NL) of the

pituitary gland, this perivascular space is bordered by the terminals of

magnocellular, neurosecretory axons (NSAs) that in turn are enveloped

by astrocyte-like cells, the pituicytes. The NSAs from the magnocellular

94
FIGURE 3. Neural lobe graft 4 weeks after transplantation. Regenerating

NSA terminal, containing small vesicles and about nine

neurosecretory vesicles, is 1.66 pm from a fenestrated vessel

(FBV). The terminal is separated from the FBV by a process,

presumably a pituicyte (Pit), subtended by basal lamina (BL).

Pituicytes often have lipid droplets. X 29,000

95
FIGURE 4. Pineal graft 2 weeks after transplantation. Regenerating NSA is

0.38 μm from capillary with fenestrae (arrowheads). Axon and

capillary lie in extracellular matrix (ECM). X 42,100

96
FIGURE 5. Adrenal medulla graft, 2 weeks old. Shortest separation of NSA

from fenestrated capillary (FBV) is 0.4 μm, but two thin cell

processes (Pit) intervene. The processes, having no BL, may

be fibroblasts. NSA and FBV lie in ECM. X 20,500

97
neurosecretory cells of the hypothalamic supraoptic and paraventricular

nuclei thus terminate as neurovascular endings.

The question posed by the lead author of this chapter is whether a regenerating

NSA becomes as closely associated with the fenestrated endothelium of other

CVOs or of endocrine glands as it does with the fenestrated vessels of its

own CVO target. To answer the question, fragments of NL (figure 3), pieces

of another CVO, the pineal gland (figure 4), and fragments of a peripheral

endocrine gland, the adrenal medulla (figure 5), were inserted into the lateral

retrochiasmatic area (Kadota et al. 1990). Through this area, NSAs from both

nuclei course as a compact bundle on their way to the NL (Palkovits 1934).

The area is anterior to the median eminence and is supplied by very few, if

any, fenestrated vessels; thus, any such vessels next to or within graft tissue

would have been introduced by the transplants. The act of inserting the grafts

inevitably damages some of the NSAs, and it was expected that collateral

sprouts from adjacent, intact NSAs would be in a position to terminate on

nearby, fenestrated vessels. A few such NSAs did penetrate each type of graft

to terminate as closely on the fenestrated vessels of the pineal and adrenal

medulla as they did on similar vessels of the NL transplants. There appeared

to be no specificity with respect to the closeness of association between NSAs

and fenestrated capillaries (figures 3 through 5) (Kadota et al. 1990).

However, there may be a chemotactic specificity between the regrowing

NSAs and either the fenestrated capillaries or the pituicytes. By 2 to 4 weeks

after transplantation, the NL grafts consisted primarily of fenestrated vessels,

pituicytes, redundant basal lamina, fibroblasts, and collagen, with the NSAs

within the NL grafts having degenerated. Although the results were not

statistically significant, there was a consistent tendency for more NSAs,

more fenestrated capillaries, and more NSA-capillary associations to be

included in the NL grafts than in either the pineal or adrenal ones (Kadota

et al. 1990). At present, the pituicyte is suspected of being the source of

the purported chemoattractant and not only may provide a tropic factor

or directional guidance to ingrowing axons and vessels but also might be

trophically “axonogenic” and vasogenic as well. If there is a vasogenic factor,

like purported adrenal and pineal factors, it might induce the formation of

fenestrae within the regenerating capillary endothelium.

THE BARRIER IN GRAFTS OF PERIPHERAL TISSUE TO BRAIN

Tissue Grafts

It is certain that the solid grafts of CVO and endocrine tissue contained some

of their intrinsic, fenestrated blood vessels. As will be shown, it is also likely

98
that the parenchymal cells of the grafts must have influenced the structure

and permeability of regenerating vessels growing into the transplants. By

means of this influence, the ingrowing capillaries came to resemble the intrinsic

ones normally supplying the grafted tissue.

A discrete, invasive means of circumventing the passive BBB is by creating

a nonendocrine, CVO-like structure in the form of a graft of peripheral tissue

to the ventricular surface of the brain. This focal circumvention can be

accomplished by transplanting solid tissues or by implanting pure populations

of cell lines. Allografts consisting of fragments of (Rosenstein and Brightman

1963) or whole (Tsubaki et al. 1987) superior cervical ganglia, when placed into

the fourth ventricle of rodents, survive indefinitely. These periventricular grafts

are permeable to exogenous protein (Rosenstein and Brightman 1963) and, as

measured by quantitative autoradiography, to a small amino acid as well

(Tsubaki et al. 1987).

The same bypass can be accomplished with isografts of skeletal muscle. Small

pieces about 1 to 2 mm2 are carefully inserted into the IV ventricle of mature

rats. As with other tissues (Rosenstein and Brightman 1983; Tsubaki et al.

1987) it is essential to force the grafts against or into the choroid plexus. In

such transplants of, for example, the superior cervical ganglion, vessels from

both pia and choroid plexus rapidly enter and anastomose with the vessels of

the graft (Tsubaki et al. 1987). Being skeletal muscle, the “milieu” imposes

permeable features on the regenerating endothelium that enters the graft.

Circulating HRP quickly crosses the permeable muscle type of capillaries

within the transplant to enter the muscle’s interstitial space. From here, the

protein enters the IF of the contiguous brain, that is, the medulla oblongata

and cerebellum (Wakai et al. 1986). The interstitial pathway taken by the

protein is longitudinal, between the dorsal, long fiber tracts of the medulla,

and may extend for about 0.6 to 1.0 mm (Wakai et al. 1986).

The muscle autografts survive indefinitely on the ependymal or pial surface

but soon die if they are placed within the neural parenchyma. The transplants

on the brain’s surface endure because they become innervated by myelinated

fibers that can form myoneural junctions (Wakai el al. 1986). Whereas most

of the muscle cells do not become innervated and undergo fatty degeneration,

some cells do receive innervation and survive. The source of the innervation

is unknown. The grafts might receive branches from one of the nearby cranial

nerves (e.g., the facial nerves). Another possible source of the myetinated

axons might be branches of the superior cervical ganglion that innervates

the pia. If so, then the IV ventricle, as a chamber for receiving grafts,

can be compared with the anterior chamber of the eye. The insertion of

intraocular grafts inevitably injures the iris and the nerves of the superior

99
cervical ganglion supplying it. One result is that collateral sprouts from

uninjured fibers or perhaps damaged but regenerating axons then Innervate

the graft.

In summary, transplants of peripheral neural or muscle tissue to the surface

of the brain survive for longer periods than those inserted into the CNS

parenchyma and are sites for the focal entry into the brain’s IF of large,

hydrophilic solutes, elsewhere excluded by the BBB.

Cell Implants

Grafts of solid pieces of peripheral tissue within the brain are supplied by

vessels with the permeable characteristics of the grafts’ original vessels.

It is likely that surviving portions of the grafts’ blood vessels regenerate and

anastomose with the ingrowing endothellum of the adjacent brain (Stewart

and Wiley 1981; Tsubaki et al. 1987). However, the new vessels in the graft

are not necessarily derived, even in part, from vessels that had survived within

the graft. Rather, the vessels in and around a transplant may arise solely from

the intrinsic vessels of the surrounding brain.

Pure cultures of neoplastic cells, without any endothelial contaminant, when

introduced into the brain substance, can induce brain endothelium to take

on the permeability of the vessels once associated with the cells from which

the tumor had been originally derived. A cell line, PC12, derived from a rat

pheochromocytoma consists of replicating adrenal chromaffin-like cells

(Greene and Tischler 1976). When such dividing, naive, PC12 cells are

treated with nerve growth factor, they differentiate-they stop dividing, emit

neurites, and, like naive cells, are able to synthesize both catecholaminergic

and cholinergic neurotransmitters (Greene and Tischler 1976). The growth

factor must be continually replenished or the neurone-like cells revert to the

mitotic naive state. A longer, apparently indefinite, differentiation has been

attained by infecting naive PC12 cells with a retrovirus containing the Kirstenras oncogene (Noda et al. 1985).

When naive, mitotic PC12 cells are implanted into the brain of rats, some

vessels within or adjacent to the implant may become fenestrated, such as

those depicted in figures 3 through 5 (Jaeger 1987; Pappas and Sagen 1966).

As do the cells of brain tumors, PC12 cells appear to elaborate and release

Something that induces the formation of fenestrae and, presumably, the

associated tubulovesicular apparatus. It is the tubulovesicular complex,

rather than the diaphragm-covered fenestrae, that transfers large, hydrophilic

solutes from blood to IF (Coomber and Stewart 1988).

100
Brain Tumors

Fenestrated blood vessels supply not only CVOs but also normally developing

brain (Bar and Wolff 1972) and tumors of the brain (Groothuis and Vick 1982).

All these fenestrated vessels are structurally the same, are associated with a

generous perivascular space, and are passively permeable to circulating

molecules. In normal rat embryos at 11 to 12 days of gestation, fenestrated

capillaries lie at the border between the periventricular, neuroepithelial cell

layer and the rest of the parenchyma. By the 17th day of embryonic life, when

neuroblasts are being formed, the fenestrae disappear, presumably because

of a differentiating influence of neuroblasts and glial cells (Yoshida et al. 1988).

At least some of the vasculature supplying almost all brain tumors is

fenestrated. Brain tumors that are induced by the intracerebral inoculation of

virus (Vick 1980) or the administration of nitrosourea compounds (summarized

by Groothuis and Vick 1982), as well as primary and metastatic gliomas in

human brains (Hirano and Matsui 1975), all contain some vessels that are

fenestrated. The number and location of fenestrated microvessels in

experimentally induced and in spontaneously arising brain tumors are highly

variable within individual tumors and between tumors (Groothuis and Vick

1982). Whereas these vessels are permeable to x-ray contrast media and

chemotherapeutic agents, neighboring vessels within the tumor or near its

border with normal brain are barrier vessels. Consequently, the BBB must

be opened (e.g., osmotically [Neuwelt and Papoport 19841) to attain a greater

entry of agents into brain tumors.

Necrotic brain tissue does not induce the formation of fenestrae in regenerating

blood vessels. Presumably, a substance secreted by normal fetal brain cells

or a variety of neoplastic cells brings about the development of fenestrae in

growing, responsive endothelium. A substance in the form of a 30-kD protein

is elaborated by malignant glioma ceIIs in vitro (Bruce et al. 1987). The protein,

which has been designated as a vascular permeability factor (VPF), rapidly

augments the permeability of already permeable intradermal blood vessels.

This property has been taken to suggest that a VPF also may cause the

vasogenic edema often associated with brain tumors (Bruce et al. 1987).

However, an increased permeability and the development of fenestrae in

brain endothelium exposed to VPF have yet to be demonstrated.

It is conceivable that PC12 cells may secrete a substance that causes

brain endothelial cells to become fenestrated. Counter to this notion is the

observation that, in vitro, the presence of PC12 cells does not result in the

development of fenestrae in adjacent endothelial cells (Tao-Cheng et al. 1989).

101
Moreover, the implantation of PC12 cells does not always result in tumor

formation. On the contrary, if placed within the brain of otherwise normal

adult rats, naive PC12 cells may cause the development of hemorrhagic cysts

and disappear (Freed et al. 1986; Okuda et al. 1991). The hemorrhages

suggest that the endothelial cells had been damaged by the naive PC12 cells,

but endothelial cells are not damaged when cocultured with naive PC12 cells

(J.H. Tao-Cheng, 0. Okuda, L.S. Simpson, L. Chang, and M.W. Brightman,

unpublished results). If ras-oncogene, differentiated PC12 cells are implanted

into the brain opposite to the side that had received the naive cells, few if any

small hemorrhages develop, and some of the PC12 cells persist for at least

8 weeks (Okuda et al. 1991).

Do PC12 cells, like other tumor cells, secrete a VPF that converts barrier,

impermeable vessels into fenestrated, permeable ones? It may be that

cerebral vessels have to be “primed” in some fashion before they can

become responsive to factors that might be released from PC12 or glioma

cells.

If PC12 cells were a rich source of a VPF, it may be informative to determine

the conditions under which normal, barrier endothelium might be converted

to the permeable, fenestrated type. It may be anticipated that PC12 cells,

differentiated by either nerve growth factor or ras oncogene, may stop

producing or releasing VPF and that the nearby capillaries would remain

unfenestrated and impermeable to large hydrophilic molecules. However,

the number of passages of PC12 cells maintained in vitro may result in a

varying rate of fenestrae formation. Repeated cell passages also might

diminish unpredictably the amount of VPF being elaborated by the cells. The

resulting variability in the number of fenestrated vessels would make it difficult

to ascertain whether differentiated PC12 cells no longer induce the creation

of fenestrae. Nevertheless, the identification of the conditions under which

brain endothelium can become responsive to secreted permeability factors

might yield further insight into the modulation of endothelial permeability to

hydrophilic substances.

CONCLUSIONS

There is strong evidence obtained from in vivo and in vitro experiments that

the structural and functional barriers of CNS endothelium are influenced by

astrocytes. New in vitro, methodological variations have yielded a confluent,

endothelial cell layer with a high R, provided that astrocytes or their Secreted

products are present. However, cloned brain endothelial cells also may exhibit

a high R in the absence of astroglia, and in some species, brain endothelium

is permeable to large solutes, Whereas its astrocytic sheath acts as a barrier

102
between blood and brain IF. Therefore, the possibility remains that the barrier

properties of endothelium may be an intrinsic, inherent property independent of

astrocytes.

CVOs, grafts to the brain of muscle or endocrine tissue, and brain tumors

all have some vessels that are fenestrated and permeable. These vessels

enable circulating protein to bypass the BBB. Whether developing or

regenerating blood vessels become fenestrated and permeable in response

to factors secreted by certain cells, including tumor cells, has yet to be fully

explored. While regenerating NSAs of one CVO, the pituitary gland’s neural

lobe, may nonspecifically regrow in vivo to grafted fenestrated vessels from a

variety of sources, the axons may be specifically attracted to and maintained by

the lobe’s pituicytes. Further definition of how such permeable vessels are

induced and how they affect the contents of the brain’s IF may add to a

perspective of how the properties of barrier vessels may be induced and

maintained.

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AUTHORS

Milton W. Brightman, Ph.D.

Section Head

Laboratory of Neurobiology

National Institute of Neurological Disorders and Stroke

National Institutes of Health

Building 36, Room 2A-29

9000 Rockville Pike

Bethesda, MD 20892

Yoshiaki Kadota, M.D.

Neurosurgeon

Department of Neurosurgery

Juntendo University School of Medicine

Tokyo

JAPAN

107
The Blood-Brain Barrier Is Not a

“Barrier” for Many Drugs

Joseph D. Fenstermacher

INTRODUCTION

Because there is little or no movement of solute between blood and brain

through the intercellular clefts of the capillaries, drugs and other compounds

enter most brain areas by passing through the endothelial cells (the exceptions

to this are the choroid plexus and circumventricular organs, where rapid

transfer may occur by way of the intercellular junctions and/or transendothelial

fenestrations). To some extent, all solutes move across the blood-brain barrier

(BBB) by dissolving in the endothelial cell membranes and diffusing through

them and the endothelial cytoplasm. In addition, blood-brain transfer of some

solutes is facilitated by specific membrane transporters or carriers or is set, in

part, by other processes.

More than 90 years ago, Over-ton (1900) demonstrated that the permeability

of cell membranes depends on the lipid solubility and diffusability of the

penetrating solute. More recently, Orbach and Finkelstein (1980) have

shown that the permeability of planar lipid (lecithin) bilayer membranes to

nonelectrolytes is in agreement with Over-ton’s rule; that is, permeability was

proportional to the hexadecane:water partition coefficient times diffusion

coefficient (PcDc). (Incidentally, as pointed out by Orbach and Finkelstein,

lipid solubility can be approximated by solubility in many nonpolar organic

solvents, including hexadecane. olive oil, ethyl ether, and octanol.) This

suggests that BBB permeability also might follow Overton’s rule.

OVERTON’S RULE AND THE BLOOD-BRAIN BARRIER

To show that solute permeation through the BBB fits Overton’s rule, good

experimental estimates of blood-brain transfer constants are needed. Transfer

constants are determined by intravenously administering the material of interest

and subsequently measuring its time course in blood and uptake by brain.

From these data, one or more of three unidirectional transfer constants-the

108
extraction fraction (E). the influx rate constant (K,), and the efflux rate constant

(k2)—is calculated. If the rate of cerebral blood flow is known and the

experiments have been carefully designed and rigorously executed, then a

permeability-surface area (PS) product can be calculated from E, K1, or k2

and used as an indicator of BBB “permeability” (Fenstermacher et al. 1986).

With respect to the lipid solubility part of Overton’s rule, several respectable

experimental studies have indicated a linear correlation between PS product

and either olive oil:water or octanol:water Pc for cerebral capillaries

(Cornford et al. 1982; Levin 1980; Oldendorf 1974; Rapoport et al. 1979).

The applicability of Overton’s rule per se to the BBB has been examined in

several reviews (Fenstermacher 1983, 1989; Fenstermacher and Rapoport

1984). In these reviews, blood-to-brain transfer constants were determined

from published reports for 11 or more physiological substances, and PS

products were calculated from these constants. Plots of PS product vs. the

product of the octanol:water PcDc yielded a linear relation over a 10,000-fold

range. In agreement with Overton’s rule, the slope was one (1.0) on a log-log

scale. Recently, this relationship has been extended for the BBB to cover 23

physiological compounds, 76 measurements, and a millionfold range

(J.D. Fenstermacher and C.S. Patlak, manuscript in preparation).

Figure 1 is an example of such a plot with data for 11 compounds and 41

measurements (1 to 10 different measurements per compound); the slope

of the parallel lines enveloping the points is one. Transfer of these substances

across the BBB, therefore, seems to follow Overton’s rule. Incidentally, the

major cause of the variation in the PcDc product is the partition coefficient,

and a log-lot plot of PS vs. PC for these same data also yields a linear plot

with a slope of one.

The good agreement between BBB permeability and Overton’s rule implies

that the cerebral capillary wall functions like an aporous membrane. That is,

the aqueous channels through the BBB, if present, are so small that most

compounds cannot pass through them. This is consistent with earlier reports

that suggest the effective pore radius or slit width of the BBB is around 0.7 nm

(Fenstermacher and Johnson 1966; Paulson et al. 1977) but it conflicts with

the more recent estimates of an effective pore radius of 8 nm for frog cerebral

capillaries (Crone 1984) and for confluent monolayers of cerebral endothelial

cells (van Bree et al. 1988). An effective pore radius of 8 nm, however, seems

unduly large since aqueous channels of such size would yield a PS product for

sucrose, a relatively impermeable solute, twentyfold greater than measured

(figure 1) if these pores comprise as little as 0.001 percent of the BBB surface

area (Dorovini-Zis et al. 1983).

109
FIGURE 1. Log-lot plot of PS product vs. octanol:water PcDc product

for 11 physiological compounds. The PS products are from

Fens Fermacher (1983, 1989) and Fenstermacher and Rapoport

(1984). Two or more PS values are plotted for 10 of these 11

compounds because two or more separate measurements of K,

or E were found. The parallel diagonal lines enclose or envelop

38 of the 41 points and have a slope of one. The vertical spread

of the envelope is 10 mL/g/min (one order of magnitude of PS).

The influx of a highly permeable compound is almost completely limited by the

rate of blood flow or, more precisely, the product of the rate of blood flow (F)

and the distribution volume of the exchangeable material in the blood (V,). For

most of the studies providing data for the present (figure 1) and the published

PS vs. PcDc plots (Fenstermacher 1983, 1989; Fenstermacher and Rapoport

1984), the animals were anesthetized with pentobarbital, and the forebrain or

part of the forebrain was sampled. In the case of highly permeable solutes,

the blood flow-exchangeable volume product (FVf) is around 1 mL/g/min. When

E or extraction is greater than 95 percent, then tissue influx is considered to be

110
flow-limited, and the PS/FVf

 ratio is equal to or greater than 3.0 according to

the single capillary model and the Renkin-Crone equation (Crone 1965;

Fenstermacher et al. 1986; Renkin 1959). If FVf is therefore assumed to be

around 1.0mL/g/min, then PS should be greater than 3.0 mL/g/min for a

substance whose rate of influx across the BBB is set virtually completely by

blood flow. As indicated in figure 2, this condition would be expected to hold

when the PcDc product >2x10-5 cm2/sec or the octanol:water Pc>1.2 (PS vs.

PC plot not shown).

The influx of a relatively impermeable solute is virtually completely set by its

PS product at the BBB. For the permeability-limited case, the extraction fraction

is less than 0.2, and PS/FVf<0.2 (Fenstermacher et al. 1986). Since FVf=0.5

mL/g/min for relatively impermeable solutes because they mainly distribute in

plasma, then PS should be less than 0.1 mL/g/min, which occurs when the

PcDc product <6x10-7 cm2/sec (figure 2) or the octanol:water Pc<5x10-2 (graph

not shown).

For solutes with PS products between 0.1 and 3.0 mLg/min, influx depends

strongly on both PS and FVf In this situation, PcDc products would be

expected to be around 6x10-7 to 2x10-5 cm2/sec (octanol:water PC between

0.05 and 1.2). This ‘area” lies between the PS-limiting and FVf-limiting

domains in figure 2.

Carrier systems or transporters facilitate the transfer of several compounds

across the BBB. For example, the fluxes of D-glucose and L-phenylalanine

across the BBB are enhanced by the glucose transporter, GLUT-l, and the

large neutral amino acid carrier, respectively. The measured PS products of

such compounds would be considerably higher than those predicted on the

basis of their PcDc products and the relationship shown in figure 2 if the

transporter-mediated flow accounts for a major portion of the flux. As previously

indicated (Fenstermacher 1983), the PS product of D-glucose is 50 to 100 times

higher than that predicted by the PS vs. PcDc plot and that of L-glucose, whose

transcapillary flux is not facilitated, and the PS product of L-phenylalanine is

about 10 times greater than predicted. Incidentally, this analysis indicates the

usefulness of PS vs. PcDc plots in assessing the relative importance of simple

diffusional (lipid-mediated) and carrier-mediated flows of solutes across the

BBB.

DRUG INFLUX ACROSS THE BBB

Acceptable estimates of transfer rate constants and PcDc products have been

found not only for the physiological compounds indicated above (figure 1) but

111
PcDc Product (cm2

/sec)

FIGURE 2. This graph is identical to figure 1 except the names of the

compounds have been deleted and dashed lines have been

added marking the putative permeability (PS)-limiting and blood

flow (FVf-limiting domains. These domains are defined as

PS<0.10 mL/g/min and Ps>3.0 mL/g/min, respectively.

also for 22 drugs; subsequently, the PS products of these drugs have been

calculated and tabulated (Fenstermacher 1989). The PS and PcDc products

of these 22 drugs and the relationship between PS and PcDc, shown in figure

2, indicate several points.

First, the experimentally derived PS products of most of these drugs are less

than the PS products estimated from their partition and diffusion coefficients.

That is, the experimental PS products were around or below the lower diagonal

line in figures 1 and 2. Figure 3 shows this relationship for a representative set

of 12 of these 22 drugs. Since none of the experimental PS products were

above the upper diagonal line, carrier- or transporter-mediated flux does not

appear to be involved with the blood-to-brain transfer of any of these drugs.

112
FIGURE 3. This graph is similar to figure 2. The 41 PS products in figures 1

and 2 have been dropped, and PS products for 12 representative

drugs have been plotted. These values have been taken from

Fenstermacher (1989). The drugs’ names and abbreviations are

Baker’s antifol (BkAf), methotrexate (MTX),dianhydrogalactitol 

(DAG), 5-fluorouracil(5-FU). Adriamycin (Adrmy), procarbazine

(Prorb), valproic acid (VaA), morphine (Morphn), codeine

(Codn), nicotine (Nice), amphetamine (Amph). and lidocaine

(Lido).

Second, the lipid solubility is very low for only three of these drugs—BkAf, MTX,

and DAG—and their PcDc products place them in the “PS-limiting” domain

(figure 3). In other words, the BBB would be expected to limit markedly the

influx of only 3 of these 22 drugs. Incidentally, the experimental PS product of

BkAf falls within the envelope formed by the parallel diagonal lines in figure 3;

it may be one of the few drugs in this set whose rate of influx depends almost

entirely on its ability to dissolve in and diffuse through the BBB. For DAG, MTX,

and most of the other drugs, different processes may be important in setting

their rates of influx across the BBB.

113
Third, the PcDc products of 13 of these 22 drugs are greater than 2x10-5

cm2/sec, which roughly corresponds to an octanol:water Pc greater than 2;

therefore, in accordance with the preceding suggestion, the rates of influx of

these drugs would be anticipated to be set mainly by the rate of blood flow

or FV (figure 3). The PS products of five drugs with PcDc products between

2x10-5 and 1x10-3 cm2/sec are indicated in figure 3; the data for three other

drugs in this group, BCNU, propranolol, and phenobarbital, are not presented

in figure 3. The PcDc products of the five remaining drugs in this set—

phenytoin, vincristine, pyrimethamine, diazepam, and CCNU—are equal to

or greater than 3x10-3cm2/sec. The PS products of these five drugs ranged

from 0.006 (vincristine) to >3 (diazepam) mL/g/min and are not plotted in

figure 3. The PS products of flow-limited solutes at the BBB will be around 2

to 3 mL/g/min because higher values cannot be accurately determined when

PS>3FVf (Fenstermacher 1989). The drugs with measured PS products in this

range are nicotine, lidocaine, and diazepam; thus, the major process in setting

the influx rate of these three drugs is the rate of delivery of exchangeable drug

to the microvessels. The PS products for the remaining 10 drugs in the FVf-

limiting group lie considerably below the lower line of the diagonal envelope in

figure 3, and processes other than blood flow and dissolving and diffusing

through the BBB are likely to be important in setting influx.

Fourth, the remaining 6 of these 22 drugs are moderately lipid soluble

(0.08<Pc<10) and have PcDc products between 8x10-7 and 2x10-6 cm2/sec.

According to figure 2, the influx rate of these drugs would be expected to

depend on both PS and FVf, and their PS products should be between 0.1

and 1.2 mL/g/min. The data for four of the six drugs in the moderately lipidsoluble group are given in figure 3. The measured PS products of these six

drugs are either somewhat below the lower diagonal line of the envelope

(valproic acid, procarbazine, tegafur [Ftorafur], and 5-fluorouracil) or 1 to

2 orders of magnitude below this line (dibromodulcitol and doxorubicin

hydrochloride [Adriamycin]). This suggests that distributional processes

other than passage through the BBB and blood flow affect the influx of these

six drugs and, especially, strongly affect that of Adriamycin. Incidentally, the

experimentally measured PS products for two additional “drugs” with PcDc

products in this range, caffeine and ethanol, fit within the diagonal envelope

(figure 1); and their rates of influx appear to depend on PS and FVf in

accordance with the equation for the single capillary model (Renkin 1959;

Crone 1965) and Overton’s rule.

As is apparently true for many physiological materials, the measured PS

products at the BBB of six drugs fit Over-ton’s rule as set forth in figures 1

114
and 2. These six drugs are ethanol and caffeine (figure 1), nicotine, lidocaine,

and BkAf (figure 3) plus diazepam. The PS products of two other drugs, DAG

and valproic acid (figure 3) appear to fit Over-ton’s rule fairly closely. The

measured PS products of the remaining drugs in this group, most of which

are moderately to highly lipid soluble, are less than would be predicted from

Overton’s rule (figure 3). Instead of questioning the broad applicability of

Over-ton’s rule to drug permeation through the BBB. two other possibilities are

worthy of consideration: (1) the incorrect measurement of transfer constants

and PS products and (2) the involvement of other physiological and

pharmacological processes.

MEASUREMENT PROBLEMS

Since the PS products of 16 of these drugs were low relative to their

lipid solubility and diffusability, their transfer constants may have been

underestimated. Low transfer constants are obtained when the amount of

drug that has influxed into the brain is underevaluated and/or when the driving

force for influx, the concentration-time integral of exchangeable drug within the

perfused capillaries, is overestimated (Fenstermacher et al. 1986).

Underestimation of drug influx arises most commonly from not properly

accounting for drug efflux during the course of the experiment. With some

techniques, it is assumed that all drug that has entered the brain during the

experiment is still there at the time of tissue sampling. For a permeable drug

that does not instantly and virtually completely bind within the capillaries and

parenchyma, drug efflux may be appreciable after only a few seconds. With

these approaches and such drugs, tissue samples must be obtained within 5 to

15 seconds after drug administration. For other techniques, tissue is sampled

several times, a compartmental model is assumed, and influx and efflux rate

constants are estimated by compartmental analysis. The results obtained are

model-specific and depend strongly on accurate and extensive sampling of

tissue and blood. With such approaches, erroneous values of K, are obtained

readily and often for drugs with complicated distribution kinetics.

The concentration-time integral within the capillaries can be overestimated

easily with drugs that bind to plasma proteins and/or interact with blood cells.

The transfer constants should be determined on the basis of drug concentration

in the fluid (blood or infusate) flowing into the capillaries. The value obtained

will then be correct with respect to this part of the calculation of E or K1, but

the accurate evaluation of PS from these transfer constants requires additional

data plus a more complex model and equation than the single capillary model

115
and the Renkin-Crone equation (Pardridge and Landaw 1984; Robinson and

Rapopori 1986). Further analysis of this problem is beyond the scope of this

review, and plasma protein binding and blood cell interaction are discussed only

in a simple, qualitative sense below.

OTHER PROCESSES INVOLVED WITH BLOOD-BRAIN TRANSFER

Over the past decade, various observations have indicated several processes

that may be involved in limiting or lowering blood-to-brain transfer of drugs. In

addition to plasma protein binding and red cell interaction, these processes

include metabolism within the endothelial cell and transfer from the endothelial

cell back into the blood.

Many drugs bind to plasma proteins (Cornford et al. 1983, 1985; Dubey et al.

1989; Hamberger et al. 1987; Pardridge et al. 1983; Rapoport et al. 1979).

More than half of the 22 drugs mentioned above bind to plasma proteins;

incorrect accounting for this can lead to sizable (more than 1,000-fold)

overestimation of the drug’s effective concentration-time integral. In recent

years, several groups have produced evidence that some drugs rapidly

dissociate from plasma proteins and are available for blood-to-brain exchange.

For example, using the brain uptake index technique, Pardridge and colleagues

(1983) have shown that propranolol and lidocaine dissociate from plasma

proteins during a single capillary passage and enter brain tissue, and

Hamberger and colleagues (1987) have observed that some of the progabide

bound to plasma proteins and contained in red blood cells exchanges across

the BBB. Clearly, binding to plasma proteins and availability for exchange

across cerebral capillaries is a complicated and variable process for many

drugs, and careful examination of this is necessary to understand blood-to-brain

transfer. The same thing holds for drug uptake and release from blood cells.

Ghersi-Egea and colleagues (1988) have reported measurable levels of four

drug-metabolizing enzymes in microsomal fractions from endothelial cells

obtained from rat brain microvessels. These enzymes are cytochrome

P-450-linked monooxygenases, epoxide hydrolase, NADPH:cytochrome

P-450 reductase, and 1-naphthol NDP-glucuronosyl transferase. Lipophilic

xenobiotics are substrates for these enzymes. Other studies also have

indicated the presence of dopa-decarboxylase and monoamine oxidase in

cerebral capillaries (Kalaria et al. 1988; Wade and Katzman 1975a). The

capacity of these systems to reduce the penetration of their substrate drugs

has not been examined in vivo; and the fate of these metabolites have not

been determined, with the exception of L-dopa and the dopa-decarboxylasemonoamine oxidase system (Wade and Katzman 1975a, 1975b).

116
Brain capillaries recently have been shown to express or contain P-glycoprotein

(Pgp or P-170), the multidrug resistant (MDR) transporter that mediates the

efflux of various natural-product drugs (e.g., vincristine and Adriamycin) from

drug-resistant cultured tumor cells (Cordon-Cardo et al. 1989; Thiebaut et al.

1989). The blood-to-brain transfer of these drugs is notoriously slow and may

be limited by the MDR transporter moving them from endothelial cells back into

blood.

The distribution of drugs between blood and brain involves several different

processes. Some of these processes (e.g., cerebral blood flow and local BBB

permeability) are known to vary among brain areas. In addition, local variations

in some of these other disposition processes-for instance, monoamine oxidase

activity (Kalaria et al. 1988)-have been suggested. Drug influx almost

certainly varies widely among brain areas because of these dissimilarities.

CONCLUSION

The BBB does not restrict the entry of many addictive drugs, including alcohol,

nicotine, and caffeine. In fact, the initial distribution of such drugs in the brain is

likely to reflect blood flow, whereas the later phase(s) of distribution strongly

depends on the fate of the drug within the brain parenchyma (e.g., metabolism

and receptor binding). The somewhat lower influx rates of such drugs as

codeine and phenobarbital may be more the result of plasma protein binding

and blood cell interaction than of modest BBB permeability. Thus, for many

drugs, the BBB is not much of a “barrier,” and this aspect of their blood-brain

distribution contributes to their popularity in this impatient age and disenchanted

society.

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L-dopa following carotid injection. Am J Physiol 228:352-359, 1975a.

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ACKNOWLEDGMENT

Preparation of this chapter was supported in part by National Institutes of Health

grants NS-21157, NS-26004, and HL-35791.

119
AUTHOR

Joseph D. Fenstermacher, Ph.D.

Professor

Department of Neurological Surgery

State University of New York at Stony Brook

Health Science Center, T12-080

Stony Brook, NY 11794-8122

120
Drug Delivery to the Brain: Barrier

Modification and Drug Modification

Methodologies

Stanley I. Rapoport

INTRODUCTION

Recent advances in the fields of pharmacology and molecular biology have

led to a greater understanding of disease processes, allowing development of

new classes of therapeutic agents that can interact with specific intracellular

and extracellular targets (Greig et al. 1990a; Pomponi et al. 1990). Several

drugs, peptides, biological response modifiers, and monoclonal antibodies are

available and have proven of value (1) in inhibiting a variety of malignant and

infectious diseases; (2) in ameliorating neurotransmitter, enzyme, or growth

imbalances in culture systems; or (3) in animal models using direct intracranial

administration. However, therapeutic efficacy in vivo, particularly with regard to

the central nervous system (CNS), frequently is diminished or prevented by the

inability of the agent to reach and maintain active concentrations in the brain for

an appropriate length of time. Frequently, the molecule is too large or has polar

functional groups, so that its access to the brain target organ is limited by the

blood-brain barrier (BBB).

The BBB is a system of layers of cells-at the cerebral capillary endothelium,

the choroid plexus epithelium, and the arachnoid membranes-that are

connected by tight junctions (zonulae occludens) and that together separate

the brain and cerebrospinal fluid (CSF) from blood (Rapoport 1976). The

junctions confer on each layer the properties of an extended cell membrane,

as they limit paracellular exchange of water-soluble nonelectrolytes, ions,

proteins, and peptides, forcing transcellular exchange. The cell membranes

at each of the layers are lipoid in nature, so that the property that generally

governs passive penetration of a drug into the brain, via these layers, is the

extent of its lipid solubility, measured as the octanol:water partition coefficient.

Figure 1 illustrates that cerebrovascular permeability is roughly proportional to

lipid solubility for nonelectrolytes whose molecular weights are less than 1,000

121
Octanol:Water Partition Coefficient

FIGURE 1. Relation between cerebrovascular permeability and octanol:water

partition coefficient of several agents (asterisk designates

anticancer drug)

SOURCES: Ohno et al. 1978; Greig et al. 1990a, 1990b

daltons, including anticancer drugs. The permeability coefficient, P cm•sec-1,

is defined by the following operational equation for unidirectional flux from blood

to brain across cerebral capillaries and can be determined experimentally by

appropriate quantitative techniques (e.g., Ohno et al. 1978; Takasato et al.

1984):

dCbrain/dt=PA fCplasma (1)

C-plasma is the net plasma concentration at any time t after drug administration;

Cbrain is the brain parenchymal concentration (excluding intravascular material);

A is the surface area of the capillary bed (about 180 cm2•g brain-l for gray

matter); and f is the steady-state fraction of drug unbound and un-ionized in

plasma. In equation 1, the ionized form of a dissociable drug, whose plasma

fraction depends on plasma pH (see below), is ignored because its permeability

122
at the BBB is about one-thousandth of the permeability of the unionized form

(Rapoport 1976). Equation 1 holds following an intravenous (IV) bolus injection

of a labeled drug, where it is not so long as to allow sufficient accumulation

of drug in the brain to provide significant back-diffusion to blood (Ohno et al.

1978). However, to calculate the time-dependent concentration profile in the

brain for any administration regimen one needs to know the extended plasma

concentration profile; the concentration profile of the agent in CSF; and the

exchange coefficients between blood and brain, between CSF and brain,

and between extracellular and intracellular (or bound) brain compartments.

Appropriate equations derived for a bolus injection, continuous IV infusion,

and a step elevation in blood concentration of drugs have been derived

(Rapoport et al. 1982).

Exceptions to the proportionality relation in figure 1 have been noted for several

compounds. For example, the lipophilic anticancer vinca alkaloids vinblastine

and vincristine are much less permeable than expected from their high octanol:

water partition coefficients (Greig et al. 1990b). For such relatively large

compounds (vincristine has a molecular weight of 825 daltons; vinblastine,

814 daltons), steric hindrance and molecular charge distribution also influence

cerebrovascular permeability. The author suggests that the additive rules that

govern bulk phase octanol:water partition coefficients (Hansch 1972) break

down for large molecules in which distances between hydrophobic and ionic

regions within the molecule become significant with respect to the distance of

the membrane-water interface and of the thickness of the biological membrane

(about 100 A).

For organic acids, as well as for such bases as morphine, the extent of

dissociation in blood may markedly influence access to the brain, as the unionized form usually has an octanol:water partition coefficient 1,000 times

greater than the ionized form (Rapoport 1976). Thus, for morphine, a pKa of

7.93 at 37 oC causes its rate of entry into the brain from blood to increase with

metabolic alkalosis by as much as fourfold between a blood pH of 7.2 and 7.6

(Shulman et al. 1984).

Availability of a drug for entry into the brain depends not only on its plasma

concentration profile (Rapoport et al. 1982) but also on the extent and

kinetics of its binding with and release from plasma proteins and other

plasma constituents (Robinson and Rapoport 1986). Equation 1 indicates

that only unbound drug in plasma is available for entry into the brain.

However, recent studies have challenged this “free drug hypothesis,” at

least with regard to some agents (Greig et al. 1990a). Experiments using

palmitate, arachidonate, phenytoin, diazepam, and other benzodiazepines

have demonstrated that these latter agents, unlike such others as bilirubin,

123
erythrocin B, and phenobarbital, are not restrictively bound to plasma

constituents and that their extraction from blood into brain is greater than

predicted by their steady-state free plasma concentrations, fCplasma (Rapoport

1983a; Cornford 1984; Levitan et al. 1984; Jones et al. 1988). The rates of

release of these drugs (off-rates) from plasma protein are quite rapid, allowing

them to be stripped and made available for diffusion into brain during the

second or two that blood passes through brain capillaries (circulation time).

Their extraction fraction (upper bound) is given as follows:

E=1-e-(1-dk3*t (2)

where E is the fraction of total drug that is extracted during passage of blood

through brain; t is transit time of blood passing through brain capillaries; k3’ is

effective rate constant for drug uptake into brain; and a is equilibrium fraction

of total drug bound to protein in arterial plasma.

In addition to the proportionality between permeability and octanol:water

partition that characterizes simple passive diffusion between blood and brain

(figure 1), entry into the brain in some important cases depends on specific

transport mechanisms at the BBB. For example, there are several facilitated,

stereospecific, and saturable transport systems at cerebral capillaries for

amino acids, peptides, small organic acids, and monosaccharides, whose

affinity characteristics can be exploited to enhance drug entry into the brain

(see below) (Rapoport 1976; Greig et al. 1987; Smith et al. 1989). Furthermore,

receptor-mediated transfer of such proteins as transferrin and of such peptides

as insulin at the cerebrovasculature has been demonstrated, as well as

enhanced transcytosis of positively charged compared with neutral or negatively

charged proteins and large polysaccharides (Fishman et al. 1987; Armstrong

et al. 1989; Pardridge et al. 1990). Proteins also can gain access to the brain

after first entering CSF at the choroid plexus (Felgenhauer 1974; Rapoport

1983b), and their rates of entry are greater if they are more positively charged

(Griffin and Giffels 1982). Finally, enzymes existing at the cerebral capillaries

can be inhibited to enhance entry of drugs sensitive to metabolism. Thus,

administration of inhibitors of capillary dopa-decarboxylase enhances brain

uptake of L-dopa In the treatment of Parkinson’s disease (Sourkes 1981).

This chapter examines studies on enhancing drug entry into the brain,

which were conducted, to a large extent, at the Laboratory of Neurosciences

in Bethesda, MD. Two approaches are discussed, one based on the structure

and vulnerability of the BBB to osmotic exposure, the other on drug modification

with a view to enhance brain uptake by any of the following factors: prolonging

half-life in blood, increasing lipid solubility to enhance passive diffusion

according to the principle of figure 1, or enhancing transport of the drug via

established carrier mechanisms for amino acids.

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OSMOTIC OPENING OF THE BBB

The cellular nature of the BBB at cerebral capillaries, first demonstrated by

Reese and Karnovsky (1967), suggested to the author’s group (Rapoport

1970; Rapoport et al. 1972) that the BBB might be made more permeable by

applying hypertonic solutions on either side of the vascular wall and thereby

osmotically shrinking vascular endothelial cells. Accordingly, this group

investigated effects of hypertonic solutions, applied topically (extravascularly)

for 10 minutes to the arachnoid surface of the rabbit brain, on the permeabilities

of pial arterioles and venules to intravascular Evans blue-albumin. Minimal

(threshold) concentrations were determined for various electrolytes and

nonelectrolytes for increasing the permeability of the pial vessels to this tracer.

When Evans blue was administered intravenously, 30 minutes after a threshold

concentration of a particular test solution was applied to the brain surface for

10 minutes, the absence or presence of its extravasation demonstrated whether

osmotic BBB opening was reversible.

An inverse relation between threshold concentration and octanol:water

partition coefficient was demonstrated for reversibly acting agents, that is,

those agents less lipid-soluble than ethylene glycol (octanol:water partition

coefficienL0.012) (Rapoport et al. 1972; Rapoport 1976). This relation was

interpreted to indicate that osmotic BBB opening was mediated by shrinkage

of cerebrovascular endothelial cells, stressing and thereby widening

interendothelial tight junctions. This suggested mechanism subsequently

was confirmed, using electronmicroscopy and an electron dense tracer

(horseradish peroxidase or ionic lanthanum) following intracarotid infusion of

hypertonic urea or arabinose in animals (Brightman et al. 1973; Dorovini-Zis et

al. 1983). Suggestions that osmotic BBB opening was mediated by increased

transcellular vesicular transport later were shown to be unlikely (Rapoport

and Robinson 1986; Robinson and Rapoport 1987). Indeed, tight junctional

widening without stimulation of pinocytosis has been demonstrated at

monolayers of cerebrovascular endothelia exposed to hypertonic arabinose

solution (Dorovini-Zis et al. 1984).

In a series of quantitative studies in rabbits, rats, mice, and rhesus monkeys,

the author’s group and others showed that the BBB could be reversibly opened

without damaging the brain by infusing any of several osmotic solutions into

the carotid circulation, while maintaining appropriate oxygenation and blood

flow to the brain. Two concentrated solutions proved most useful: 1.6 molal

arabinose and 1.4 molar (1.6 molal) mannitol solution (Rapoport et al. 1980;

Neuwelt 1989). At the author’s laboratory, concentrated arabinose is used

rather than mannitol solution in small animal experiments because of its

lesser viscosity and lesser tendency to form crystals that produce embolic

125
neuropathology during the infusion procedure (Tomiwa et al. 1982; Rapoport

and Robinson 1991). Postmortem studies demonstrated the absence of

neurological abnormalities or neuropathology following the osmotic procedure

(Rapoport and Thompson 1973; Tomiwa et al. 1982; Neuwelt et al. 1983;

Cosolo et al. 1989) provided care was taken with regard to solution

temperature, filtration, and animal maintenance. When this proscription

was ignored, embolic brain damage occurred (Suzuki et al. 1988; Salahuddin

et al. 1988). Brain edema follows osmotic treatment, as expected from the

protective role of the intact BBB against edema (Rapoport 1985) but it is

absent within 24 hours after the procedure (Rapoport et al. 1980).

Using a sensitive quantitative procedure to examine regional permeability of

the BBB to [14C]sucrose, Rapoport and colleagues (1980) showed that the

BBB in pentobarbital-anesthetized rats was maximally opened at a threshold

arabinose concentration of 1.6 molal for 30 seconds of intracarotid infusion.

Higher concentrations had no incremental effect on BBB permeability, whereas

1.4 molal arabinose was minimally effective. However, the osmotic threshold is

sensitive to the solute employed, to the animal species studied, as well as to

the type of anesthetic used (Rapoport and Robinson 1991). Thus, 1.4 molar

mannitol solution produces a maximal opening when infused into the carotid

circulation of rats anesthetized with phenobarbital, ketamine-xylazine, or

isoflurane but has a minimal BBB effect when methoxyflurane or fentanyldoperidol is the anesthetic. The latter anesthetics make animals either more

hypotensive or tachycardiac than does phenobarbital (Gumerlock and Neuwelt

1990).

The [14C]sucrose method also was used to demonstrate that BBB opening is

essentially reversed within 2 hours (Rapoport et al. 1980). Thus, the elevation

of cerebrovascular permeability to [14C]sucrose, following 1.6 molal arabinose

infusion, has fallen dramatically by about 10 minutes after the procedure

(suggesting that if drugs of similar size are to be employed they should be

administered within this time). Furthermore, by using normally impermeant

radiotracers of differing sizes, the author’s group showed that the rate of

decline of the permeability elevation following the osmotic procedure is greater

for larger than for smaller tracer molecules. This observation is consistent with

the interpretation that osmotic BBB modification is mediated by widening of tight

junctions between endothelial cells but not by stimulation of transendothelial

vesicular transport (see above) (Rapoport and Robinson 1986; Robinson and

Rapoport 1987). It is likely that these open junctions (pores) are negatively

charged, as permeability is increased more to positively charged than to neutral

macromolecules (Armstrong et al. 1989). This suggests that entry of antibodies

or other macromolecules into the brain following the osmotic procedure can be

enhanced further by manipulating their charge.

126
In a series of animal studies, the authors group and collaborators also showed

that the osmotic technique could be used to open the BBB to agents relevant

to CNS disease: neutralizing antibodies to measles virus (Hicks et al. 1976);

[

3H]norepinephrine and [125I]albumin (Chiueh et al. 1978); lysosomal enzymes

(Barranger et al. 1979); mothotrexate (Ohata et al. 1985); bilirubin-albumin

(Levine et al. 1985); and human interferon alpha (Greig et al. 1988).

Clearances from brain, in relation to disease models, were determined for

methotrexate and for bilirubin (Levine et al. 1985; Ohata et al. 1985) and

illustrated the contribution of intracerebral binding or uptake on brain

pharmacokinetics and pharmacodynamics. With regard to cancer

chemotherapy, Neuwelt and colleagues (1983) evaluated the neurotoxicity

of a number of antineoplastic agents that were allowed into the rat brain by

the osmotic procedure; they concluded that doxorubicin, cis-platinum,

bleomycin, 5-fluorouracil, and mitomycin are neurotoxic, whereas

cyclophosphamide and methotrexate are not. Neuwelt (1989) summarizes

additional studies on drug entry into the brain using the osmotic procedure.

Neuwelt and colleagues (1980) were the first to apply the osmotic procedure

in the clinic for treating brain tumors (for a review, see Neuwelt 1989). They

employed a combination of drugs—methotrexate, procarbazine, and

cyclophosphamide—with the osmotic method. Their data provide an upper

limit for morbidity and mortality associated with the osmotic procedure in

humans as results were obtained on patients with brain tumors using

anticancer drugs. When infusing a filtered, 1.4 molar mannitol solution at

37 °C into the carotid circulation, the incidence of permanent neurological

damage equaled 0.8 percent (3 of 384 procedures) and that of seizures

equaled 14 percent (55 of 384 procedures). No death could be related

directly to the osmotic procedure (Rapoport 1988; Rapoport and Robinson

1990). Furthermore, in the absence of radiotherapy, there was no evidence

that the procedure accelerated cognitive deterioration (Crossen et al., in

press). Retinal pigment epithelial damage occurred in the eye unilateral

to perfusion, as predicted from animal experiments, but visual loss was

insignificant (Okisaka et al. 1976; Millay et al. 1986). Neuwelt and coworkers

(1986, 1991) also reported, from Phase II studies, that the osmotic procedure

with the aforementioned drugs significantly prolonged mean survival duration

of patients with primary CNS lymphoma.

CHEMICAL MODlFlCATlON OF DRUGS

Lipophllicity and Passive Diffusion

Although a certain degree of lipid solubility is essential for a drug to cross the

BBB, an agent that distributes excessively in octanol compared with water has

127
some pharmacokinetic limitations. Uptake by brain as well as pharmacodynamic

action may become limited if the octanol:water partition coefficient of the drug

exceeds 100 (log10Partition>2) (Hansch 1972; Rapoport 1976; Greig et al.

1990a). An excessively lipid-soluble agent may be difficult to solubilize and,

when soluble, be heavily bound to plasma proteins. Indeed, studies by Sholtan

(1968) demonstrate a linear relation between the binding of various drugs to

human serum albumin and their lipophilicity. Furthermore, a very lipid-soluble

drug may be rapidly cleared from plasma, as it is taken up into lipid

compartments In body organs.

Modification studies by the author’s group on the anticancer agent chlorambucil

illustrate how the principle of lipid solubllity can be employed with regard to drug

pharmacokinetics and pharmacodynamics. As shown in figure 2, chlorambucll

is a bifunctional, nitrogen mustard alkylating agent; it has proved useful in

treating chronic lymphocytic leukemia and Hodgkin’s disease (Greig et al.

1990a). However, it is largely ionized at physiological blood pH. As a charged

anion, it minimally enters the brain, so that it is ineffective against potentially

sensitive brain-sequestered tumors.

Chlorambucil

Chlorambucil-Tertiary Butyl Ester

FIGURE 2. Chemical formulas of chlorambucil and of chlorambucil-terliaty

butyl ester

128
Postulating that masking the ionizable carboxylic acid moiety of chlorambucil

by a lipophilic ester would increase its access to the brain but would not

necessarily destroy its antitumor alkylating activity, Greig and colleagues

(1990c, 1990d) reacted chlorambucil with many alcohols to form corresponding

ester derivatives, without modifying its alkylating moieties. Each ester

synthesized possessed significant intrinsic alkylating activity, although less

than that of chlorambucil. However, none achieved or maintained greater

concentrations in the rat brain after equimolar administration than did

chlorambucil, with one exception, chlorambucil-tertiary butyl ester (figure 2).

All but this latter ester were rapidly cleaved in plasma by esterases or were

so lipophilic that they became tightly bound to plasma proteins (Sholtan 1968).

thereby restricting their access into brain (equation 1). However, the steric

hindrance of the branched chain chlorambucil-tertiary butyl ester reduced

its rate of hydrolysis in plasma. Furthermore, despite its high lipophrlicity

(log10Partition=3.2), it can be readily formulated for IV administration, and

a significant unbound fraction f exists in plasma. As illustrated in figure 3,

administration of this ester to rats, compared with an equimolar amount of

chlorambucil, resulted in significant uptake within brain.

With regard to pharmacodynamics, furthermore, the maximum tolerated dose of

chlorambucil-tertiary butyl ester in rats was found to be sixfold higher than that of

chlorambucil. Unlike chlorambucil, high doses of the ester did not cause

seizures. Finally, chlorambucil-tertiary butyl ester, unlike chlorambucil.

FIGURE 3. Concentrations of chlorambucil (left) and of chlorambucil-tertiary

butyl ester (right) in plasma and brain following equimolar IV

administration in rats (10mg/kg and 13mg/kg, respective/y)

SOURCE: Greig et al. 1990a

129
significantly prolonged survival of rats with brain-implanted tumors and was

found to be effective against human brain tumors in vitro that are insensitive to

BCNU, the mainstay of brain tumor chemotherapy (Greig et al. 1990a; N.H.

Greig, S. Genka, S.I. Rapoport, and F. Ali-Osman, unpublished observations).

Drug Modification for Carrier-Mediated Transport

An alternative goal of drug modification might be to synthesize an active

compound that structurally resembles an endogenous agent that shares a

facilitated transport system at the BBB. This principle has been demonstrated

with regard to melphalan, the anticancer drug that is a nitrogen mustard

derivative of L-phenylalanine (figure 4). Greig and colleagues (1987) showed

that melphalan is transported into the CNS via the capillary carrier system for

large neutral amino acids; its transport can be inhibited by L-phenylalanine,

and it is concentration-dependent and saturable, with an affinity, however, that

is less than those of the natural large neutral L-amino acids.

To more rationally identify which properties of large neutral L-amino acids

determine affinity for the common carrier at the cerebrovasculature, Smith

and coworkers (1987) examined affinities 1/Km, where Km equals half

saturation concentration) of each of 14 neutral L-amino acids at cerebral

capillaries of pentobarbital-anesthetized rats, using an in situ brain perfusion

technique (Takasato et al. 1984). As illustrated by figure 5, the affinity of each

L-amino acid was found to be proportional to the octanol:water partition

coefficient, a measure of side-chain hydrophobicity (Yunger and Cramer 1981).

Thus, although preferring the L-form of the amino acid, the carrier has weak

requirements with regard to side-chain structure and size, other than how they

influence hydrophobicity.

Under normal in vivo conditions, circulating large neutral L-amino acids

compete with each other for the common carrier, which is saturated and has

a high transport capacity. The very high affinity amino acids L-leucine and

L-phenylalanine together occupy more than 50 percent of vascular carrier sites.

Smith and coworkers (1989) later used the relation of figure 5 to design and

study more than 100 synthetic amino acids of varying lipid solubilrties to

determine if some would have sufficiently high affinities to displace even

L-phenylalanine and L-leucine from the carrier. They measured the ability of

each amino acid to inhibit transport into rat brain of [1-14C]L-phenylalanine,

using their in situ perfusion technique. Even for the synthetic L-amino acids,

affinity depended over a 10,000-fold range on side-chain hydrophobicity.

Amino acid analogs with large hydrophobic side chains (e.g., L-napthylalanine)

had much higher affinities than even L-phenylalanine (296 mM-1 compared

with 95 mM-1 for phenylalanine) for the large neutral L-amino acid carrier.

130
Saline Perfusion

Compound

PAx104,

S

-1 K

m

Tryptophan 966.7 0.009

Phenylalanine 790.0 0.010

Leucine 411.5 0.026

Tyrosine 356.0 0.050

lsoleucine 209.8 0.05 1

Methionine 77.3 0.075

Melphalan 10.8 0.150

Km=μmoles/mL

Phenylalanine

Melphan

FIGURE 4. Permeability-surface area products and ha/f-saturation

concentrations of large neutral L-amino acids and of melphalan

at the BBB, as measured by the in situ brain perfusion technique

of Takasato and colleagues (1984). Structures of phenylalanine

and of melphalan are illustrated.

SOURCE: Greig et al. 1987

On the basis of these studies, it should be possible to design drugs in the form

of large neutral L-amino acids with hydrophobic side chains to take advantage

of the high-capacity, large neutral L-amino acid carrier at the BBB. Similar

approaches might be made with regard to other carrier systems, such as those

subserving peptide transport at the BBB (Banks and Kastin 1990), although

these usually have a lower transport capacity than does the large neutral

L-amino acid carrier (Bradbury 1989).

131
Octanol:Water Partition Coefficient

FIGURE 5. Relation between 1/Km (Km=ha/f-saturation concentration) and

0ctanol:waterpartition coefficient for 14 large neutral L-amino

acids. Asn=asparagine; Ala=alanine; Cyc=cycloleucine;

Gbglutamine; His=histidine; Ile=isoleucine; Leu=leucine;

Met=methionine; Phe=phenylalamine; Sekserine;

Thr=threonine; Trp=tryptophan; Tyr=tyrosine; Vakaline

SOURCE: Smith et al. 1987

In conclusion, understanding the structure and function of the BBB can allow

ways to be devised to increase BBB permeability to drugs or other agents or to

design synthetic approaches to modify drugs so as to enhance their penetration

into the brain passively or by means of existing carrier mechanisms.

132
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AUTHOR

Stanley I. Rapoport, M.D.

Chief

Laboratory of Neurosciences

National Institute on Aging

National Institutes of Health

Building 10, Room 6C-103

Bethesda, MD 20892

137
Cultured Brain Microvessel

Endothelial Cells as In Vitro Models

of the Blood-Brain Barrier

Yoshinobu Takakura, Kenneth L. Audus,

and Ronald T. Borchardt

INTRODUCTION

A central nervous system (CNS) site of action generally requires that, following

entry into the systemic circulation, substances must interact with the blood-brain

barrier (BBB) en route to brain tissue targets. Many substances with CNS

activity, including drugs of abuse, cross the BBB by simple passive diffusion

(Oldendorf 1974; Levin 1980; Cornford et al. 1982). Accordingly, the BBB

plays an important role in regulating access of drugs of abuse to the brain.

Recent evidence suggests that some drugs of abuse may alter BBB

permeability characteristics. The permeability-altering activity of drugs

of abuse at the BBB has been reported in chronic amphetamine intoxication

(Rakic et al. 1989), acute ethanol exposure (Gulati et al. 1985), and opiate

treatment (Baba et al. 1988). Ethanol addiction also has been reported to

alter opiate interactions with the transport systems of the BBB (Banks and

Kastin 1989). On the basis of these reports, one might infer that interactions

of drugs of abuse with the BBB may have implications for the observed

pharmacological and toxicological manifestations of exposure to these

substances. These implications remain to be explored and confirmed.

Consequently, the development of a fundamental understanding of the

permeability and metabolic features of the BBB may contribute to a broader

knowledge of the activity of drugs of abuse In the CNS.

The complexity of the whole animal as an experimental model has certain

limitations when specific events at the cellular or molecular level are examined.

In vitro models (i.e., brain microvessel suspensions or cultured brain endothelial

cells) offer a promising alternative for the investigation of cellular- or molecularlevel characteristics of the BBB (Audus et al. 1990; Audus and Borchardt

1987). The purpose of this chapter is to describe studies of the transport

138
and metabolism characteristics of the BBB that employ cultured brain

microvessel endothelial cells as in vitro BBB models. This chapter does not

describe isolated suspensions of brain microvessel endothelial cells as in vitro

models, since this subject has been reviewed by Betz and Goldstein (1984)

and more recently by Takakura and coworkers (1991 a). In addition, this

chapter does not describe how cultured brain microvessel endothelial cells

have been used to study the development and regulation of the BBB and

pathological changes in the BBB, since these topics are discussed in other

chapters in this monograph and have been reviewed previously by Takakura

and coworkers (1991 a).

ESTABUSHMENT AND CHARACTERIZATION OF CULTURED BRAIN

MICROVESSEL ENDOTHELIAL CELLS AS IN VITRO BBB MODELS

Since Panula and colleagues (1978) demonstrated that rat brain microvessel

endothelial cells could be maintained in tissue culture, various kinds of both

primary and passaged cultures of isolated brain microvessel endothelial cells

have been established from mouse, rat, bovine, human, canine, and porcine

brain (for a review, see Audus et al. 1990).

In general, either enzymatic or mechanical dispersal, or a combination of both

techniques, followed by either filtration or centrifugation steps are employed to

isolate a homogeneous population of brain microvessel endothelial cells from

the extremely heterogeneous population of cells found in brain tissues. For

example, isolation of a viable, homogeneous population of brain capillary

endothelial cells for establishment of a tissue culture system is accomplished

by a two-step enzymatic digestion with dispase and a dispase/collagenase

mixture of cerebral gray matter and successive centrifugation over dextran

and percoll gradients (Bowman et al. 1983; Audus and Borchardt 1986a,

1987). In the authors’ laboratories, primary cultures of bovine brain microvessel

endothelial cell monolayers have been shown to retain morphological and

biochemical properties typical of the BBB in vivo. These include tight junctions,

attenuated pinocytosis, lack of fenestra, and the presence of proteins (e.g., yglutamyl transpeptidase, alkaline phosphatase, angiotensin-converting enzyme,

factor VIII antigen) enriched in the endothelium of the BBB (Audus and

Borchardt 1986a, 1987).

Basically, two types of experimental systems have been employed for study of

transport phenomena using cultured brain microvessel endothelial cells: The

first is the uptake study, and the second involves a transcellular transport study.

The former system uses microvessel endothelial cell monolayers grown in

culture dishes (Scriba and Borchardt 1989a, 1989b). Uptake experiments can

be performed also using cerebral microvessel endothelial cells cultured on

139
microcarriers (e.g., dextran beads) (Bottaro et al. 1986; Kempski et al. 1987).

These systems allow examination of the first step of the transport process,

that is, the uptake of the solutes into the brain capillary cells from the luminal

side. The most sophisticated in vitro system for transport studies consists of

cultured brain microvessel endothelial cell monolayers grown on microporous

membranes. Transport studies can then be conducted in side-by-side diffusion

cells or the cell-insert system (Audus et al. 1990). These systems afford an

opportunity to look at bidirectional transendothelial movement (transfer from

brain to blood and that from blood to brain) of solutes across the BBB in vitro

since, at least for primary cultures of bovine microvessel endothelial cells, the

cells are shown to be morphologically and functionally polarized in terms of ricin

recycling (Raub and Audus 1990), transferrin transport (Newton and Raub

1988), and angiotensin II (Ang II) responsiveness (Guillot and Audus 1990,

1991a). The purity of these monolayers is greater than 95 percent (Gurllot et

al. 1990). and the transelectrical resistance is 160±18 ohm/cm2 (T.J. Raub,

unpublished results).

Although bovine brain microvessel endothelial monolayers retain tight junctions,

the tight junctions are not identical to those observed in vivo with regard to

extent and complexity. This leads to higher leakiness in vitro than in vivo,

which is a disadvantage in the use of cultured endothelial cells alone to study

transcellular transport.

More impermeable monolayers of cultured brain microvessel endothelial cells

may be developed in the future by exploiting the regulatory role of astrocytes in

endothelial cell growth and development. It is widely accepted that the brain

microvessel endothelial cells in vivo form the structural and functronal bases

of the BBB; however, some of the BBB functions are known to be regulated

by astrocytes, which encircle the microvessel endothelial cells with their foot

processes In vivo. Similar regulatory effects of astrocytes on the permeability

properties of cultured endotheltal cells in vitro have been reported. For

example, Cancilla and DeBauIt (1983) demonstrated that contact with glial

cells (a rat line of neoplastic astrocytes designated as C6 glioma cells) or

exposure to glial-conditioned media enhances neutral amino acid uptake by

passaged mouse cerebral endothelial cells in culture. Conditioned media

prepared from astrocytes and C6 glioma cells have been shown to stimulate

glucose uptake in passaged mouse cerebral endothelial cells (Maxwell et al.

1989) and in primary cultures of bovine brain microvessel endothelial cells

(Takakura et al. 1991 b). Conditioned media from rat astrocytes or C6 glioma

cells also ,have been shown to decrease the permeability of various solutes

across monolayers of bovine brain microvessel endothelial cells grown on

microporous membranes (Trammel and Borchardt 1989; Raub et al. 1989).

Recently, Dehouck and colleagues (1990) observed decreases in permeability

140
and increases in electrical resistance using an in vitro model system consisting

of passaged brain microvessel endothelial cells grown on one side of a filter and

astrocytes on the other side of the filter.

UPTAKE AND TRANSPORT STUDIES

Nutrients

Since glucose is an important source of energy for the brain, the mechanism

of glucose transport across the BBB has been particularly well studied in

vivo (Pardridge 1983). These studies support the concept that glucose is

transported through the cerebral microvessel endothelium mainly by the

mechanism of carrier-mediated facilitated diffusion. Glucose transport

characteristics also were studied in cultured brain microvessel endothellal

cells. For example, Vinters and coworkers (1985) demonstrated that the

properties of 3-O-methylglucose (3MG) and 2-deoxyglucose uptake in

established lines of cultured mouse cerebral microvessel endothelium are

similar to those observed in vivo. Recently, characteristics of both uptake and

transendothelial transport of 3MG were studied in primary cultures of bovine

brain microvessel endothelial cells (Takakura et al. 1991c). The uptake

characteristics of 3MG were shown to be identical to those observed in vivo

and in vitro using isolated capillaries. Transport rates from the luminal to

abluminal side and from the abluminal to luminal side, measured across the

brain microvessel endothelial cells grown onto polycarbonate membranes,

were nearly identical, suggesting symmetrical glucose transport across the

monolayer of endothellal cells.

The passage of amino acids across the BBB was found to be saturable and

stereospecific in vivo (Oldendorf 1971). Cancilla and DeBault (1983) showed

the presence of A- and L-systems for uptake of neutral amino acids in cultured

mouse cerebral endothelial cells. Using primary cultures of bovine brain

microvessel endothelial cell monolayers grown onto microporous membranes,

transport of a large neutral amino acid, leucine, was shown to be saturable,

bidirectional, competitive with other amino acids, and energy independent

(Audus and Borchardt 1986b). The kinetic parameters for leucine transport

appear to be in good agreement with true kinetic parameters of the in vivo

BBB. The transport of several amino acid drugs-including baclofen (van

Bree et al. 1988), a-methyldopa (Chastain and Borchardt 1989). and acivicin

(Chastain and Borchardt 1990)-by the amino acid carrier also has been

explored in this system.

Because of a limited capability for de novo synthesis of choline, an important

precursor to acetylcholine and phospholipid, the brain must depend on the

141
blood for its supply of choline. Cornford and colleagues (1978) demonstrated

the saturability of brain uptake of choline after intracarotid injection in rats.

Recently, Estrada and associates (1990) studied choline uptake by bovine

cerebral capillary endothelial cells in culture, demonstrating that these cells

were able to incorporate choline by a carrier-mediated mechanism. The

choline uptake was temperature dependent and was inhibited by choline

analogs but was not affected by ouabain or dinitrophenol. Transendothelial

transport of choline in cultured bovine brain microvessel endothelial cells

has been characterized (Trammel and Borchardt 1987); it has been shown

that the transport is saturable and is insensitive to ouabain and sodium azide,

suggesting a facilitated diffusion mechanism.

Since the brain is one of the most active tissues for carrying out nucleotide

and nucleic acid synthesis, the transport of nucleosides and purine bases

across the BBB has been of interest. By employing the intracarotid injection

technique in rats, Cornford and Oldendorf (1975) demonstrated the presence

of two independent carrier systems for nucleic precursors: a nucleoside carrier

and a purine base carrier. Beck and coworkers (1983) have described a

carrier-mediated uptake of adenosine into mouse cerebral capillary endothelial

cells in tissue culture. Characterization of the nucleoside uptake (e.g.,

adenosine, thymidine) into monolayers of cultured bovine brain endothelial

cells also was studied, and the results suggested the presence of a carriermediated uptake of adenosine and thymidine (Shah and Borchardt 1989).

Adenosine uptake is primarily via the carrier-mediated pathway, whereas

thymidine enters by both a carrier-mediated and a passive pathway. Both

nucleosides are extensively metabolized (e.g., phosphorylated) in the cultured

bovine endothelial cells.

Peptides and Proteins

Although the physiological role of insulin in the regulation of brain functions

remains to be elucidated, van Houten and Posner (1979) revealed that blood

vessels throughout the CNS of the rat bind plasma insulin rapidly and with

considerable specificity in vivo. The binding and receptor-mediated endocytosis

of insulin and insulin-like growth factor I (IGF-I) also were studied using cultured

bovine brain microvessel endothelial cells (Keller and Borchardt 1987; Keller et

al. 1988). Rosenfeld and colleagues (1987) showed the similarity between the

characteristics of the specific receptors for IGF-I and insulin-like growth factor II

in the cultured bovine brain microvessel endothelial cells and in isolated rat

brain microvessels.

Using monoclonal antibodies to the transferrin receptors, Jefferies and

coworkers (1984) first reported that rat and human brain capillary endothelia

142
have receptors for transferrin, an iron-transport protein in the circulation.

Newton and Raub (1988) characterized the transferrin receptor in primary

cultures of brain capillary endothelial cells, indicating saturable binding and

internalization. They also demonstrated the transcytosis and the polarized

efflux of transferrin using brain microvessel endothelial cells grown onto

polycarbonate filters; these findings are in good agreement with the in vivo

observations of Fishman and coworkers (1987) and Banks and coworkers

(1988).

Atrial natriuretic factor (ANF), which is a 28-amino acid peptide produced by

cardiac myocytes and released in response to increases in atrial pressure,

expresses its natriuretic, diuretic, and hypotensive effects by acting on renal

and vascular tissues. A specific receptor for ANF also was identified using

primary cultures of bovine brain capillary endothelial cells (Smith et al. 1988).

The binding of ANF was specific, saturable, and reversible. ANF also was

shown to be rapidly internalized by a temperature-dependent process, A

specific receptor for brain natriuretic peptide (BNP), which was found in the

brain and has similarity to ANF in its structure and biological activities (Sudoh et

al. 1988), was identified recently in primary cultures of bovine brain microvessel

endothelial cells (M. Fukuta, M. Nonomura, Y. Takakura, and R.T. Borchardt,

unpublished results). These studies suggested that ANF and BNP share the

same receptor in bovine brain microvessel endothelium.

Speth and Harik (1985) reported that Ang II binds to microvessels isolated from

dog brain in a specific, saturable, and reversible manner and with high affinity.

It was suggested that specific Ang II receptor-binding sites are present in brain

microvessels and that these receptors may have an important role in regulating

the microcirculation of the brain. Work in the authors’ laboratories indicates that

bovine brain microvessel endothelial cell monolayers retain a high-affinity Ang II

binding site that can be competed for by Ang II peptides (Guillot and Audus

1991b).

Recently, the transport of leu-enkephalin across the BBB by a carrier-mediated

mechanism has been demonstrated in vivo (Zlokovic et al. 1987). In addition,

Thompson and Audus (1989) showed that leu-enkephalin transfer across

monolayers of brain microvessel endothelial cells occurs at a relatively high

rate, which is consistent with a facilitated diffusion mechanism.

Vasopressin transport across the BBB has been examined with primary

cultures of brain microvessel endothelial monolayers (Reardon and Audus

1989). Results suggest the existence of facilitated transport of the peptide from

the abluminal to the luminal side of the monolayers. This finding is consistent

with the in vivo characterization of a vasopressin BBB transport system (Banks

143
et al. 1987). Van Bree and colleagues (1989) also have studied transport of

vasopressin using an in vitro system and suggested that no carrier mediation is

involved over a higher concentration range. Confirmatory studies are required

to clarify the transport mechanism in detail.

Raeissi and Audus (1989) characterized the BBB permeability to delta sleepinducing peptide (DSIP) in cultured microvessel endothelial cells. The results

support in vivo observations indicating that intact DSIP crosses the BBB by

simple transmembrane diffusion (Banks and Kastin 1987). Recently, Zlokovic

and coworkers (1989) presented evidence in support of a facilitative BBB carrier

for DSIP in vivo. Further work is ongoing concerning the solution structure of

DSIP, which may help to explain its ability to readily penetrate the BBB (Audus

and Manning 1990).

Native albumin, which is an acidic protein in plasma, is considered to pass

through the BBB very slowly (Pardridge et al. 1985). However, increased

BBB uptake and transport recently has been reported when it is chemically

modified. Kumagai and colleagues (1987) demonstrated the enhanced binding

and adsorptive-mediated endocytosis of cationized albumin by isolated bovine

brain capillaries. The binding was saturable and inhibited by other polycations

(e.g., protamine, protamine sulfate, and polylysine). Similar results have

been reported for other types of polycationic proteins, including cationized

immunoglobulin G (Triguero et al. 1989) and histone (Pardridge et al. 1989).

Smith and Borchardt (1989) studied the binding, uptake, and transcellular

transport of bovine serum albumin (BSA), cationized BSA (cBSA), and

glycosylated BSA (gBSA) in cultured bovine brain microvessel endothelial cells.

This study demonstrated that cBSA and gBSA bind to the cells specifically and

are transported by an adsorptive-phase endocytotic mechanism. The use of

cationized albumin in directed delivery of peptides through the BBB was

examined by coupling ß-endorphin to cationized albumin via a disulfide linkage

(Kumagai et al. 1987).

Drugs

In contrast to water-soluble nutrients and peptides, which are transported

by specific carrier- or receptor-mediated systems as mentioned above, most

water-soluble solutes, including drugs, pass through the BBB by a passive

diffusion mechanism. From in vivo studies, it has been well established that

the permeability of these molecules across the BBB depends directly on their

lipophilicity and inversely on their molecular size (Oldendorf 1974; Levin 1980;

Cornford et al. 1982). Rim and coworkers (1986) and Shah and coworkers

(1989) established a positive correlation between lipid solubility of a drug and

its permeability across bovine brain microvessel endothelial monolayers grown

144
onto microporous membranes. As mentioned earlier in this chapter,

brain microvessel endothelial monolayers are leakier than the BBB in vivo.

Therefore, this leakiness in the monolayers must be corrected for by using

impermeant marker molecules (e.g., sucrose, fluorescein, inulin, and dextran)

when drug transport studies are conducted (Shah et al. 1989).

IN VITRO STUDIES ON BBB METABOLISM

Several different enzyme systems have been established in primary cultures

of bovine brain microvessel endothelial cell monolayers. Both biochemical and

histochemical techniques have been used to follow the expression of marker

and catecholamine-metabolizing enzymes associated with these monolayers.

For instance, enzyme systems considered markers for the BBB, glutamyl

transpeptidase and alkaline phosphatase, and for the endothelium, angiotensinconverting enzyme, are retained in the monolayers (Baranczyk-Kuzma et al.

1986). In addition, the brain microvessel endothelial cell monolayers retain the

ability to degrade catecholamines through expression of monoamine oxidases A

and B, catechol O-methyltransferase, and phenol sulfotransferase (BaranczykKuzma et al. 1986, 1989a; Scriba and Borchardt 1989a. 1989b). Using

monolayers of bovine brain microvessel endothelial cells, the metabolism of

catecholamine esters (Scriba and Borchardt l989a) and of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) has been studied (Scriba and Borchardt

1989b).

The lysosomal compartment of endothelia is an important component of the

transcytosis of macromolecules (Simionescu 1979). Current interest by the

pharmaceutical industry in the delivery of therapeutic biotechnology products

to the CNS (Audus et al. 1990) suggests a need for considering the activity of

the lysosomal compartment of the BBB. However, little work has focused on

lysosomal or acid hydrolase compartments within the BBB in vivo. By both

quantitative and qualitative measures, bovine brain microvessel endothelial

cells possess a lysosomal compartment that expresses typical acid hydrolases,

acid phosphatase, ß-galactose, and sulfatases (Baranczyk-Kuzma et al. 1989b).

Peptidase activity has not yet been examined in the lysosomal fraction;

however, aminopeptidase activity is characteristic of membrane and cytosolic

fractions of the cells (Baranczyk-Kuzma and Audus 1987). Corresponding

quantitative studies in in vivo enzyme systems and compartments have not

been performed and may not be feasible. Therefore, the in vitro model may be

a reasonable system in which to initiate further studies on the contributions of

the lysosomal compartment to the fate of endocytosed peptides and proteins at

the BBB (Baranczyk-Kuzma et al. 1989b).

145
CONCLUSIONS

The development of in vitro BBB models consisting of cultured brain

microvessel endothelial cells has made possible the study of BBB transport

and metabolism phenomena at the cellular level. Basic characteristics of

BBB transport and metabolism of endogenous and exogenous solutes and

their biochemical, pharmacological, ontogenic, and pathological regulation

mechanisms have been investigated. This information has led not only to a

better understanding of BBB transport but also to the construction of strategies

for improving drug delivery to the CNS for diagnosis and therapeutics. To

elucidate the complexity of BBB transport, in vivo studies are always necessary

at some point; however, in vitro systems can be useful complements to in vivo

systems. The tissue culture systems seem to be especially important in the

clarification of cellular, biochemical, and molecular features of BBB transport.

Appropriate systems should be selected or combined, depending on the

purpose of the investigation.

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Triguero, D.; Buciak, J.B.; Yang, J.; and Pardridge, W.M. Blood-brain barrier

transport of cationized immunoglobulin G: Enhanced delivery compared to

native protein. Proc Natl Acad Sci U S A 86:4761-4765, 1989.

van Bree, J.B.M.M.; Audus, K.L.; and Borchardt, R.T. Carrier-mediated

transport of baclofen across monolayers of bovine brain endothelial cells

in primary culture. Pharm Res 5:369-371, 1988.

van Bree, J.B.M.M.; de Bore, A.G.; Verhoef, J.C.; Danhof, M.; and Breimer,

D.D. Transport of vasopressin fragments across the blood-brain barrier:

In vitro studies using monolayer cultures of bovine brain endothelial cells.

J Pharmacol Exp Ther 249:901-905. 1989.

van Houten, M., and Posner, B.I. Insulin binds to brain blood vessels in vivo.

Nature 282:623-625, 1979.

Vinters, H.V.; Beck, D.W.; Bready, J.V.; Maxwell, K.; Berliner, J.A.; Hart, M.N.;

and Cancilla, P.A. Uptake of glucose analogues into cultured cerebral

microvessel endothelium. J Neuropathol Exp Neural 44:445-458, 1985.

Zlokovic, B.V.; Lipovac, M.N.; Begley, D.J.; Davson, H.; and Rakic, L.

Transport of leucine-enkephalin across the blood-brain barrier in the

perfused guinea pig brain. J Neurochem 49:310-315, 1987.

Zlokovic, B.V.; Susie, V.T.; Davson, H.; Begley, D.J.; Jankov, R.M.; Mitrovic,

D.M.; and Lipovac, M.N. Saturable mechanism for delta sleep-inducing

peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea

pig brain. Peptides 10:249-254, 1989.

ACKNOWLEDGMENTS

The authors’ research on bovine brain microvessel endothelial cells as a model

of the BBB was supported by grants from the Upjohn Company, Merck Sharp &

Dohme-INTER, Corp., the American Heart Association, and the American

Heart Association-Kansas Affiliate.

AUTHORS

Yoshinobu Takakura, Ph.D.

Assistant Professor

Kenneth L. Audus, Ph.D.

Associate Professor

151
Address all correspondence to:

Ronald T. Borchardt, Ph.D.

Summerfield Professor and Chairman

Department of Pharmaceutical Chemistry

University of Kansas School of Pharmacy

Malott Hall, Room 3006

Lawrence, KS 66045

152
Opioid Peptide Drug Development:

Transport of Opioid Chimeric Peptides

Through the Blood-Brain Barrier

William M. Pardridge

INTRODUCTION

There currently is a need for developing new drugs for the treatment of

heroin addiction. Ideally, newer agents will lack the side effects of physical

dependence or respiratory depression that are typical of l-related opioid

peptides but will still block heroin withdrawal symptoms. Among the three

classes of opioid peptides (dynorphins, ß-endorphin, and enkephalins),

dynorphin analogs have shown some promise. These compounds have a

high degree of -opioid receptor specificity (Goldstein et al. 1988). Moreover,

studies over the past several years have shown that dynorphin 1-13 acts at

spinal cord sites to suppress withdrawal symptoms in morphine-dependent

rats and to play a role in tolerance and dependence mechanisms (Wen and

Ho 1982; Wen et al. 1985; Green and Lee 1988; Stevens and Yaksh 1989).

In addition, the termination of dynorphin treatment is not associated with

withdrawal symptoms (Smith and Lee 1988). However, the use of opioid

peptides in drug design is complicated by three principal problems:

(1) rapid degradation of the native analogs by tissue peptidases, principally

aminopeptidase; (2) lack of transport of peptide pharmaceuticals across

the gut intestinal barrier; and (3) lack of transport of water-soluble peptides

through the brain capillary endothelial wall, which makes up the blood-brain

barrier (BBB) in vivo (Pardridge 1988).

With regard to the rapid inactivation of opioid peptides by tissue

aminopeptidase, analogs have been synthesized that render the peptide

much less resistant to aminopeptidase inactivation. For example, the

substitution of the second amino acid with a D-Ala2 results in significant

resistance to aminopeptidase activity (Pert et al. 1979). With respect to

circumventing the gut barrier, peptides may be administered by an intranasal

insufflation route. For more than 10 years, this approach has been used

successfully for the treatment of diabetes insipidus with a metabolically stable

analog of arginine vasopressin (Cool et al. 1990). The vasopressin analog is

153
only nine amino acids in length. However, peptides as large as insulin, which

is approximately 50 amino acids in length, also have been successfully

administered by intranasal insufflation (Salzman et al. 1985). Indeed, a

series of peptides and even larger proteins have been shown to gain access

to the bloodstream following nasal administration (Chien and Chang 1987).

The availability of the transnasal route obviates the difficult problem of

designing analogs that survive the acidity of the stomach and are transported

through the gut intestinal barrier.

Since the problems of preparing peptidase-resistant analogs and of

circumventing the gut barrier have existing solutions (Veber and Freidlinger

1985; Chien and Chang 1987) the major difficulty in opioid peptide drug

design is the BBB transport problem. If BBB could be solved, there would be

advantages to using opioid peptides in drug design, such as (1) no kidney or

liver damage as in the case of nonpeptide benzomorphan or other nonpeptide

drugs (Rapaka 1986) (2) easy synthesis of analogs in high yield that are

peptidase resistant (Rapaka 1986) and (3) minimization of side effects by

design of receptor-specific analogs with conformational restriction (Hruby

and Mosberg 1982).

OVERVIEW OF STRATEGIES AVAILABLE FOR BBB DRUG DELIVERY

There are three principal approaches to delivery of drugs through the BBB:

(1) invasive or neurosurgical-based, (2) pharmacologic-based, and (3)

physiologic-based. Neurosurgical-based strategies include (1) the opening

of the BBB following the intracarotid injection of hypertonic media and (2) the

placement of intraventricular delivery catheters and continuous intraventricular

drug infusion. Repetitive opening of the BBB with the injection of intracarotid

hypertonic media has been associated with a high incidence of seizures as well

as chronic neuropathologic changes in the brain (Neuwelt and Rapoport 1984;

Salahuddin et al. 1988). The delivery of drugs by intraventricular infusion is

complicated by the steep gradients of drug concentrations that are established

in the brain with this procedure (Blasberg et al. 1975). These gradients arise

from the rapid bulk flow of cerebrospinal fluid (CSF) through the ventricular

spaces and absorption into the peripheral circulation at the superior sagittal

sinus, relative to the slow rates of peptide diffusion down into brain parenchyma

from the surface of the brain. For example, the CSF compartment in the human

brain contains approximately 140 mL and is turned over every 4 hours by

absorption at the arachnoid granulations into the superior sagittal sinus and

peripheral bloodstream. Conversely, diffusion rates are relatively slow. For

example, given a peptide with a molecular weight of 10,000 and a diffusion

coefficient (D) of 1 x10-6cm2/sec, it may be calculated that it takes this peptide

2.9 days to diffuse 5 mm (Pardridge 1991). Thus, the peptide is more likely

154
to leave the brain into the peripheral circulation following release into the

ventricular compartment than it is to diffuse down into the brain parenchyma.

Moreover, as soon as the peptide enters brain parenchyma, it is subject to

sequestration by the cells in brain, thus impeding its further diffusion into brain

parenchyma. Conversely, the intraventricular drug delivery approach has

proven successful in treating disorders that involve the surface of the brain.

For example, there are high concentrations of opioid receptors on the surface

of the spinal cord, and intraspinal morphine has proven effective in treatment

of chronic pain syndromes (Coombs et al. 1983). Indeed, the intraventricular

injection of ß-endorphin in rodents produces analgesia, probably via the release

of spinal methionine enkephalin and the interaction of this analog with -opioid

receptors (Suh and Tseng 1990). However, the treatment of drug dependency

syndromes likely involves significant peptidergic pathways in the cerebral

cortex, and intraventricular drug administration would not allow for adequate

drug delivery to these structures within the brain.

Pharmacologic-based approaches for BBB drug delivery Include lipidization

of peptides or the use of liposomes. Peptide lipidization involves the blocking

of polar functional moieties on the peptide backbone with groups that greatly

enhance the lipid solubility of the peptide. For example, carboxyl groups or

hydroxyl groups on peptides may be blocked by esterification. Indeed, the

high addictive properties of heroin (diacetyl morphine) depend on the extremely

rapid rate of transport of heroin through the BBB owing to the acetylation of

the two hydroxyl groups on the morphine nucleus (Oldendorf et al. 1972).

The permeability of the BBB is enhanced a log order by the blockade of a

single hydroxyl group on the drug nucleus (Pardridge 1988), and the rapid

transport of heroin across the BBB relative to morphine contributes to the

addictive properties of heroin relative to the parent morphine compound.

The problem with peptide lipidization is that there is a size exclusion property

of BBB lipid-mediated transport. For example, cyclosporin is a highly lipidsoluble, 11-amino acid peptide compound. The octanol/Ringer’s partition

coefficient of cyclosporin is approximately 1,000 and is equal to that of highly

lipid-soluble steroid hormones that penetrate the BBB as fast as heroin or

ethyl alcohol (Cefalu and Pardridge 1985). However, the BBB transport of

cyclosporin is paradoxically low and, in the presence of plasma lipoproteins,

is essentially negligible. Another lipid-soluble peptide, minisomatostatin,

is composed of eight amino acids; and this peptide also has a relatively

high degree of lipid solubility owing to the formation of internal hydrogen

bonding within the cyclic peptide structure. However, the BBB transport

of minisomatostatin is essentially nil, owing to the high molecular weight

(approximately 1,000) of this compound (Pardridge et al. 1990a). Therefore,

it seems unlikely that the lipidization of peptides that are composed of more

than five to six amino acids is a feasible strategy for enhancing BBB drug

155
transport, owing to the size exclusion property of lipid-mediated transport

through the BBB (Pardridge 1988). This size exclusion property is also

the principal reason why liposomes have not proven to be an efficacious

mechanism for drug delivery across the BBB. Initial liposomes were large

and found to be rapidly destabilized by plasma proteins and to be rapidly

taken up by cells comprising the reticuloendothelial system (Gregoriadis

1976). Subsequently, small unilamellar vesicles were developed on the order

of 50 nm in size. It may not be possible to synthesize liposomes that are much

smaller than 20 to 50 nm in size (Freeman and Mayhew 1986). Nevertheless,

this is still a relatively large size. For example, lipoproteins are some of the

larger molecules in the bloodstream, and these proteins are on the order of

20 nm, or approximately 10 times the diameter of a peptide such as cyclosporin,

which itself is subject to significant size exclusion with regard to BBB transport.

The prototypic physiologic-based strategy for BBB drug delivery is the use of

L-dopa in the treatment of Parkinson’s disease (Nutt et al. 1984). Dopamine,

a catecholamine, is not transported through the BBB. Therefore, brain

dopamine levels, which are greatly depressed in the caudate putamen

nucleus in individuals with Parkinson’s disease, cannot be repleted by

dopamine administration. However, the precursor to dopamine, L-dopa, is

transported through the BBB by virtue of its affinity for the neutral amino acid

transporter within the BBB (Oldendorf 1971). That is, a knowledge of the

underlying transport physiology of the BBB leads to the development of new

strategies for drug delivery through this barrier. This principle has been

extended further with regard to peptide transport into brain. Evidence

accumulated over the past 5 years has demonstrated that the BBB is endowed

with receptors for certain circulating peptides or plasma proteins, and these

receptors act as transport mechanisms in a process called receptor-mediated

transcytosis (Pardridge 1986). Subsequently, it was demonstrated that certain

ligands that enjoy receptor-mediated or absorptive-mediated transcytosis

through the BBB could function as drug transport vectors and ferry across

peptide pharmaceuticals that by themselves are not capable of transport

through the BBB. This approach, called the chimeric peptide strategy, is

discussed in the next section of this chapter.

CHIMERIC PEPTIDE STRATEGY FOR BBB TRANSPORT OF PEPTIDE

NEUROPHARMACEUTICALS

BBB Drug Transport Vectors

Chimeric peptides are composed of a nontransportable peptide pharmaceutical

that is covalently coupled to a transportable peptide that is capable of receptormediated or absorptive-mediated transcytosis through the BBB. Receptor156
mediated drug transport vectors include insulin, transferrin, and, possibly,

insulin-like growth factor (IGF-2) (table 1). The discovery of a receptormediated BBB drug transport vector requires, first, the demonstration of a

saturable receptor for the peptide on the brain capillary and, second, the

demonstration that the receptor acts as a transport system allowing the

net transcytosis of the peptide from blood into brain interstitial fluid. The

simplest approach for identifying receptors on the BBB for a variety of

peptide compounds is the use of the isolated brain capillary preparation.

Capillaries may be isolated from either the brains of laboratory animals or

the autopsied human brain using a relatively simple 3-hour procedure. As

shown in figure 1, human brain capillaries can be isolated in high yield, free

of contiguous brain tissues. These capillaries may then be used along with a

radiolabeled peptide and standard radioreceptor methodology to quantify the

binding characteristics of the BBB peptide receptor. As shown in table 1, the

dissociation constant (KD) and maximal binding capacity (Bmax) have been

measured for human brain capillary insulin, transferrin, and IGF-1 and IGF-2

receptors. The demonstration that these receptors may act as transport

systems was first reported for insulin using morphologic techniques, including

thaw-mount autoradiography (Pardridge 1986; Duffy and Pardridge 1987),

and, subsequently, for transferrin using cell isolation techniques (Fishman

et al. 1987). The transcytosis of peptides or plasma proteins through the

BBB may be quantified using an internal carotid artery perfusion/capillary

depletion method recently developed (Triguero et al. 1990).

TABLE 1. BBBpeptide receptors

Origin of Peptide or

Brain Capillary Protein

KD

nM)

B MAX

Pmol mgp -1

Human Insulin 1.2±0.5 0.17±0.08

IGF-1 2.1±0.4 0.17±0.02

IGF-2 1.1±0.1 0.21±0.01

Transferrin 5.6±.4 0.10±0.02

Bovine Histone

Cationized IgG

CatIonized

albumin

15,200±2,800 7,700±1,000

900±370 1,400±400

2,300±1,100 6,200±2,600

SOURCE: Pardridge et al. 1985, 1989, 1990b; Kumagai et al. 1987; Duffy et al.

1988; Triguero et al. 1989

157
FIGURE 1. Isolated human brain capillaries

SOURCE: Pardridge et al. 1985. copyright 1985, Raven Press

There are two principal concerns in using natural peptides as drug transport

vectors. First, the vector itself WiII have biologic activity. For example, the side

effects of using insulin as a drug transport vector could include hypoglycemia.

The use of transferrin may alter iron homeostasis, and the use of IGF-2 as a

drug transport vector is complicated by the avid binding of this peptide by

specific binding proteins in plasma (Duffy et al. 1988). Second, the interaction

of peptide ligands with receptor binding sites is generally highly specific, and the

addition of a nontransportable pharmaceutical onto an insulin or transferrin

vector may result in markedly reduced rates of affinity of the modified insulin or

transferrin for its native receptor. However, these two concerns are addressed

with the use of polycationic proteins as BBB drug transport vectors. It has been

known for many years that the cationization of proteins, in general, enhances

the cellular uptake of these macromolecules (Basu et al. 1976; Shen and Ryser

1978). This property was demonstrated for BBB transport processes when it

was shown that cationized albumin is transported across the BBB in vivo

(Kumagai et al. 1987). Cationized albumin is prepared by converting surface

carboxyl groups on the albumin molecule to extended primary amino groups

158
using such derivatives as hexamethylenediamine. This raises the isoelectric

point of the protein from approximately four to nine and causes the modified

albumin to undergo rapid absorptive-mediated transcytosis through the BBB

(Triguero et al. 1990). Native cationic proteins, such as histone, have been

shown to undergo absorptive-mediated transcytosis through the BBB and to

have saturable binding sites on brain capillaries (Pardridge et al. 1989) similar

to cationized albumin or native peptides (table 1). However, histone has been

shown to be toxic to the BBB and to cause changes in BBB permeability at

relatively low doses. This property has not been observed following the infusion

of cationized albumin (Triguero et al. 1990). Nevertheless, two concerns with

regard to using cationized albumin as a drug transport vector are the potential

toxicity of this polycationic protein and the immunogenicity of the cationized

form of the native protein. These issues have been addressed recently in a

study wherein 1 mg/kg of cationized rat albumin was administered daily to rats

for periods of up to 8 weeks (Pardridge et al. 1990b). During the course of

these studies, organs were analyzed by tissue histology, and serum chemistries

were monitored. After 8 weeks, no discernible toxicity was found in 10 organs

examined in the rat, and approximately 20 different metabolic, kidney, and

hepatic function tests were found to be normal in the treated animals as

compared with those animals receiving equal doses of native rat albumin.

Moreover, the antibody response to the cationized rat albumin in rats was

minimal owing to the use of a homologous system (i.e., the administration

of cationized rat albumin to rats) (Pardridge et al. 1990b). The use of

heterologous cationic proteins has been shown in previous studies to result

in marked immunogenicity (Muckerheide et al. 1987). Nevertheless, the use

of cationized homologous proteins has not been found to generate a significant

immune response. On the basis of these other studies, cationized rat albumin

is believed to be a suitable vector for the delivery of peptides across the BBB

in a physiologic setting and to provide a model for pharmacologic paradigms

involving repetitive drug administration (Pardridge et al. 1990b).

ß-Endorphin Chimeric Peptides

A ß-endorphin-cationized albumin chimeric peptide was prepared using

the disulfide-based cross-linking reagent N-succinimidyl-3-(2-

pyridyldithio)propionate (SPDP) (Carisson et al. 1978). Disulfide-based crosslinkers were chosen because previous studies have shown that disulfide bonds

are relatively stable in plasma but are labile in cells (Letvin et al. 1986) which is

the necessary criterion that must be established for a drug delivery vehicle. The

ß-endorphin was coupled via N-terminal and lysine s-amino groups on the

opioid peptide to extended primary amino groups on the cationized albumin as

follows (Kumagai et al. 1987):

159
Cationized

albumin

-C0NH(CH2)6NHC0(CH2)2-S-S-(CH2)2C0NH-ß-endorphinj

The cationized albumin-ß-endorphin chimeric peptide was radiolabeled by first

labeling the ß-endorphin moiety with [125l]-iodine or PH] prior to synthesis of the

chimeric peptide. This allowed for the study of the transport and metabolism of

the ß-endorphin chimeric peptide by following the fate of the radiolabel.

The transport of chimeric peptides through the BBB is viewed within the context

of four individual steps that are depicted in figure 2. The first step is absorptivemediated endocytosis at the blood side of the BBB. The absorptive-mediated

endocytosis of the cationized albumin-ß-endorphin chimeric peptide was

demonstrated with isolated brain capillaries using a mild acid wash technique

(Kumagai et al. 1987).

The second step in the overall process is absorptive-mediated exocytosis at the

brain side of the BBB, which completes the transcytosis of the chimeric peptide

through the BBB, allowing for distribution of the chimeric peptide into brain

interstitial fluid. This process was demonstrated using an internal carotid artery

infusion/capillary depletion method (Pardridge et al. 1990c) recently developed

in the author’s laboratory (Triguero et al. 1990). Moreover, these studies

showed that the rate of transcytosis of the 6-endorphin-cationized albumin

chimeric peptide through the BBB was essentially identical to the rate of

transcytosis of the unmodified cationized albumin. These data supported the

initial hypothesis that vectors that traverse the BBB via absorptive-mediated

mechanisms (as opposed to receptor-mediated mechanisms) would be better

able to tolerate the addition of bulky pharmaceuticals to the transport vector

without significant impairment of BBB transport rates. This property is likely due

to the fact that absorptive-mediated transport is triggered by the electrostatic

interaction between cationic groups on the drug transport vector and anionic

groups on the surface of the microvascular endothelium.

The third step in the overall chimeric peptide strategy is the cleavage of the

disulfide bond joining the drug transport vector and the peptide pharmaceutical

(figure 2). Moreover, it is necessary that the rate of cleavage of this disulfide

bond is substantially faster than the rate of degradation of the pharmaceutical

peptide while it is still attached to the drug transport vector. In the setting of

slow cleavage of the disulfide bond by brain, there would be little free

pharmacologically active peptide generated to interact with the respective

peptide receptor on brain nerve endings. However, recent studies have shown

that brain is endowed with high activities of the disulfide reductase enzymatic

activity that cleaves the j3-endorphin-cationized albumin chimeric peptide

160
Delivery of Chimeric Peptides

Through the Blood-Brain Barrier

FIGURE 2. Chimeric peptide drug delivery strategy. Scheme for delivery

of chimeric peptides through the BBB shows four steps in the

overall process: (1) receptor-mediated endocytosis of the

blood-borne chimeric peptide, (2) receptor-mediated exocytosis

of the chimeric pepride into the brain interstitium, (3) cleavage

of the disulfide bond liberating the unconjugated transport vector

and the pharmacologically active peptide, and (4) binding of

pharmacologically active peptide with its respective receptor on

brain cells. A=BBB transport vector (e.g., cationized albumin);

B=nontransportable (pharmacologically active) peptide (e.g.,

ß-endorphin); A-R=peptide A receptor: B-R=peptide B receptor.

SOURCE: Pardridge 1991, copyright 1991, Raven Press

(Pardridge et al. 1990c). As shown in figure 3, the conversion of the ßendorphin-cationized albumin chimeric peptide into the free ß-endorphin occurs

at a rate that is approximately 50 percent complete within 1 minute at 37 

Moreover, the formation of free endorphin in brain occurs at a rate substantially

faster than the subsequent degradation of the ß-endorphin (figure 3).

The fourth and final step of the chimeric peptide strategy depicted in figure 2

is the interaction of free pharmacologically active peptide with its respective

receptor on brain nerve endings. However, significant care must be

taken in choosing coupling strategies that allow for the generation of a

161
FIGURE 3. Demonstration of cleavage of ß-endorphin chimeric peptide by

rat brain. Superose 12-HR fast protein liquid chromatography

gel filtration of [125l]-[D-Al2]-ß-endorphin-cationized albumin

chimeric peptide following incubation with rat brain homogenate

for 0, 1, 15, or 60 minutes at 37 °C The data show that

monoiodotyrosine, which migrates at 20 mL, is formed

subsequent to the formation of the unconjugated ß-endorphin

that is cleaved from the chimeric peptide by rat brain enzymes.

SOURCE: Pardridge et al. 1990c, copyright 1990, The Endocrine Society

162
pharmacologically active peptide subsequent to the cleavage of the peptide

from its drug transport vector. For example, the principal mode of attachment

of a pharmaceutical peptide through the cationized albumin vector is via free

amino groups on the pharmacologically active peptide. Subsequent to the

cleavage of the chimeric peptide, there is a mercaptopropionate group

remaining on the amino group that participated in the coupling reaction:

HS(CH2)

2CONH-ß-endorphin

Therefore, the construct for opioid peptide drug design must consider the

structural requirements that are necessary for preservation of opioid peptide

biologic activity following cleavage from the drug transport vector (see next

section).

Chimeric Peptide Coupling Strategies

To prepare opioid chimeric peptides that are pharmacologically active following

cleavage of the disulfide bond, there are several structural requirements that

must be incorporated into the parent opioid peptide (Pardridge 1991).

1. The peptide must be relatively resistant to rapid aminopeptidase

inactivation. This requires a substitution of glycine at the number two

position with a D-alanine. The disadvantage of this substitution is that it

causes the loss of K-opioid peptide receptor selectivity (Goldstein et al.

1988). However, this substitution is essential for in vivo studies because

the cerebral vasculature is endowed with high quantities of aminopeptidase

(Pardridge and Mietus 1981).

2. The N-terminal a-amino group must be protected during the coupling

procedure that converts free amino groups to pyridyl dithiopropionate

(PDP) moieties. This procedure converts amino groups to active PDP

groups that will then participate with free sulfhydryls on the transport vector

to form the disulfide bond. In the case of opioid peptides that lack a

cysteine sulfhydryl, PDP derivatives must be formed by reacting SPDP with

free amino groups other than the N-terminal a-amino group, since this latter

group must be free to maintain biologic activity of the opioid peptide

(Bewley and Choh 1993). A blockade of the a-amino group with citraconic

anhydride is a possibility: however, it is likely that this procedure will also

block the internal (e.g., lysine s-amino group) amino groups; and this, in

turn, would prevent subsequent formation of the PDP derivative. This

problem may be solved by preparing N-terminal t-butoxycarbonyl (hoc)

163
3.

derivatives of the opioid peptide by aminolytic cleavage of the peptide from

the solid-phase resin using 1,5-diaminopentane, as described by Goldstein

and coworkers (1988). This results in selective protection of the N-terminal

a-amino group during the formation of the PDP derivative, and the hoc

group may then be cleaved with exposure to acid pH.

It is important to be able to radiolabel the opioid peptide so that following its

conversion into a chimeric peptide, its transport through the BBB in vivo

may be followed, and the pharmacokinetics of the clearance of the opioid

chimeric peptide from blood in vivo may be determined. Injaddition,

radiolabeled opioid chimeric peptide is needed to perform in vitro

radioreceptor assays to assess biologic activity of the opioid peptide

following its cleavage from the transport vector. The N-terminal tyrosine

could be oxidatively iodinated directly. However, such [iodo-Tyr1]

derivatives of opioid peptides exhibit reduced biologic actrvity (Li and

Chung 1976). Therefore, an optimal approach for radiolabeling opioid

peptrides is to initially use boc derivatives of diiodotyrosine in the solid-state

synthesis of opioid peptide. These iodo groups may then be removed by

catalytic reduction, yielding high specific activity [3H]-labeled opioid

peptides (Miller et al. 1978).

4. The opioid peptide must be pharmacologically active following its release

from the drug transport vector after cleavage of the disulfide bond. This

requirement necessitates that the “molecular baggage” that is left on the

opioid amino group following its cleavage from the chimeric peptide is

placed in a portion of the molecule that is not involved in biologic activity

and receptor binding. In the case of dynorphin derivatives, this problem

may be solved by replacing lysine groups with arginines and preparing an

analog with a C-terminal extended amino group that may participate in the

coupling reaction, but that is not important for receptor binding site activity

(Goldstein et al. 1988).

SUMMARY AND CONCLUSIONS

The use of chimeric peptides and physiologic-based strategies for drug delivery

through the BBB may be applied to opioid peptides (dynorphins, endorphin

analogs, and enkephalin analogs), and these agents have considerable

advantages in drug development with respect to drug addiction. Recant

research has allowed for the development of efficacious BBB drug transport

vectors, as well as the demonstration that these vectors allow for shuttling of

opioid peptides through the BBB In vivo. The major challenge to future

research is the development of coupling strategies that allow for the release of

biologically active opioid peptide from the drug transport vector following its

164
cleavage by disulfide reductase enzymes, which are abundant in brain. These

coupling strategies must be developed in advance since the amino groups

necessary for coupling are incorporated into the opioid peptide at the level of

solid-state synthesis.

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AUTHOR

William M. Pardridge, M.D.

Professor

Department of Medicine

Brain Research Institute

University of California, Los Angeles School of Medicine

Los Angeles, CA 90024-1682

168
Redox Approaches to Drug Delivery to

the Central Nervous System

Marcus E. Brewster and Nicholas Bodor

INTRODUCTION

Targeted drug delivery is a highly desirable goal in the practice of medicinal

chemistry. The preparation of molecular systems that act to concentrate

a pharmacologically active agent at its pathophysiologically relevant site

would provide a variety of advantages over agents lacking this specificity.

For example, since a greater portion of the administered dose of a drug is

sequestered at a particular locus, the delivery system should be highly

efficacious and the drug dose could be reduced. In addition, nontarget

site toxicities would be attenuated since lower concentrations of the drug

are present. The lowering of the effective dose and the increasing of the

dose of drug required to initiate toxicity results in a significant improvement

in the therapeutic index. This ratio is arguably the most important parameter

that must be optimized during the drug design and testing process.

One organ system for which drug targeting would be beneficial is the central

nervous system (CNS). The reason for this importance, aside from the vital

role that the CNS plays in body functioning and homeostasis, is that a variety

of drugs are barred from entering the brain, making the treatment of cerebral

disease difficult. The basis of this impermeability is the blood-brain barrier

(BBB) (Neuwelt 1989; Rapoport 1976; Suckling et al. 1986), which acts as

a functional barricade to separate the brain from the systemic circulation.

Structurally, the component endothelial cells of the CNS capillaries are joined

tightly together, precluding intercellular bulk transport (Reese and Karnovsky

1967). This architecture forces compounds to diffuse directly through the

cell membranes if they are to gain access to the brain parenchyma; and

as the cell membranes are phospholipoidal in nature, only lipophilic agents

may breach the BBB, meaning that many water-soluble substances are

excluded. In the periphery, capillaries are leaky, allowing nonspecific

intercellular transport. Brain capillaries also are characterized by a high

concentration of various enzymes that help to maintain the delicate cerebral

milieu by preventing the uptake of blood-borne neurotransmitters and other

169
substances. Enzymes found in high concentration in the cerebral vascular

system include monoamine oxidase, catechol-O-methyltransferase, y-glutamyl

transpeptidase, y-aminobutyric acid transaminase, aromatic amino acid

decarboxylase, various enkephalinases, and others (Pardridge et al. 1975;

Levin 1977).

These anatomical and enzymatic differences explain the barrier properties

of the BBB but incompletely describe the system. Many essential nutrients

such as glucose and metabolic wastes such as acetate must be taken up

or expelled. These compounds, because of their high polarity, would not be

expected to rapidly diffuse through cell membranes, and thus, an apparent

problem arises. This paradox is explained by the presence of specialized

carrier systems that are located in the BBB (Neuwelt 1989; Pardridge 1981).

These protein macromolecular systems are characterized by saturability and

high molecular selectivity. Carriers are facilitative in nature and have been

identified for hexoses; neutral, acidic, and basic amino acids; monocarboxylic

acids; nucleosides and nucleoside bases; choline; and various other biologically

important molecules. This avenue, therefore, provides for the bidirectional

movement of selective molecules; but with few exceptions (Greig et al. 1987)

these systems are not relevant to drug delivery. Thus, the CNS has evolved

to exclude polar metabolites and other compounds that may prove harmful.

Unfortunately, this barrier system also may exclude many potentially useful

therapeutic agents.

Therefore, general methods for improving drug flux into the brain would be

useful. Brain uptake of compounds can be improved by derivatization of the

drug molecules via prodrug formation (Stella 1975; Bodor 1981; Bodor and

Kaminski 1987; Bundgaard 1985). A prodrug is a pharmacologically inactive

compound that results from transient chemical modification of a biologically

active species. The chemical change is designed to improve some deficient

physicochemical property of the drug, such as membrane permeability or

water solubility. After administration, the prodrug by virtue of its improved

characteristics is brought closer to the receptor site for longer periods where it

can convert to the active species. Prodrugs usually require a single activating

step. When the BBB is considered, increased drug penetration is usually well

correlated with the lipophilicity or the octanol:water partition coefficient of a

drug (Levin 1980; Fenstermacher 1985). To improve the entry of a hydroxy-,

amino-, or carboxylic-acid-containing drug, esterification or amidation may be

performed. This greatly enhances the lipid solubility of the drug, and as a

result, the drug can better enter the brain parenchyma. Once the drug is in

the CNS, hydrolysis of the lipophilicity-modifying group will release the active

compound.

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Unfortunately, simple prodrugs suffer from several important limitations.

Whereas increasing the lipophilicity of a molecule may improve its movement

through the BBB, the uptake of the compound into other tissues is likewise

augmented, leading to a generally greater tissue burden (Stella and

Himmelstein 1980). This nonselectivity of delivery is especially detrimental

when potent drugs such as steroids or cytotoxic agents are considered

because nontarget site toxicities are exacerbated. In addition, although drug

uptake into the CNS may be facilitated by increasing the lipophilicity of a drug,

its efflux also is enhanced. This results in poor tissue retention of the drug and

short biological action. Finally, whereas the only metabolism associated with

prodrugs should be by conversion to the parent drug, other routes can occur

and may contribute to the toxicity of the compounds (Gorrod 1980). These

effects (i.e., poor selectivity, poor retention, and the possibility for reactive

catabolism) often conspire to decrease, not increase, the therapeutic index

of drugs when masked as a prodrug.

CHEMICAL DELIVERY SYSTEMS

Some of the limitations associated with the prodrug approach are derived

from the fact that only one chemical conversion occurs in the activation of the

compound. In many circumstances, multiple, facile conversions may not only

lead to selectivity in delivery but also may act to decrease the toxicity of a drug

as well as sustain its action. A chemical delivery system (CDS) is defined as a

biologically inert molecule that requires several steps in its conversion to the

active drug and that enhances drug delivery to a particular organ or site (Bodor

and Brewster 1983; Bodor et al. 1981; Bodor 1987). In designing a CDS for the

CNS, the unique architecture of the BBB was exploited. As with a prodrug, a

CDS should be sufficiently lipophilic to allow for brain uptake. Subsequent to

this step, the molecule should undergo an enzymatic or other conversion to

promote retention within the CNS but, at the same time, to accelerate peripheral

elimination of the entity. Finally, the intermediate should degrade, releasing the

active compound in a sustained manner.

One system that possesses these attributes is summarized in figure 1. In

this CDS, a carrier molecule is used as a lipophilicity modifier. Although many

moieties may serve such functions, 1,4-dihydrotrigonellinates have proved

to be the most useful. In this approach, a hydroxy-, amino-, or carboxylicacid-containing drug is esterified, aminated, or otherwise covalently linked to

nicotinic acid or a nicotinic acid derivative. This compound is then quaternized

to generate the 1-methylnicotinate salt or trigonellinate and chemically reduced

to give the 1,4-dihydrotrigonellinate or CDS. This dihydro moiety greatly

enhances the lipophilicity of the drug to which it is attached. Upon systemic

administration, the CDS, due to its enhanced lipophilicity, can partition into

171
FIGURE 1. The CDS is a reduced 1-methyl nicotinic acid conjugate of an

active drug. Systemic administration of the dihydronicotinate

or nicotinamide results in relatively extensive distribution due to

the lipophilicity of the species. Subsequent to distribution, the

labile dihydronicotinamide undergoes oxidation resulting in the

formation of a nicotinate salt conjugate (kox), This more polar

species is rapidly eliminated from the periphery but is retained in

the CNS (k815 is rapid). In the CNS, the locked-in salt can slowly

hydrolyze, liberating the drug of interest as well as the spent

carrier.

172
several body compartments, some of which are inaccessible to the

unmanipulated compound. At this point, the CDS is working as a lipoidal

prodrug. The carrier molecule is specially designed, however, to undergo

an enzymatically mediated oxidation that converts the membrane-permeable

dihydrotrigonellinate to a hydrophilic, membrane-impermeable trigonellinate

salt. This conversion occurs ubiquitously. The now polar, oxidtzed carrier-drug

conjugate is trapped behind the lipoidal BBB and is, in essence, depoted in the

CNS. Any of this oxidized salt that is present in the periphery will be rapidly lost

as it is now polar and an excellent candidate for elimination by the kidney and

liver. The conjugate that is trapped behind the BBB can then slowly hydrolyse

to give the active species in a slow and sustained manner. By the system

design, concentrations of the active drug are very low in the periphery, reducing

systemic, dose-related toxicities. In addition, the drug in the CNS is present

mostly in the form of an Inactive conjugate, thus lowering any central toxicities.

This approach should allow for a more potent compound in that a larger portion

of the administered dose is shunted to its site of action. In addition. this system

should allow for increased dosing interval.

Since it was first proposed in 1978, the brain-targeting CDS has been applied

extensively to various pharmacologically active agents. Reviews of work

conducted prior to 1983 and 1986 are available (Bodor and Brewster 1983:

Bodor 1987).

BRAIN-ENHANCED DELIVERY OF ANTIVIRAL AGENTS

Viral encephalitic diseases are major health problems in the United States

and throughout the world. These diseases have the reputation of being

extraordinarily pernicious and difficult to treat. The basis of this refractoriness

can be traced to several factors, not the least of which is the inability of many

potentially useful drugs to penetrate into brain tissue. This section examines

the application of the CDS to antiviral agents-specrfically, drugs useful in the

treatment of acquired immunodeficiency syndrome (AIDS), herpes encephalitis,

cytomegaloviral encephalitis, and Japanese encephalitis.

Zidovudine or azidothymidine (AZT) is the first approved drug for the treatment

of AIDS. In a few cases, this modified riboside has been shown to be useful in

improving the neuropsychiatric course of AIDS encephalopathy, but the doses

required to elicit this improvement precipitate severe anemia (Yarchoan et al.

1987). This usually leads to cessation of therapy. When the drug is withdrawn

in response to this neutropenia, all the abated symptoms promptly return.

Interestingly, AZT does enter the cerebrospinal fluid (CSF) after oral or

intravenous (IV) dosing, and it achieves signtficant concentrations (Balis et al.

1989; Klecker et al. 1987; Blum et al. 1988). Unfortunately, these CSF levels

173
appear to greatly overestimate brain parenchymal and neuronal levels as AZT

poorly penetrates the BBB (Terasaki and Pardridge 1988). In an effort to

ameliorate the prognosis of AIDS encephalopathy, the CDS approach was

applied to AZT (Little et al. 1990; Brewster et al. 1988a; Gallo et al. 1989; Gogu

et al. 1989; Torrence et al. 1988; Chu et al. 1990; Aggarwal et al. 1990). This

antiviral agent has a primary alcohol functionality in the 5’-position, which was

considered for carrier attachment. As shown in figure 2, AZT was treated with

nicotinoyl chloride hydrochloride in pyridine to yield the 5'-nicotinate. This ester

was subsequently quaternized with methyl iodide to yield the 5'-trigonellinate

(1-methylnicotinate) and reduced in basic aqueous sodium dithionite to give the

5'-(1,4-dihydrotrigonellinate) derivative of AZT (AZT-CDS).

The AZ-T-CDS is a crystalline solid that is stable at room temperature for

several months when protected from light and moisture. The lipophilicity of

the AZT-CDS and its metabolites (AZT and the AZT-5'-trigonellinate salt [AZTQ

+

]) is important in determining the efficiency of the CDS. The n-octanol:water

partition coefficient (log P) was determined to be 0.06, -2.00, and 1.5 for AZT,

AZT-Q+, and AZT-CDS, respectively, as shown in figure 2. This indicates that

FIGURE 2. Preparation of an azidothymidine-CDS, as well as log P values for

selected species

174
the CDS is 34-fold more lipophilic than the parent riboside and more than 3,900-

fold more lipophilic than the AZT-Q+. These parameters should correlate with

rapid brain uptake of the CDS (log P> 1.0) and rapid systemic elimination of the

AZT-Q+ (Greig 1987).

The in vitro stability of the AZT-CDS and its metabolite/precursor, AZT-Q+, was

determined in pH 7.4 phosphate buffer, 20 percent rat brain homogenate, and

whole rat blood. The CDS is relatively stable in buffer but rapidly oxidizes in

enzymatic media. In addition, the depot form AZT (i.e., the ATT-Q+) was shown

to gradually release the parent compound.

The CDS form of AZT was subsequently examined in a rat model. This study

compared the CNS delivery of AZT after dosing animals with either the parent

drug or the AZT-CDS. Figure 3 gives the brain and blood concentration

of AZT after an IV dose of 0.136 mmol/kg AZT. As shown, blood levels were

initially high but disappeared rapidly, with a half-life of approximately 20

minutes. By 2 hours, no AZT was detected. In the brain, AZT levels never

surpassed blood concentrations and were at their peak at the first sampling

point. AZT was not found in the brain at 60 minutes. The profile of drug

distribution after CDS administration was significantly different. In blood,

high levels of the AZT-CDS occurred initially but disappeared rapidly, with

a

t

b1⁄2 of 5 minutes (figure 4). The AZT-Q+

 also was present in high levels

at early time points, but the concentration of this also fell rapidly (t1⁄2=7

minutes). Finally, low levels of AZT could be detected in blood, but the

drug was completely cleared from the body by 2 hours. The amount of

AZT present in the blood after AZT-CDS administration was much lower

than that generated after AZT dosing. In brain (figure 5) no CDS was

detected consistent with its in vitro lability. AZT-Q+ concentrations were

characterized by an appearance phase reaching a Cmax at 15 minutes and

then by a slow decline. The appearance phase is typical for CDS derivatives

and is a consequence of the in vivo conversion of the CDS to its corresponding

pyridinium salt. AZT-Q+ was detectable in the brain at the last time point

examined (4 hours). These sustained levels of AZT-Q+ were associated with

release of AZT in the CNS, which reached peak levels at 30 minutes post-drug

administration and were detectable up to 120 minutes. When the parent drug

was considered, the areas under the brain AZT concentration curve were

threefold higher after AZT-CDS dosing compared with AZT administration

(figure 6). This was a consequence not only of a higher Cmax, value but also

of a longer CNS mean residence time. This is important in that the estimated

virustatic concentration of AZT (1.0 μmol/L) is maintained twice as long after

AZT-CDS administration compared with AZT dosing. This, in combination

with the lower blood levels of AZT, results in a favorable increase in the

brain:blood ratio for the parent compound when administered as its CDS.

175
Time (minutes)

FIGURE 3. Brain and blood concentrations of AZT (mea±SEM) after an IV

dose of 0.736 mmol/kg AZT using dimethyl sulfoxide (DMSO) (0.5

mL/Kg) as a vehicle

Similar distribution studies have been completed by Chu at the University

of Georgia using a mouse model (Chu et al. 1990). In the evaluation, National

Institutes of Health-Swiss mice were dosed with either AZT (50 mg/kg) or

equimolar AZT-CDS (72.7 mgkg) via the tail vein. Animals were sacrificed at

various times post-drug administration; and brain and serum were analyzed

for AZT in the case of AZT dosing and for AZT, AZT-Q+, and AZT-CDS in the

case of the AZT-CDS experiments. Whereas similar areas under the serum

concentration-time curves (AUC) were observed for AZT when administered

either as such or as its CDS, the brain AUC for AZT was almost tenfold higher

after systemic use of the AZT-CDS. This dramatic increase was related with a

long t1⁄2 for AZT in the CNS subsequent to AZT-CDS dosing compared with

AZT administration (15.8 hours and 0.54 hours, respectively).

The behavior of the CDS for AZT also has been examined in the rabbit.

This model is useful in that simultaneous brain, CSF, and blood data can be

obtained in terminal samples. AZT administration generated a distributional

176
5 15 3 0 60 120 240

Time (minutes)

FIGURE 4. Blood concentrations of AZ-CDS and its metabolites, AZT-Q+

and MT, after an IV dose of 0.136 mmol/kg of AZT-CDS

profile similar to that observed in rats (figure 7). Administration of the AZTCDS generated a somewhat different profile. In rabbits, AZT-CDS was not

detectable at the first sampling point (15 minutes). This is slightly different

from previously obtained data in rats, which showed that, at least in blood,

AZT-CDS was present through 30 minutes. The quaternary metabolite was

found in brain and blood, although in much lower concentrations than reported

in other animals models. In brain, AZT-Q+ salt levels peaked at 30 minutes and

then decreased to undetectable levels at 3 hours. In blood, significant levels

occurred only at the first time point (15 minutes). When AZT was considered,

relatively high and sustained levels of the nucleoside were generated in brain

tissue. Blood levels, while high initially, fell below brain levels by 30 minutes.

CSF levels were significantly lower than brain or blood levels throughout the

study (figure 8). When AZT levels were compared subsequent to either AZT or

AZT-CDS treatment, a clear increase in the area under the brain concentration

curves was evident. The inversion in the magnitude of brain:blood ratio for AZT

after AZT-CDS administration resulted in a more favorable value compared with

177
5 15 3 0 60 120 240

Time (minutes)

FIGURE 5. Brain concentrations of AZT and AZT-Q+ after an IV dose of 0.136

mmol/Kg of AZT-CDS

AZT dosing. As illustrated in figure 9, AZT brain:blood ratios reached three by

60 minutes after AZT-CDS treatment but were consistently less than one after

AZT treatment.

Subsequent to the investigations in rodents, AZT-CDS was studied in a

canine model. In a preliminary study, a mongrel dog (SW=20 kg) was given

11 μmol/kg AZT in a vehicle containing 2.5-percent DMSO in polyethylene

glycol (400) followed 1 week later with a dose of 11 μmol/kg AZT-CDS. In both

administrations, samples of CSF and blood were obtained. Blood levels of AZT

after IV administration of either the AZT or the AZT-CDS are given in figure 10.

After administration of the parent compound, blood levels of AZT disappeared in

an apparent biphasic manner, as previously reported. Blood levels of AZT after

the CDS were much lower initially than those obtained after the parent drug. By

100 minutes, the blood concentration curves were superimposed after the two

treatments. In the CSF (figure 11), AZT administration produced significant

178
FIGURE 6. Brain /eve/s of AZT in rat after a 0.136 mmol/kg dose of either

AZT or AZT-CDS

levels of AZT at early times. However, these levels were below the 1.0 μmol/L

therapeutic threshold for virustatic activity. In addition, these levels fell quickly,

with an estimated half-life of 30 minutes. Equimolar AZT-CDS generated

therapeutically significant concentrations of AZT in the CSF for up to 3 hours

postdosing. These data indicate that the area under the AZT concentration

curve in CSF was doubled after AZT-CDS dosing compared with AZT

administration.

A variety of in vitro studies also have suggested that AZT-CDS may be a useful

compound in the treatment of AIDS encephalopathy. Aggarwal and colleagues

(1990), for example, found that the delivery system was more active in inhibiting

human immunodeficiency virus replication than AZT and similarly was less

cytotoxic. In addition, the uptake of the AZT-CDS into H9 cell was significantly

greater than that of the AZT.

179
FIGURE 7.

Time (minutes)

Brain, blood, and CSF concentrations of AZT in rabbits (μg/ml)

after an 17.2 mgkg dose of AZT

FIGURE 8. Brain, blood, and CSF concentrations of AZT in rabbits (μg/mL)

after a dose of AZT-CDS equimolar to 17.2 mg/kg AZT

180
FIGURE 9. Brain to blood ratio of AZT after an IV dose of AZT or equimolar

AZT-CDS

Ribavirin (RV) was first synthesized in 1972 and, since that time, has been

shown to be a useful, broad spectrum antiviral agent (Conner 1984; Huggins

et al. 1984; Sidwell et al. 1979; Canonico et al. 1988). This riboside has been

demonstrated to inhibit the cytopathicity of various RNA viruses and has been

found clinically useful in the treatment of respiratory syncytial virus (Rodriguez

and Parrott 1987) and Lassa fever (McCormick et al. 1988). However, RV is

highly water soluble and poorly lipophilic (log P=-2.06) (Sidwell et al. 1979).

This property renders this potentially useful drug useless in the treatment

of RNA viral encephalitic diseases, such as Japanese B encephalitis, Rift

Valley fever, and Dengue fever. Application of the CDS to RV may, therefore,

improve its spectrum of activity. RV has three hydroxy groups (2’-, 3’-, and

S-positions) that are amenable to derivatization. The brain-targeting 1,4-

dihydrotrigonellinate moiety can be placed at any or all of these loci. In

addition, the underivatized hydroxy groups can be masked with lipophilicity

modifying ester to optimize delivery. The simple 5’-1,4-dihydrotrigonellinate

derivative of RV was prepared according to figure 12. RV was protected as the

181
Time (minutes)

FIGURE 10. Blood levels of AZT in dog after an 11 μmol/kg dose of either

AZT-CDS or AZT

2’,3’-acetonide, This compound then was nicotinoylated with nicotinic anhydride

and deprotected using formic acid. The 5’-nicotinate then was methylated to

give the 5’-trigonellinate of RV (RV-Q+) and reduced in aqueous dithionite to

give the RV-5’-CDS. This compound, independently prepared, was tested in

C57BL/6 mice that had been infected intraperitoneally (i.p.) with 10 LD,s of the

Peking strain of Japanese encephalitic virus. The CDS was administered i.p.

at doses of 45 mg/kg/day for 9 days. A survival rate of 40 to 50 percent was

described even though vehicle or RV itself were totally ineffective (100 percent

mortality) (Canonico et al. 1988).

A variety of other systems were generated. Acylation of the 2’- and 3’-hydroxy

groups of RV-CDS produces a family of triacylated derivatives, including the

2’,3’-bis-O-acetate, 2’,3’-bis-O-isobutyrate, 2’,3’-bis-0-pivaloate, and 2’,3’-bis-Obenzoate adducts of 5’-(1,4-dihydrotrigonellinate) RV. In addition, placement of

the dihydrotrigonellinate targeting group at the 2’- or 3’-locus was considered.

The most efficient way to produce these compounds appeared to be

182
Time (minutes)

FIGURE 11. CSF levels of AZT in dog after an 17 μmol/kg dose of either

AZT-CDS or AZT

nonselective benzoylation followed by nicotinoylation. In this way, the

2’-O-(1,4-dihydrotrigonellinate)-3’benzoate and 2’-O-benzoate-3’-O-( 1,4-

dihydrotrigonellinate) derivatives of RV were obtained. Several of these

compounds were found to exert antiviral activity in a murine model in which

Punta Toro virus (Balliet strain) was inoculated intracranially into Swiss

Webster mice (figure 13). The 2’-O-(1,4-dihydrotrigonellinate)-3’-O-benzoate

derivative of RV caused a significant increase in animals surviving the viral

challenge compared with vehicle controls as well as a decrease in brain viral

titres. A variety of other compounds, including the bis-O-pivaloate and bis-Oisobutyrate derivatives of RV-CDS, acted to significantly increase the mean

survival time of animals inoculated with Punta Toro virus.

BRAIN-SUSTAINED DELIVERY OF ESTROGENS

Estrogens are lipophilic steroids that are not impeded in their entry to the CNS.

These compounds readily penetrate the BBB and achieve high central levels

after peripheral administration. Unfortunately, estrogens are poorly retained by

183
FIGURE 12. Synthesis of an W-CDS, RV was protected as a 2’,3’-0-

acetonide, reacted with nicotinic anhydride, the acetonide group

removed, and the resulting nicotinate quatemized with methyl

iodide and reduced using sodium dithionite.

184
FIGURE 13. Antiviral efficacy of selected W-CDS using a murine Punta Two

viral model

185
the brain. This circumstance requires that frequent doses of these steroids be

administered to maintain therapeutically significant concentrations. Constant

peripheral exposure of estrogen has been related, however, to several

pathological conditions, including cancer, hypertension, and altered metabolism

(Fotherby 1985; Kaplan 1978). As the CNS is the target site for many of the

actions of estrogens, a brain-targeted delivery form of these compounds may

provide for safer and more effective estrogens. A CDS for 17ß estradiol was

generated by treatment of estradiol with nicotinoyl chloride hydrochloride to

give the 3,17-bis nicotinate (figure 14). Treatment of this ester with methanol

potassium bicarbonate resulted in selective cleavage of the phenolic nicotinate.

The obtained secondary nicotinate was then quaternized and reduced to

provide the 17-(1,4-dihydrotrigonellinate) ester of ß-estradiol or E2CDS (Bodor

et al. 1987; Brewster et al. 1987).

FIGURE 14. Sythesis of an estradiol-CDS. Estradiol was bis-acylated using

nicotinoyl chloride, the phenol ester cleaved in base, and the

resulting 17-nicotinide methylated with methyl iodide and

reduced with sodium dithionite to give the CDS.

186
FIGURE

Brain Kidney Heart Lung Testis Eye Fat

Tissues

15. Half-life of elimination of E2-Q+ formed after E2COS

administration in brain, kidney, heart, lung, testis, eye, and fat

The E2CDS was shown to be readily converted to its corresponding quaternary

salt (E2-Q+) subsequent to systemic administration in rats, dogs, and monkeys.

The rate of loss of the E2-Q+ was found to be relatively slow from the CNS

compared to peripheral tissues (figure 15) (Mullersman et al. 1988). The

high, sustained E2-Q+ levels in the CNS were associated with a prolonged but

significant release of the parent compound (Sarkar et al. 1989). This release

was sufficient to reduce luteinizing hormone (LH) secretion in ovariectomized

rats for periods over 1 month subsequent to a single 1 mg/Kg dose (Simpkins et

al. 1986). The LH suppression occurred even though serum levels of estradiol

were low and, in fact, identical with those obtained after estradiol treatment.

Estradiol resulted only in a transient effect (24 days) on LH levels (Estes et al.

1987). The E2CDS was also shown to be efficient in stimulating male sexual

activity in castrate rats (Anderson et al. 1987), in decreasing weight gain in

rodents (Estes et al. 1988), and in reducing food intake in rats (Simpkins et al.

1989). All of the parameters are thought to be CNS-mediated effects.

187
Given the highly successful animal pharmacology of this compound, toxicity

studies and human clinical trials were planned and carried out. Prior to clinical

evaluation, extensive safety studies were performed on E2CDS to ensure, as

much as possible, that the drug would not provoke untoward reactions in

humans. The E2CDS formulated in a water-soluble vehicle (2-hydroxypropylß-cyclodextrin, E2CDS-CD) was shown to lack mutagenic potential in bacteria

as measured in a standard Ames test. This excipient was chosen as it is

not parenterally toxic and gives adequate aqueous solubility for the E2CDS

(Brewster et al. 1988b, 1990a). The E2CDS-CD system was then tested in

Sprague-Dawley rats and cynomolgus monkeys over a period of either 14

(subacute) or 90 (subchronic) days. In the subacute studies, groups of 10 rats

(5 males and 5 females) or 2 monkeys (1 male and 1 female) were injected IV

with normal saline, 2-hydroxypropyl-ß-cyclodextrin in sterile water, or E2CDS

in the cyclodextrin vehicle at doses of 0.025, 0.25. 1.5, and 5.0 mg/kg. The

treatments were administered every second day, resulting in cumulative doses

of 0.2,2, 12, and 40 mg/kg. Twenty-four hours after the eighth dose, animals

were sacrificed and necropsied. Histopathological samples were prepared from

various organs, and blood was drawn for hematologic and clinical chemistry

evaluation. No effects of the vehicle were apparent. The estrogen caused

expected changes in hematology (increased neutrophils) in both species. In

rats, various expected estrogenic reactions were observed, Including weight

loss and pituitary hypertrophy. In the subchronic paradigm, 20 rats (10 males

and 10 females) and 8 monkeys (4 males and 4 females) were used. Again,

one group received normal saline and another the cyclodextrin vehicle at a

dose of 200 mg/kg. E2CDS was administered to monkeys at doses of 1.0 and

5.0 mg/kg and to rats at a dose of 2.5 mg/kg. Drug treatment was performed

every other day, resulting in cumulative doses of 46 and 230 mg/kg in monkeys

and 115 mg/kg in rats. Twenty-four hours after the 46th dose, animals were

necropsied and biological samples obtained for evaluation. There were no

mortalities in this study as in the subacute trials, and no abnormal cageside

behaviors were observed. Again, various estrogen-sensitive parameters were

significantly altered as a result of E2CDS dosing, including decreased body

weight and white blood cell count and increased pituitary weight in rats and

decreased protein and glucose concentrations with increased triglycerides in

monkeys. The toxicological evaluation of the data obtained from the subchronic

studies indicated that the no-observable adverse effect dose was the maximum

dose used in the protocol (i.e., 5 mg/kg in monkeys and 2.5 mg/kg in rats).

Since the CDS, by its design, targets drugs to the CNS, the neurotoxicological

potency of these compounds should be examined. This was done for E2CDS

by evaluating the effect of the CDS on central neurochemical function (Brewster

et al. 1988c). In the experiment, E2CDS, saline, or the cyclodextrin vehicle was

administered to conscious, restrained cynomolgus monkeys IV every other day

188
for 2 weeks. The cumulative dose of E2CDS ranged from 0.2 to 40 mg/kg, and

two monkeys (one male and one female) were included in each group. Twentyfour hours after the eighth dose, animals were euthanized and samples of

striatum removed and frozen. During the study, animals were observed twice

a day for such locomotor impairments as rigidity, akinesia, and hypoactivity.

No altered motor activity was observed. Neurochemical analysis of brain

samples was performed by high-performance liquid chromatography with

electrochemical detection, as earlier described. The results demonstrate

that E2CDS did not affect striatal dopaminergic concentration. The unaffected

dopamine levels and the lack of behavioral changes suggest neurotoxicological

safety of these materials.

The first human examination of E2CDS was designed as a rising dose

safety study (Howes et al. 1988; Brewster et al. 1990b). In this evaluation,

menopausal women served as the test population in that plasma LH and

follicle-stimulating hormone (FSH) could be readily assayed as indicators of

pharmacological action. The initial report evaluated 10 subjects. In the

protocol, all subjects were healthy, postmenopausal females as defined by

age and time of last menstrual period, which was confirmed by plasma FSH

and LH levels. Each subject received a single dose of E2CDS as an IV

injection of the drug dissolved in 20 percent (w/v) 2-HPCD. Subjects

remained for 48 hours in the clinic, and subjective side effects and vital

signs were recorded. Blood samples were obtained for analysis of LH, FSH,

and 17-ß-estradiol at 15 minutes, at 30 minutes, and at 1, 2, 4, 8, 24, and 48

hours after drug administration. Subjects then were released from the clinic

but were asked to return on the mornings of days 4 and 7 for additional blood

sampling. In addition, prestudy and poststudy blood samples were taken for

clinical evaluation.

In the study, all subjects met the entry criteria for inclusion. There were no

dropouts, and all subjects completed the protocol. No adverse effects were

reported that could be attributed to the E2, and all hematological and clinical

chemistry values were unaffected by drug administration. In examining the

data, the results appeared to cluster into three groups: 10 to 40 μg dose (n=3),

which showed minimal changes; 80 to 640 μg (n=5), which showed threshold

effects; and 1,280 μg (n=2), which showed substantial and sustained decreases

in plasma LH levels. Grouping the data for interpretation also had the benefit of

reducing the effects of interindividual variation as only one subject was given a

particular dose of drug in most cases.

Figure 16 illustrates the relationship between these three dose groups and

the percent maximum fall in LH value. Mean peak decreases of 11, 34, and

50 percent were noted for the 10 to 40 μg, 80 to 640 μg, and 1,280 μg dosing

189
FIGURE 16. Effect of various grouped doses of E2CDS on mean LH

suppression relative to baseline values in postmenopausal

women

groups, respectively. This fall in LH favorably compares with mean peak

decreases of 28 to 36 percent seen after 1 month of dosing with estradiol

transdermal patches (Judd 1987; Steingold et al. 1985).

Figure 17 illustrates the duration of LH changes as a function of the magnitude

of the LH decrease (i.e., at least 10-, 20-, or 40-percent decreases from

baseline values). The duration of the effect also shows a substantial dosedependent relationship (e.g., a >20-percent decrease lasted for up to 48

hours after 1,280 μg, 18 hours at 80 to 640 μg, and 0 hour at 10 to 40 μg). In

these studies, it was difficult to quantitate the magnitude of improved action

obtained using the CDS relative to the natural hormone estradiol, in that no

studies had been published that specifically examined the effect of moderate IV

dose of E2 on serum LH and E,. This lack of experimental data was not totally

unexpected in that estradiol is poorly water soluble. This makes parenteral

dosing especially difficult when moderate to high E2 doses are considered. The

190
FIGURE 17. Effect of various grouped doses of E2CDS on the duration of

action of LH suppression. Duration is measured by return to

baseline values in at /east two consecutive measurements.

Suppression of 10, 20, and 40 percent of LH values is

presented.

recent development of nontoxic, highly water-soluble modified ß-cyclodextrins

has allowed for the IV administration of highly water-insoluble compounds such

as sex steroids. The paucity of historic controls and the timely development of

safe, useful, and water-soluble inclusion complexes of estradiol led to an IV

study using this technology to examine E2 and LH levels with time after

moderate doses of estradiol.

Figure 18 illustrates the effect of E2 and E2CDS. both administered In an

aqueous-modified ß-cyclodextrin vehicle, on LH levels at various times

posttreatment in postmenopausal human volunteers. In this study, the dose

of E2 was 900 μg and the equimolar dose of E2CDS was 1,280 μg. The data

were reported as percent of control where control values were obtained by

averaging the LH levels obtained at time 0 and on the day prior to treatment.

As illustrated, LH is dampened in a clinically meaningful way through 96 hours

191
FIGURE 18. Effect of E2CDS (1,280 μg) and E2

 (900 μg) on LH values in a

human postmenopausal volunteer. Data are reported as percent

of control.

after E2CDS treatment, whereas LH values have already returned to control by

24 hours after E2 dosing. The LH suppression induced by E2CDS returns to

control values by 168 hours. As would be expected for a long-acting drug such

as E2CDS, the ratio of the areas under the LH suppression curve obtained after

either E2CDS or E2 dosing increases as a function of time. In addition, if the

initial LH drop that is common to both treatments is discounted, the apparent

E2CDS/E2 enhancement is even greater. Thus, the suppression of LH elicited

by E2CDS was 12.6-fold greater than that produced by E2 between 24 and 168

hours.

The effect of E2 and E2CDS on serum E2 is given in figure 19. Administration

of estradiol in an HPCD vehicle rapidly generates high serum E2 levels as

expected, and these levels fall rapidly. In contrast, E2CDS produces relatively

low initial levels of estradiol. These concentrations also are cleared rapidly.

Past 8 hours, the profiles of E2 elimination are superimposed after either E2

192
FIGURE 19. Effect of E2CDS or E2

 on E2 levels in a human postmenopausal

volunteer. The data point at 15 minutes obtained after E2

treatment represented E2 levels, which were too high to be

calculated with the standard curve used in the assay (>6000

pg/mL). This point was estimated using a curve-fitting procedure.

The model used was y=a+b/x+c/x2, which gave an r2>0.999.

or E2CDS administration. The relative E2CDS areas under the estradiol

concentration curve increase with time starting at 0.2 at 4 hours and approach

1.0 at 168 hours.

Two important pieces of information are derived from this study. One,

the initial levels of plasma E2 generated by E2CDS are fivefold to tenfold

less than those generated after estradiol. This dampened surge should

improve the therapeutic index of E2CDS relative to estradiol, especially in

circumstances where estrogens are counterindicated. such as in breast

cancer. The levels of E2 following E2CDS treatment, however, do seem to

be sufficient to support bond development In postmenopausal women. Two,

LH is clearly suppressed at 24, 48, and 96 hours in a clinically relevant manner

193
after E2CDS treatment, whereas no LH suppression is evident at 24 hours

and beyond after E2 treatment. This occurs in spite of the fact that the serum

levels of immunoreactive estradiol are similar for the two groups. This strongly

suggests that E2CDS is indeed working via brain-mediated mechanism in that

serum differences in E2 cannot account for the LH suppressron.

BRAIN-ENHANCED DELIVERY OF AMINO ACIDS AND PEPTIDES

L-tryptophan is an essential amino acid that is used pharmacologically as a

nutrient and, more recently, as an antidepressant. Several reports have

indicated that acute administration of tryptophan can lower blood pressure in

spontaneously hypertensive and other rat models (Wolf and Kuhn 1984a, 1984b;

Sved et al. 1982; Fuller et al. 1979). In addition, administration of this amino

acid to humans has shown a hypotensive effect (Feltkamp et al. 1984). Many

mechanisms have been proposed for the antihypertensive effect of tryptophan,

including changes in NaCl ingestion, increased serotonin turnover, and alteration

of angiotensin II binding (Ito and Schanberg 1974; Fuller et al. 1979; Wurtman

1981). In any case, the brain figures highly in most of these hypotheses.

Tryptophan is transported into the CNS by a stereospecific large neutral amino

acid carrier (Pardridge 1985). In addition, tryptophan is the only amino acid that

significantly binds to serum albumin. These mechanisms attenuate transport of

the amino acid to the CNS and require high systemic doses to significantly alter

brain levels. Unfortunately, there is evidence that tryptophan can cause bladder

cancer and blood dyscrasias, especially eosinophilia, after large oral doses

(Caterall 1988).

Synthesis of a nicotinamide CDS for tryptophan therefore was considered (Pop

et al. 1990). Tryptophan esters were treated with nicotinoyl chloride to give

the corresponding amide-esters (figure 20). Quaternization and reduction of

these derivatives produced the Try-CDS. The compounds were found to be

of sufficient lipophilicity to pass the BBB readily and also to convert to the

corresponding quaternary salt. The Try-CDS containing an ethyl ester was

tested in a deoxycorticosterone acetate (DDCA) model of hypertension (Fregly

and Fater 1986; Fregly et al. 1987, 1988). Although vehicle or tryptophan

was ineffective in changing blood pressure, an IV dose of the Try-CDS of 14.2

mg/kg significantly reduced blood pressure in rats, 14 percent by 3 hours and

25 percent by 4 hours. Lowering the dose by 63 percent generated an equivalent

hypotensive effect at 3 hours that was not as prominent at 4 hours postdosing

(figure 21).

The inability to deliver peptides to the CNS is due to two characteristics of the

BBB: its exclusion of hydrophilic substances and its histochemistry. This latter

point is important in that many peptides degrade in transit through the vascular

194
FIGURE 20. Synthesis of tryptophan-CDS. Various alkyl esters of tryptophan

were condensed with nicotinic acid giving the corresponding

nicotinamides. Quaternization and reduction afforded the CDS.

epithelium. Even lipophilic peptides such as cyclosporin may breach the

luminal vascular membrane but then are degraded rapidly by amino and

other peptidases. Direct introduction of peptides to the CSF does not seem

feasible, as the low diffusion coefficient for peptides in brain tissue would

restrict distribution to little more than 1.2 mm over a period of 1 hour. Given

the active catabolic processes available in the CNS, it is unlikely any peptide

would traverse the necessary distance intact to be of much use. Other

approaches for increasing peptide delivery include osmotic opening of the

BBB using hypertonic nonelectrolytes, cationic liposomes, or chemical

manipulation.

Application of the CDS to peptides is possible and, in fact, has been completed

in the case of a pentapeptide. Results indicate significant activity and high

concentrations of the corresponding quaternary salt in the CNS.

195
FIGURE 21. Effect of CDS for tryptophan on systolic blood pressure in a

DOCA-induced hypertension rat model

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AUTHORS

Marcus E. Brewster, Ph.D.

Director of Research

Pharmatec, Inc.

P.O. Box 730

Alachua. FL 32615

Nicholas Bodor, Ph.D., DSc.

Graduate Research Professor and Director

Center for Drug Discovery

University of Florida

College of Pharmacy

P.O. Box J-497, JHMHC

Gainesville, FL 32610

201
Drug Delivery to the Brain Using an

Anti-Transferrin Receptor Antibody

Philip M. Friden, Lee Walus, Marjorie Taylor, Gary F. Musso,

Susan A. Abelleira, Bernard Malfroy, Fariba Tehrani, Joseph B.

Eckman III, Anne R. Morrow, and Ruth M. Starzyk

INTRODUCTION

The blood-brain barrier (BBB) poses a formidable obstacle to the effective

treatment of neurological disorders. Access to the brain by nonlipophilic drugs

is blocked by the endothelial cells that compose the vascular network within

the brain (Goldstein and Betz 1986; Pardridge 1986). These cells, unlike those

in other organs, are joined together by complex, tight intercellular junctions

that form a continuous wall against the passive movement of substances from

the blood to the brain (Brightman 1977; Reese and Karnovsky 1967). Brain

capillary endothelial cells are also unusual in that they have few pinocytic

vesicles that would allow somewhat nonselective transport across the capillary

wall. Moreover, continuous gaps or channels connecting the luminal and

abluminal membranes are lacking. Because the brain requires, in a controlled

manner, many blood-borne compounds (such as glucose, amino acids, and

hormones), brain capillary endothelial cells possess an array of transport

systems that allow selective passage across the BBB (Pardridge 1986).

Together, these features of brain capillary endothelial cells ensure that the

homeostasis of the brain is maintained.

In shielding the brain from toxic substances as well as fluctuations in the levels

of blood-borne compounds, such as ions and hormones, the BBB also restricts

the entry of many drugs. Highly lipophilic drugs, such as chloramphenicol and

heroin, readily pass through the membranes of brain capillary endothellal cells

and enter the brain unaided (Goldstein and Betz 1986). However, the majority

of drugs with potential for treating neurological disorders are water soluble

and, therefore, require some assistance in reaching their site of action within

the brain. Current methods for the delivery of nonlipophilic drugs to the brain

include disruption of the tight junctions between endothelial cells through

the intracarotid injection of hyperosmolar sugar solutions (Cosolo et al. 1989;

Neuwelt et al. 1982) and more invasive means such as intracerebroventricular

202
injection of drug into the cerebrospinal fluid or implantation of drug-releasing

polymers during neurosurgery. All these procedures are quite hazardous and

have many drawbacks. The development of a noninvasive method for the

delivery of drugs to the brain would have a significant impact on the treatment

of neurological disorders.

One potential solution to this problem involves the use of a redox delivery

system (Bodor 1987). In this scheme, the drug to be delivered is modified

by the attachment of a lipophilic group that enables the drug to pass freely

through the membranes of the brain capillary endothelial cells. In addition, the

chemical modification that allows the drug to enter the brain is designed to be

rapidly converted to a charged, hydrophilic moiety, thus trapping the modified

drug within the brain. The modifying group is subsequently released, leaving

active drug within the brain. Some success has been reported in enhancing

the delivery of certain drugs to the brain using this approach, but there are

several limitations. Such a delivery system appears to be best suited for

small drugs, and then only for those that can accommodate the required

chemical modifications. Also, because there is no selectivity in this system,

the enhanced lipophilicity of the modified drug allows it to more readily

penetrate all cellular membranes.

Another approach to drug delivery to the brain utilizes cationic and cationized

proteins as drug carriers (Pardridge et al. 1989; Triguero et al. 1989). Proteins

with a net positive charge, such as cationized immunoglobulin G (IgG) or

histone, can interact with the negatively charged surface of the capillary

endothelial cells. Absorptive-mediated endocytosis at the luminal membrane

and exocytosis at the abluminal membrane are thought to mediate the

transcytosis of these compounds across the brain capillary endothelial cells

and, thus, the BBB. In vitro and in vivo data suggest that positively charged

proteins traverse the endothelial cell layer upon intracarotid injection and gain

access to the brain parenchyma. However, the attachment of “passenger”

drugs to cationic or cationized proteins will no doubt have some effect on the

net charge as well as other physical/chemical properties of the carrier protein,

possibly making it difficult to use this system for the delivery of small drugs,

which would require a high drug-to-carrier ratio to achieve therapeutic doses

in the brain. Cationization of an antibody for imaging, on the other hand, may

be a more appropriate use for this system, provided that the cationization

procedure does not alter functional properties of the protein and that sufficient

quantities cross the BBB. Other potential problems with this approach are

enhanced immunogenicity due to the cationization and renal toxicity (Lambert

et al. 1984; Muckerheide et al. 1987).

203
Other types of carrier-based systems for the delivery of drugs across the BBB

include antibodies or ligands that bind to receptors on brain capillary endothelial

cells or, to achieve higher levels of targeting, antibodies that recognize brainspecific antigens. The authors have explored one of these possibilities by

examining whether an antibody, which recognizes the transfernn receptor

(a protein known to transport its ligand, transferrin, across brain capillary

endothelial cells), can function as a carrier for drug delivery to the brain.

The transferrin receptor is an integral membrane glycoprotein consisting of two

identical 95,000 dalton subunits that are linked by a disulfide bond (McClelland

et al. 1984; Omary and Trowbridge 1981). It has been reported in the literature

that brain capillary endothelial cells have a high density of transferrin receptor

on their membrane surface, which is unusual for cells in a nonproliferative

state (Jefferies et al. 1984). This observation may be due to the fact that the

transcytosis of iron-loaded transferrin across the EBB is the primary, if not the

only, method for the delivery of iron to the brain parenchyma (Fishman et al.

1987; Pardridge et al. 1987). Therefore, to ensure that the proper levels of iron

are maintained within the brain, capillary endothellal cells must be capable of

binding and transporting sufficient transferrin to satisfy their own needs as well

as those of other cells within the brain such as neurons and astrocytes.

The uptake of iron by individual cells is a multistep process. Iron-loaded

ferrotransferrin binds to the receptors on the cell surface and is Internalized

by endocytosis (Klausner et al. 1984). In most cells, the endocytic vesicles

then are acidified to a pH of less than 5.5 (Dautry-Varsat et al. 1983). This

process results in the release of free Iron from transferrin. The iron then is

separated from the apotransferrin-receptor complex, which recycles to the cell

surface where the apotransferrin is released and the receptor is made available

for another round of endocytosis. Brain capillary endothelial cells must possess

an alternative pathway for iron uptake (Pardridge 1986). A substantial portion

of the endocytic vesicles containing ferrotransferrin-receptor complexes must

escape acidification so that the iron-loaded transferrin will be transported to and

released at the abluminal surface instead of within the endothelial cells. Once

inside the barrier, the ferrotransferrin can be taken up by cells within the brain

following the pathway utilized by cells in other tissues. Thus, it may be possible

to exploit this endogenous transport system for the delivery of drugs to the

brain.

IMMUNOHlSTOCHEMlCAL STUDIES

The system chosen for testing the feasibility of using anti-transferrin receptor

antibodies for the delivery of drugs to the brain was the rat, utilizing the anti-rat

transferrin receptor antibody OX-26 (Jefferies et al. 1985). It has been reported

204
in the literature that this murine monoclonal antibody preferentially binds to

brain capillary endothelial cells following in vivo administration (Jefferies et al.

1984). lmmunohistochemistry on fresh-frozen brain sections prepared from

rats injected intravenously with OX-26 was used to confirm the selectivity of

this antibody (Friden et al. 1991). The tail vein was chosen as the site of

injection as it more closely mimics the route of administration that is envisioned

for treatment in humans, as opposed to injection into the carotid artery, for

example. The avidin:biotinylated horseradish peroxidase (HRP) complex

technique was used with 3, 3’-diaminobenzidine as the chromogenic substrate

for the detection of OX-26 in tissue sections.

As can be seen in figure 1, very intense staining of the brain vasculature,

indicative of the presence of the OX-26 antibody, was observed 1 hour

following the injection of 0.5 mg of OX-26 via the tail vein. A dose as low

as 0.05 mg of OX-26 per rat gave rise to detectable staining in the brain

vasculature. Increasing doses of antibody from 0.05 to 2.0 mg per rat resulted

in increased staining, with saturation occurring at approximately 0.5 mg OX-26

per rat. Injection of comparable amounts of a control murine monoclonal

antibody of the same subclass as OX-26 (IgG2a) did not lead to detectable

immunostaining of the brain vasculature.

A time course experiment in which animals were sacrificed at various times

postinjection of OX-26 was performed to determine how soon after injection

the antibody could be found in the brain vasculature as well as how long after

injection it persisted. Uniform staining of capillaries was observed throughout

the brain as early as 5 minutes postinjection of OX-26 and became more

intense over time. Between 2 and 4 hours postinjection, the staining pattern

developed a punctate appearance that lasted until approximately 8 hours

postinjection. By 24 hours postinjection, the staining pattern for OX-26

returned to a fainter, more uniform pattern.

The changes in the pattern of localization of OX-26 in the brain suggest a

time-dependent sequestration of the antibody followed by eventual dispersal.

The punctate staining observed with OX-26 is similar to the results obtained

by Broadwell and colleagues (1966) following the injection of a wheat germ

agglutinin (WGA)-HRP conjugate into the jugular vein of rats. Electron

microscopy was used by these researchers to demonstrate that the punctate

staining observed between 6 and 24 hours after the injection of WGA-HRP was

due to the accumulation of the conjugate in pericytes. These cells, which are

embedded in the basement membrane surrounding brain capillary endothelial

cells, are thought to have some macrophage-like properties and may represent

a second line of defense against unwanted compounds entering the brain

(Farrell et al. 1987; Van Deurs 1976). If the punctate staining observed with

205
FIGURE 1. lmmunohistochemical detection of OX-26 in rat brain sections

following intravenous (IV) administration via the tail vein. Freshfrozen brain sections (-30 μm) were prepared from rats sacrificed

1 hour following the injection of 0.5 mg of 0X-26. Biotinylated

horse anti-mouse IgG antisera and an avidin/biotinylated HRP

mixture with diaminobenzidine as the chromogenic substrate were

used for immunodetection. Larger vessels as well as capillaries

show the dark staining indicative of the presence of OX-26

(X 100).

SOURCE: Friden et al. 1991

206
OX-26 indicates uptake into pericytes, it would suggest that the antibody

has traversed the endothelial cell layer that composes the BBB. Electron

microscopy experiments are under way to confirm this.

If an anti-transferrin receptor antibody is to function as a drug carrier, it

must retain its ability to target to brain capillary endothelial cells in vivo

when conjugated with passenger molecules. As a model for a small drug

passenger, methotrexate (MTX) was conjugated to OX-26 via a hydrazone

linkage (approximately six molecules of MTX per antibody) (G. Musso and

S. Abelleira, unpublished manuscript). To prepare this conjugate, MTX

y-hydrazide was reacted with aldehydes that were formed by the oxidation of

the carbohydrate groups located in the Fc portion of the antibody. By directing

the attachment of the drug in this manner, the chance of interference with the

antigen binding sites should be decreased. Following in vivo administration via

the tail vein, anti-mouse IgG or anti-MTX antisera were used to localize the

carrier or the passenger portions of the conjugate, respectively, in fresh-frozen

brain sections as described above. Patterns of immunolocalization identical to

that of unconjugated antibody were obtained using either antisera, suggesting

that there is colocalization of the carrier and the drug in the brain vasculature

and that the attachment of drug molecules to the antibody does not alter its

targeting to the brain. Free MTX injected intravenously did not localize to the

brain vasculature. Similar results were obtained with conjugates of OX-26 to

other small drugs.

The ability of the anti-transferrin receptor antibody OX-26 to deliver large

molecules, such as proteins, also was examined. HRP was chosen

as a prototype protein passenger because it is easily detected using

immunohistochemical methods and it is of average size (40 kD). Conjugates

of HRP and OX-26 (approximately 2:1 molar ratio) were synthesized using a

periodate linkage that crosslinks oxidized carbohydrate groups on HRP with

free amino groups on the antibody. Targeting of the OX-26-HRP conjugate

to brain capillary endothelial cells following IV administration was assessed

immunohistochemically either directly for HRP or indirectly for the carrier using

anti-mouse IgG antisera. Identical patterns of localization were obtained using

either detection method; as for the drug conjugate, the patterns were the same

as those obtained for unconjugated antibody. These results suggest that OX26 can deliver proteins to brain capillary endothelial cells via IV administration.

Both the MTX and the HRP conjugates of OX-26 displayed the punctate

staining observed with unconjugated antibody at similar times postinjection.

This indicates that the process responsible for the punctate staining patternwhich the authors believe to be accumulation in pericytes-is unaffected by

the presence of passenger molecules on 0X-26. Another interesting

207
observation in this regard is that neither F(ab’)2

 nor Fab fragments of

0X-26, which do bind to brain capillary endothelial cells following in vivo

administration, display the punctate staining. These results suggest that

the process responsible for this phenomenon is mediated by the Fc portion

of the antibody, which the antibody fragments lack.

QUANTlTATlVE STUDIES

The results of the immunohistochemistry experiments show qualitatively that

the anti-rat transferrin receptor antibody OX-26 can bind to brain capillary

endothelial cells following administration via the tail vein and that the binding

is not affected by the attachment of either drugs or proteins to the antibody.

However, these experiments do not provide any information as to the quantity

of antibody or conjugate that reaches the brain or conclusive evidence that any

of the material that binds to the capillaries ever crosses the BBB. Two types

of experiments were utilized to address these questions.

In the first type of experiment, the amount of antibody that is specifically

associated with the brain was measured in brain homogenates following the IV

administration of radiolabeled 0X-26. The antibody was labeled with either

3H-succinimidyl propionate or 14C-acetic anhydride (Kummer 1986; Montelaro

and Rueckert 1975). In addition, a control antibody (3H-labeled in the case of

14C-labeled OX-26 or vice versa) was coinjected with the anti-transferrin

receptor antibody so that nonspecific accumulation in the brain due to

contaminating blood could be determined.

A peak level of OX-26 associated with the brain (approximately 0.9 percent

of the injected dose [%ID]) was reached by 1 hour postinjection and remained

relatively constant until 4 hours postinjection (figure 2). The amount of OX-26

associated with the brain then decreased until approximately 48 hours

postinjection, at which time it leveled off at approximately 0.3 %ID. The

association of radiolabeled OX-26 with the brain was significantly reduced

by the coinjection of unlabeled 0X-26.

In contrast to the brain level, the blood level of OX-26 dropped quite rapidly,

such that by 1 hour postinjection, approximately 80 percent of the labeled

antibody had been cleared from the circulation. Gel electrophoresis of serum

IgG followed by autoradiography demonstrated that the radiolabeled OX-26

remaining in the blood at 48 hours following injection was intact.

The receptor-mediated transcytosis of blood-borne compounds across the BBB

is proposed to consist of the following steps: (1) binding of the blood-borne

molecule to its receptor on the luminal surface of the brain capillary endothelial

208
Time (hours)

FIGURE 2. Brain uptake of 14C-labeled OX-26 in rats. The %ID of

radiolabeled antibody per brain and per 100 μL of blood is plotted

vs. time postinjection. All injections were made via the tail vein.

The amount of antibody due to blood associated with the brain

has been subtracted. The values shown are means±EM (n=3

rats per time point). These results are representative of other

studies that have been done.

209
cell, (2) internalization of occupied receptors into endocytic vesicles that

are directed to the abluminal membrane of the cell, and (3) fusion of the

vesicle with the abluminal endothelial cell membrane and release of receptorbound material (Broadwell et al. 1988; Pardridge 1986). The results of the

immunohistochemistry experiments indicate that step 1-binding to receptors

on brain capillary endothelial cells-does occur.

Capillary depletion experiments also were performed to address the question of

whether the anti-transferrin receptor antibody or antibody conjugates are taken

up by the endothelial cells and cross the BBB. This procedure was used by

Triguero and colleagues (1990) to study the transcytosis of cationized proteins

across the BBB. As for the experiments described above, homogenates were

prepared from brains taken from animals injected with radiolabeled antibody.

The homogenates then were depleted of vasculature by centnfugation through

dextran to yield a vascular pellet and a supernatant that consisted of the

brain parenchyma. This method removes greater than 90 percent of the

microvasculature from the homogenate. Because there is no enzymatic

digestion of the homogenate prior to centnfugatlon, the pericytes are included

in the vascular pellet. Based on the results of the immunohistochemistry

experiments, high levels of radiolabeled OX-26 should be associated with the

capillary fraction of the brain at early times postinjection. If transcytosis of the

antibody does take place, the amount of OX-26 associated with the brain

parenchyma fraction should increase, whereas the amount associated with the

capillary fraction should decrease over time. If the antibody that binds to the

brain capillary endothelial cells is not transported across the BBB, the amount

of radiolabeled OX-26 associated with the brain parenchyma fraction should

not increase.

The results of these experiments were consistent with the transcytosis of

OX-26 across the BBB (figure 3). At 30 minutes postinjection. more of the

radiolabeled OX-26 was associated with the vascular fraction of the brain

(0.47 %ID) than with the parenchyma fraction (0.29 %ID). By 24 hours

postinjection, the distribution had changed such that the majority of the

radiolabeled antibody was associated with the parenchyma fraction

(0.43 %ID vs. 0.12 %ID for the capillary fraction).

Similar experiments were performed with radiolabeled conjugates of OX-26

with either MTX or HRP to determine if drugs or proteins can be delivered

across the BBB using an anti-transferrin receptor antibody. Methotrexate

labeled at the 3', 5', and 7 positions with 3H was conjugated with OX-26 using

the y-hydrazone linkage as described above. At 30 minutes postinjection, there

was approximately twofold more of the labeled conjugate in the capillary fraction

of the brain as compared with the parenchyma fraction (figure 4). At 24 hours

210
FIGURE 3.

Time

Time-dependent changes in the disposition of 14C-labeled OX-26

between brain parenchyma and vasculature. Capillary depletion

was performed on homogenates prepared from brains taken from

animals 30 minutes and 24 hours postinjection of radiolabeled

0X-26. The %IDs of antibody for the whole homogenate, brain

parenchyma fraction, and vascularpellet are shown. The amount

of antibody due to blood associated with the brain has been

subtracted. The values shown are means±SEM (n=3 rats per

time point). These results are representative of other studies that

have been done.

211
Time

FIGURE 4. Enhanced delivery of MTX across the BBB using OX-26 as a drug

carrier. Capillary depletion experiments were used to compare

the brain distribution of an OX-26MTX conjugate to that of free

drug. The data are expressed as in figure 3. The values shown

are mean±SEM (n=3 rats per time point). These results are

representative of other studies that have been done.

SOURCE: Friden et al. 1991

212
postinjection, the distribution was reversed, with approximately fivefold more of

the radioactivity in the parenchyma fraction. These time-dependent changes in

the distribution of the OX-26-MTX conjugate were similar to those observed for

antibody alone and suggested that transcytosis of the conjugate across the

EBB had occurred.

To ensure that the 3H-MTX that accumulated in the brain parenchyma was

derived from the OX-26-MTX conjugate and not from residual free 3H-MTX

associated with the conjugate or 3H-MTX that had been cleaved from the

conjugate in the blood, a co-mix of 3H-MTX and OX-26 having the same molar

composition as the conjugate was injected into rats for capillary depletion. The

amount of 3H-MTX in the different brain fractions was significantly lower for the

co-mix as compared with the conjugate with OX-26 (figure 4). In addition, the

3H-MTX in the co-mix did not show the same time-dependent changes in

distribution within the brain as were seen with the conjugate or antibody alone.

These results indicate that the delivery of MTX to the brain parenchyma was

facilitated greatly when administered in the form of a covalent conjugate with

an anti-transferrin receptor antibody.

Capillary depletion experiments also were performed using a conjugate of

OX-26 with HRP coupled via a periodate linkage. In this case, the label (3H)

was on the antibody and the HRP. A peak of labeled material associated with

the capillary fraction of the brain was seen early (approximately 1 hour) after

injection. This was followed by a rise in the amount of material associated

with the parenchyma fraction of the brain (figure 5). Similar results were

obtained using a conjugate in which the radioactive label was only on the

HRP portion of the conjugate. These results suggest that HRP can be

delivered across the BBB using an anti-transferrin receptor antibody as a

carrier.

CONCLUSION

Using both qualitative and quantitative in vivo assays, the authors have shown

that the anti-rat transferrin receptor antibody OX-26 binds to brain capillary

endothelial cells following in vivo administration and, over time, is transported

across the BBB. The results obtained are consistent with the proposed

mechanism of receptor-mediated transcytosis across the BBB. In addition,

the anti-transferrin receptor antibody can function as a carrier for the delivery

of both small drugs and larger molecules, such as proteins, to the brain

parenchyma. Although the transferrin receptor is found on all cell types, this

delivery system appears to be somewhat brain-selective due to a high density

of receptors located on the luminal surface of brain capillary endothelial cells. It

is envisioned that this carrier system will be somewhat generic in that it could be

213
Time (hours)

FIGURE 5. Enhanced delivery of HRP using OX-26 as a carrier. The results

of capillary depletion experiments using a 3H-labeled conjugate of

OX-26 and HRP are shown as in figure 4. The values shown are

the means of the %/D in the different brain fractions at the times

shown±SEM (n=3 rats per time point). These results are

representative of other studies that have been done.

214
used for any drug for which a suitable linker can be developed. Also,

antibody fragments or chimeric antibodies having specific physical and

biological characteristics could be constructed at the DNA level using the

tools of molecular biology. In this way, second-generation carriers could

be designed to optimize the drug delivery aspects of the antibody. Thus,

anti-transferrin receptor antibodies appear to have potential as a noninvasive

means for circumventing the BBB for the treatment of neurological diseases.

The crucial test for this drug delivery system will be to demonstrate that

therapeutic levels of a drug can be delivered to the brain. Experiments to

examine the efficacy of this carrier in an animal model of a neurological

disorder are in the planning stages.

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216
AUTHORS

Phillip M. Friden, Ph.D.

Senior Research Scientist

Lee Walus, B.A.

Senior Research Associate

Marjorie Taylor, MS.

Research Associate

Gary F. Musso, Ph.D.

Senior Research Scientist

Susan A. Abelleira, MS.

Senior Research Associate

Bernard Malfroy, Ph.D.

Director of Development

Fariba Tehrani, B.A.

Research Associate

Joseph B. Eckman III, B.A.

Research Associate

Anne R. Morrow, B.A.

Research Associate

Ruth M. Starzyk, Ph.D.

Senior Research Scientist

Alkermes, Inc.

26 Landsdowne Street

Cambridge, MA 02139-4234

217
Permeation of the Blood-Brain Barrier

for Drug Delivery to the Brain

Heidi C. Homer, Karl Barbu, Frederique Bard, Deborah Hall,

Mary Janatpour, Katy Manning, Janine Morales, Seth Porter,

Paulette E. Setler, Laura Tanner, and Lee R. Rubin

INTRODUCTION

Advances in molecular neurobiology are rapidly bringing researchers closer

to understanding the etiology of acute and degenerative diseases of the brain.

A natural consequence of this improved insight is the development of

therapeutics to treat neurological disorders and dysfunctions such as stroke,

brain tumor, Alzheimer’s disease, and substance abuse. It is quite likely that

many of these therapeutics will be drugs that do not penetrate the blood-brain

barrier (BBB). The ability to manipulate the permeability properties of the BBB

is a critical factor in treating diseases of the central nervous system (CNS).

Hence, the safe and reversible enhancement of BBB permeabilityy is essential

for therapeutic efficacy. To that end, the authors have developed an in vitro

model of the BBB and used it to begin preliminary screening of compounds

that cause enhanced penetration of normally impermeant or weakly permeant

substances. This chapter describes the authors’ in vitro model of the BBB;

the use of the model to identify second messengers that are involved with

regulating permeability at the cellular level; and finally, a screening strategy

to evaluate the in vivo efficacy of agents that are, from in vitro indications, most

likely to enhance experimental drug delivery to the brain.

More than 100 years ago Paul Ehrlich (1885) noticed that systemic

administration of basic dyes stained all tissues except the brain. However,

the idea that brain capillaries constitute the BBB only began to be fully accepted

in the late 1960s when Reese and Karnovsky (1967) and, later, Brightman

and Reese (1969) showed by electron microscopy that horseradish peroxidase

does not penetrate the endothelial tight junctions of brain capillaries. It is now

generally believed that the BBB is made up of a single layer of specialized

endothelial cells (ECs). These cells are morphologically and biochemically

distinct from their peripheral counterparts. Unlike penpheral ECs. they form

tight junctions (zonulae occludens) between apposing cells and have increased

218
mitochondrial content (Oldendorf et al. 1977) and decreased pinocytotic

activity. These hallmark features of brain capillary endothelium allow the cells

to serve as a functional barrier between the blood and brain. Some biochemical

differences, such as increased concentrations of the glucose transporter protein

and increased gamma-glutamyl transpeptidase (GTase), most likely permit

selective entry of substances to brain parenchyma. Thus, the brain is protected

from unwanted blood-borne elements while still being furnished with essential

nutrients, precursors, and cofactors.

These specialized properties are bestowed on ECs by the brain tissue.

Stewart and Wiley (1981) have demonstrated that peripheral capillaries that

vascularize brain tissue acquire the ability to exclude trypan blue, a dye that

normally penetrates peripheral tissue. These cells develop tight junctions and

have increased mitochondria. Conversely, brain capillaries that vascularize

peripheral tissue lose features characteristic of the brain EC phenotype,

including the ability to exclude trypan blue. In an extension of this work,

Janzer and Raff (1987) transplanted rat astrocytes into rat eye and onto the

chorioallantoic membrane of chick embryos. In both cases the astrocytes

became vascularized by capillaries that excluded Evans blue. Meresse and

coworkers (1989) and Meyer and coworkers (1990) have shown that ECs

isolated from brain capillaries selectively lose GTase activity as they proliferate

in culture. Several investigators (DeBault and Cancilla 1980; Beck et al.

1986; Maxwell et al. 1987; Bauer et al. 1990) have reported the induction of

endothelial GTase by astrocytes or astrocyte-conditioned medium (ACM).

Thus, there is good evidence that the functional and biochemical phenotype

of brain ECs is dependent on a brain-derived factor(s).

These are important issues to consider when establishing a cell culture system

that accurately reflects the BBB in vivo. Although several groups have reported

on various features of ECs cocultured with astrocytes or ACM (DeBault et al.

1979; Bowman et al. 1983; Larson et al. 1987; van Bree et al. 1988; Shah et al.

1989), relatively few have described cell culture systems in which ECs were

shown to maintain properties corresponding to those seen in vivo, including

the presence of high-resistance tight junctions, exclusion of small hydrophilic

molecules, and low rates of pinocytosis.

The authors’ goal was to establish a cell culture system in which a monolayer

of ECs with these properties could be grown on a permeable membrane

separating two chambers, one representing the blood and the other the brain.

Such a system serves as a relatively simple model, useful for discovering

technologies that will enhance penetration of therapeutic agents into the CNS.

219
Data on compounds that increase permeability in vitro enabled the authors to

compile a list of putative BBB permeators and to design a screening strategy

to evaluate the capacity of these agents to increase permeability in vivo.

Initially, the compounds are tested for their capacity to increase radiolabeled

sucrose and bovine serum albumin (BSA) uptake into mouse brain. If the test

agent causes increased brain uptake of these normally impermeant tracers,

it is assayed for its capacity to decrease the doses of morphine required to

elicit analgesia in mice. The compound is subsequently evaluated for effects

on the cardiovascular system, including effects on heart rate, blood pressure,

and blood gases. This approach allows the efficacy of an agent as a BBB

permeator to be assessed by a variety of different in vitro and in vivo assays

and to develop a drug to be used clinically for drug delivery to the CNS.

THE IN VITRO MODEL

As discussed above, ECs in the intact BBB possess many specialized features

that distinguish them from peripheral ECs. It appears that those features that

are responsible for their functional role as a barrier are the features necessary

to reproduce in vitro. These include (1) a homogeneous monolayer of cells

having the staining characteristics of ECs. (2) the presence of high electrical

resistance as an indication of tight junction formation, (3) the exclusion of small

hydrophilic molecules that typically distribute through peripheral ECs, and (4)

low levels of pinocytosis. Bovine brain capillaries were chosen because they

are a readily available, abundant, and inexpensive source of brain ECs. In a

few experiments, ECs were isolated from human brain biopsy samples that

were transported directly from the operating room to the laboratory. Briefly,

brain ECs were isolated from capillaries by modifications of standard protocols

(Bowman et al. 1983; Audus and Borchardt 1986) and plated on collagen/

fibronectin-coated tissue culture flasks in ACM (see below). After about 7 days,

the cells were trypsinized off the flasks and plated on Costar polycarbonate

porous membranes, also coated with collagen and fibronectin. Cells were

grown in Eagle’s Minimal Essential Medium with either 5-percent fetal calf

serum or plasma-derived horse serum. Two days after plating onto the

membranes, the cells were fed growth medium supplemented with the

chlorophenylthio derivative of cyclic 5’-adenosine monophosphate (cAMP)

and a phosphodiesterase inhibitor, PDE1. To obtain ACM, rat brain Type I

astrocytes were prepared from 1-day rat cortex, essentially as described by

Lillien and colleagues (1987). ACM was collected by feeding confluent cultures

with fresh medium and collecting after 48 hours. The ACM was mixed with an

equal volume of fresh medium before use.

The resulting monolayers are brightly stained for Factor VIII and show robust

uptake of acetylated low-density lipoprotein, consistent with their endothelial

220
phenotype. There is occasional contamination by smooth muscle as revealed

by staining with an anti-smooth muscle actin antibody. In the absence of ACM

and cAMP/PDE1 , contaminating cells proliferate and form a multilayered

membrane. Furthermore, the cells show diffuse cytoplasmic staining with

rhodamine-phalloidin, a reagent that detects the band of filamentous actin

beneath the tight junctional complex. Cells treated with ACM and cAMP/PDE1

grow as a single monolayer and display dramatic changes in morphology. The

cells become elongated, and the rhodamine-phalloidin staining is confined to

the cell periphery. In addition, the ECs stain brightly with the ZO-1 antibody, a

reagent that detects a tight junctional component in high resistance epithelial

cells (Stevenson et al. 1986). Thus, treatment with ACM and cAMP/PDE1

promotes brain EC growth as a uniform monolayer with actin and a junctional

protein circumferentially distributed in belt-like fashion, typical of that seen in

high-resistance epithelial monolayers.

The electrical resistance of cells growing on a semipermeable filter is easily

measured in vitro by passing current across the cells. Studies conducted

by Crone and Olesen (1982) in amphibian brain microvessels suggest that

resistance across the intact BBB is in the range of 1,900 ohms-cm2. In the

authors’ research, brain ECs grown in the absence of ACM and cAMP/PDE1

displayed low electrical resistance, varying from 10 to 40 ohms-cm2, which is

comparable with that of peripheral ECs. The electrical resistance of brain

ECs grown in ACM alone is typically in the 100 to 150 ohms-cm2 range.

The addition of cAMP/PDE1 and agents that increase intracellular CAMP

dramatically improves electrical resistance (table 1). Resistance in brain

ECs has reached almost 800 ohms-cm2 and is, on average, greater than 500

ohms-cm2. lsoproterenol and 5-hydroxytryptamine (5-HT), agents that bind

to G-protein coupled receptors and secondarily increase CAMP, increase

resistance to a similar magnitude as CAMP itself-that is, more than fourfold

over control. Forskolin, which stimulates adenylate cyclase directly, causes

an eightfold increase in resistance.

For the intact BBB, the penetration of a substance can be predicted by its

octanol:water partition coefficient, the measure of the degree of lipophilicity

of the compound. If the isolated brain ECs maintain their barrier characteristics

in vitro, the movement of a substance across a monolayer should obey the

principles of exclusion according to lipophilicity. That is, the more lipophilic a

molecule, the greater penetration into brain parenchyma; the more hydrophilic,

the less entry into the brain unless there is a specific transporter for the

substance (e.g., D-glucose). Even quite small molecules such as sucrose

(MW 340) cannot cross the BBB due to its hydrophilic nature. Traditionally,

cultures of brain ECs have been leaky to hydrophilic molecules, Presumably,

these low-resistance monolayers lack tight junctions that are responsible for

221
TABLE 1 . Effect of increasing brain EC levels on resistance. Bovine brain

ECs were grown to confluence on collagen/fibronecfin-coated

filters. Cells were left untreated (control) or treated for 24 hours

with 250 μM cAMP. 10 μM isoproterenol, 10 μM 5-HT, or 10 μM

forskolin. All treated cultures had 35 μM PDE 1, in addition.

Each value represents the average of three experiments. The

resistance of untreated cells was assigned a value of 700.

Treatment Relative Resistance

Control 100

cAMP 465

lsoproterenol 439

5-HT 586

Forskolin 834

restricting paracellular movement. The authors assayed the flux of free

fluorescein (MW 376) and fluorescein-conjugated dextrans of 4 kDa and 71

kDa across brain ECs in culture. In low-resistance ECs there is leakage

even of the large 71-kDa dextran. In the high-resistance ECs, flux of any of

three dextrans is not detectable. An assay of the flux of sucrose across the

monolayer revealed a tenfold reduction in paracellular movement of sucrose

in ECs treated with ACM and cAMP/PDE1 compared with untreated cultures.

Similar findings were generated for cholecystokinin, gastrin, nerve growth

factor, and BSA.

The early work of electron microscopists has revealed that another wellestablished property of brain ECs is the paucity of intracellular vesicles

compared with peripheral ECs. This presumably manifests as a low degree

of pinocytosis. The significance of pinocytosis in the normal and pathologically

disrupted BBB is still unclear. It is thought that increased plasma protein

exudation into the brain parenchyma during disruption of the BBB by ischemia

or hypertension, for example, is due to increased pinocytosis. In any case, a

functionally true” in vitro model should have a low rate of pinocytosis. Fluidphase pinocytosis was measured in brain ECs by incubating the cells in a

variety of fluorescent tracers, such as fluorescein isothiocynanate-dextran.

The amount of fluorescein isothiocynanate-dextran taken up by brain ECs

was markedly lower than that in peripheral ECs. However, this low rate

of pinocytosis in brain ECs was observed in the absence of ACM and

cAMP/PDE1 , and thus far, quantitative studies have indicated that neither

of these factors alters pinocytosis. Thus, it appears that brain ECs in culture

222
maintain this phenotypic characteristic, whereas other additions are required

to induce the formation of high-resistance tight junctions.

Having replicated in culture some of the key features of the BBB, the authors

began developing techniques for discretely modulating the permeability of

brain EC tight junctions. Based on the dramatic effect of cAMP on electrical

resistance, it appeared that agents that reduce intracellular cAMP or antagonize

the effect of CAMP should enhance permeability. We were unable to test the

effects of these agents in vitro for the following reason. Modulation of cAMP in

these cells is difficult to achieve since CAMP must be present in the cells to

maintain the brain EC phenotype. It is impossible to antagonize cAMP while it

is present in pharmacologic amounts in the cell; the removal of cAMP rapidly

causes a decrease in resistance, hence, the reversion to low-resistance “nonBBB” ECs. Therefore, tests were not conducted on the effects of these agents

on electrical resistance of ECs. Instead, this information was used to establish

a roster of putative BBB permeators to test in vivo whose receptor-mediated

effects are transduced via cAMP. There are contradictory reports in the

literature regarding the effect of cyclic nucleotides on EC permeability.

Elevations of intracellular cyclic nucleotide levels enhanced barrier properties

of ECs cultured from human umbilical vein (Stelzner et al. 1989; Yamada et al.

1990). In vivo studies by Joo and colleagues have suggested that elevated

cyclic nucleotide levels cause increased macromolecular transport across the

BBB (Joo 1972; Joo et al. 1983). In the authors’ studies, agents that increase

intracellular cyclic 5’-guanosine monophosphate (cGMP) reduce electrical

resistance. For example, 8-bromo-cGMP (500 μM) and nitroprusside (10 μM)

decrease resistance by about 40 percent in 1 hour. The discrepancies may

relate to differences in the effects of the cyclic nucleotides in different EC

types, or they could reflect inconsistency in in vitro and in vivo results. We

are pursuing the effects of the cyclic nucleotides on the permeability properties

of cultured brain ECs and have started to test the effect of these compounds

on BBB permeability in vivo.

IN VIVO EFFICACY OF POSSIBILE BBB PERMEATORS

The most careful and thorough analysis of a compound’s capacity to enhance

blood-to-brain transfer is to measure changes in either the extraction fraction

or a transfer rate constant (Fenstermacher et al. 1981). The authors reasoned

that, even after determining these parameters for each compound, it would still

be necessary to test the efficacy of the compound of interest in a behavioral

assay-that is, in an assay where peripheral administration of a CNS active

drug that normally is impermeant or weakly permeant results in some

observable behavioral change. Therefore, the screening system includes a

somewhat crude brain tracer uptake assay and at least two behavioral assays,

223
Toxicity studies are routinely conducted to determine a nondebilitating,

sublethal dose of the test compound. This dose is coadministered via the

mouse tail vein along with a tracer cocktail containing 3H-sucrose and 1251-BSA.

The animals are perfused 15 or 60 minutes later, and the brains are removed,

weighed, and assayed by liquid scintillation spectrometry. The disintegrations

per minute per wet weight is averaged for a minimum of four animals per

experimental group. Standard error of the mean (SEM) values are less than

10 percent, The between-experiment error for the control groups is also less

than 10 percent. The fold increase in tracer content is calculated by dividing

the average treated DPM by that of the control DPM. Compounds that cause

an increase of 1.5fold or more are considered candidates for testing in the

behavioral assays. These assays are designed to demonstrate delivery of a

drug into the brain parenchyma at levels sufficient to have a therapeutic effect.

Morphine and the naturally occurring peptides, endorphin and enkephalin, bind

to μ-opioid receptors in the brain and suppress the sensation of pain. This

analgesic effect can be demonstrated with mice in the hot-plate assay. Mice

are placed on a surface uniformly heated to 55 °C The time it takes for the

mouse to respond to the heat stimulus by licking its paws is measured.

Morphine (MW 700) delivered intravenously at doses of 1 to 10 mg/kg has an

analgesic effect in that it increases the latency of response to the heat stimulus

measured 15 minutes after injection. The latency is expressed as percentanalgesia. The purpose of these experiments is to test the ability of putative

BBB openers to shift the morphine dose-analgesic response curve in the

leftward direction, Indicating enhanced delivery of morphine to the brain

parenchyma as reflected by increased paw-lick latency time. Similar

experiments are conducted using the less permeant but significantly more

costly enkephalin.

Adenosine agonists that bind to the adenosine-1 receptor subtype (Al) are

negatively coupled to adenylate cyclase and, thus, should decrease intracellular

CAMP and increase permeability. Cyclopentyladenosine (CPA) (100 pgikg,

1 hour) increased sucrose and BSA uptake into the mouse brain by 2.6- and

1.9-fold, respectrvely. A second Al agonist, R-phenylisopropyladenosine (RPIA), but not its inactive stereoisomer, S-PIA (25 μg/kg, 1 hour), Increased

sucrose and BSA uptake by 2.4- and 2.2-fold, respectively. In the morphine

behavioral assay, CPA and R-PIA increased the percent-analgesia by threefold

and eightfold, respectively; the S-PIA isomer had no effect in this assay.

Agents that increase cGMP also showed positive effects in the tracer and

behavioral assay. Dipyridamole (50 mg/kg, 15 minutes) and nitroprusside (3

mg/kg, 15 minutes) caused increases in sucrose uptake by 1.7- and 2.3-fold,

respectively, and both agents increased the percent morphine-induced

analgesia. Dipyridamole also caused a 4.4-fold increase in enkephalin-induced

analgesia.

224
We also looked at the 5-HT class of compounds. Recently, it has become

clear that there is a large family of 5-HT receptor subtypes whose occupation

affects different intracellular second messengers (Schmidt and Peroutka 1989).

Occupation of 5-HT1a, 1b, and 1d receptors causes inhibition of adenylate

cyclase, whereas occupation of 5-HT3 receptors stimulates the enzyme.

Agonists acting at 5-HTlc and 5-HT2 receptors stimulate phosphatidylinositol

turnover (Litosch and Fain 1985; Sanders-Bush and Conn 1987). An effect of

5-HT on BBB permeability has been reported (Westergaard 1977; Sharma et

al. 1990). The authors’ preliminary studies suggest an effect of 5-HT on tracer

uptake. In addition, amitriptyline, which inhibits 5-HT reuptake, has been shown

to increase BBB permeability in rodents and primates (Preskorn et al. 1981,

1983; Sarmento et al. 1990). Consistent with these reports is our finding that

amitriptyline increased tracer uptake and doubled the percent increase in

morphine-induced analgesia.

Thus, the authors are presently in various stages of assaying for efficacy

of these classes of compounds. In addition to the morphine-induced

analgesia assay, a second behavioral test has been included with different

pharmacological and physiological end points. Furthermore, many of these

compounds may have profound effects on cardiovascular function, rendering

them unsuitable as clinically useful prototypical BBB permeators. Many of

these agents have multiple physiologic effects; nevertheless, the authors

have established a set of assays that, considered as a whole, will provide

meaningful leads for novel BBB permeators.

CONCLUSION

The BBB represents a major obstacle in therapeutic drug delivery to the brain.

Presently, there are two practices used for enhancing drug delivery to brain

parenchyma. One is chemical modification of the drug of interest to increase

its lipophilicity and, hence, increase its passage into the brain. Although this is

a clinically effective approach, it is not always chemically possible to achieve.

A second approach was pioneered by Rapoport and colleagues (1972) and

used clinically by Neuwelt and colleagues (1981). It entails the intracarotid

administration of hyperosmotic mannitol and has been used successfully to

deliver methotrexate to brain tumors.

The authors are interested in developing a noninvasive technique that

would enable the delivery of a variety of drugs (i.e., avoiding the need for

individual drug modification) and have developed an in vitro model that allows

assessment of a critical component of drug delivery to the brain: changes in

resistance as a function of changes in tight junction formation. Key features

of this model are the requirements for ACM and cAMP/PDEl. The dependence

225
of the brain EC phenotype on cAMP has focused the investigation on the

receptor-mediated modulation of cAMP to reveal putative BBB permeators.

Furthermore, the BBB model allows examination of the novel aspects of

brain ECs at the molecular level. Such studies will provide the ability to

more precisely antagonize the intracellular events that regulate tight junction

formation and disruption.

The acid test for a true BBB permeator is efficacy at the physiological level.

The authors are examining, by a variety of in vivo assays, the use of A1 and

5-HT receptor agonists and substances that increase cGMP as putative BBB

permeators. These studies, in conjunction with information about brain ECs

derived from the in vitro model, serve two related purposes. One is to remove

a major obstacle in the drive to discover new agents for treatment of brain

dysfunction, and the second is to provide a noninvasive, safe, and reversible

technology that is clinically useful for drug delivery to the CNS.

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228
AUTHORS

Heidi C. Homer, Ph.D.

Scientist

Kari Barbu, B.S.

Research Associate

Frederique Bard, Ph.D.

Scientist

Deborah Hall, Ph.D.

Scientist

Mary Janatpour, B.S.

Research Associate

Katy Manning, B.S.

Research Associate

Janine Morales, B.S.

Research Associate

Seth Porter, Ph.D.

Scientist

Paulette E. Setler, Ph.D.

Executive Vice President of Research and Development

Laura Tanner, Ph.D.

Scientist

Lee R. Rubin, Ph.D.

Staff Scientist

Athena Neurosciences

800F Gateway Boulevard

San Francisco, CA 94080

229
Pathways Into, Through, and Around

the Fluid-Brain Barriers

Richard D. Broadwell

INTRODUCTION

As reflected in the biomedical literature, the past 25 years have witnessed a

burgeoning interest in delivering blood-borne, non-lipid-soluble micromolecules

and macromolecules (e.g., peptides, proteins) and chemotherapeutic

substances into the brain to combat infections, tumors, enzyme and

neurotransmitter deficiencies, toxins, and the like associated with disease or

dysfunction of the central nervous system (CNS). Coupled to this interest are

the CNS entry of psychotherapeutic drugs and the overwhelming health and

socioeconomic consequences of drug abuse and addiction. The scientific

investigations focus on the fluid-brain barriers, cells that inhibit the ready

passage of non-lipid-soluble molecules between the environment external

to the CNS (i.e., air, blood) and the CNS milieu. These fluid-brain barriers

include (1) the blood-brain barrier (BBB) associated with capillary, venule, and

arteriole nonfenestrated endothelia of cerebral blood vessels; (2) the bloodcerebrospinal fluid (CSF) barrier associated with epithelia of the choroid plexus

and other circumventricular organs (e.g., median eminence, area postrema);

(3) the nose-brain barrier associated with epithelia of the nasal mucosa; and

(4) the arachnoid mater-CSF barrier. Each of the barriers is believed to be

attributed to circumferential belts of intercellular tight junctional complexes that

preclude the extracellular movement of non-lipid-soluble micromolecules and

macromolecules bidirectionally between the external environment and the CNS.

Despite reports advocating experimental manipulation to open the fluid-brain

barriers transiently, most notably the BBB (Brightman et al. 1973; Rapoport

1985, 1988) these barriers under normal conditions are not absolute. Each is

circumvented in a noninvasive fashion by large-molecular-weight endogenous

and exogenous proteins moving within patent extracellular routes and/or

traversing intracellular pathways related to adsorptive and receptor-mediated

endocytic processes. These potentially viable intracellular and extracellular

avenues are considered in the following discussion. The cellular secretory

process (Palade 1975) and membrane behavior from a cell biological

230
perspective are emphasized with regard to fission and fusion of cell membranes

among constituents of the endomembrane system of organelles (e.g., Golgi

complex, endosomes, lysosomes, and the plasmalemma).

DISTINGUISHING CHARACTERISTICS OF THE FLUID-BRAIN BARRIERS

The BBB and the blood-CSF barrier are the two most well-studied fluid-brain

barriers in mammals. Circumferential belts of tight junctional complexes

among nonfenestrated cerebral endothelia (Reese and Karnovsky 1967) and

among choroid plexus epithelia (Brightman 1968) represent the distinguishing

characteristic of these two cellular barriers. Similar junctional complexes

exist among cells of the arachnoid mater (Balin et al. 1986; Broadwell and

Sofroniew, submitted for publication; Nabeshima et al. 1975); however,

junctional complexes among epithelial cells of the nasal mucosa and median

eminence are believed not to be circumferentially tight to the extracellular

passage of many molecules (see below). In vitro preparations suggest that

tight junctions among cerebral endothelia may be initiated developmentally by

astrocytes (see Brightman and Kadota, this volume).

Cells comprising the individual fluid-brain barriers all internalize or retrieve

their cell surface membrane, a normal cell biological event for exchanging

or recycling old, worn-out plasmalemma for newly synthesized plasma

membrane. This cell function is associated with fluid- or bulk-phase

endocytosis and permits extracellular macromolecules not binding to the

plasmalemma to enter the barrier cells nonselectively and indiscriminately

within 40 to 70 nm-wide endocytic vesicles of plasmalemmal origin (figure 1).

The fate of such endocytosed macromolecules (e.g., horseradrsh peroxidase

[HRP], serum proteins, uncharged molecules) is degradation within acid

hydrolase-containing secondary lysosomes (Balin et al. 1986, 1987: Balin

and Broadwell 1988; Broadwell 1989; Broadwell and Salcman 1981). The

transcellular transport or transcytosis of fluid-phase macromolecules does

not occur through the fluid-brain barriers (for a discussion, see Balin and

Broadwell 1988; Broadwell 1989). The “enzymatic” barrier provided by

intracellular secondary lysosomes is enhanced by extralysosomal enzymes

like monoamine oxidase; this particular enzyme in BBB endothelia hydrolyzes

dopamine derived by decarboxylation of blood-borne L-dopa within the

endothelium, thus denying entry of dopamine to the CNS through the BBB

(Bertler et al. 1963, 1966).

Although the recognized fluid-brain barrier cells are not categorized functionally

as phagocytes per se, other cell types located behind and intimately associated

with the fluid-brain barriers are likely phagocytic in function. These additional

cell types serve as an auxiliary line of defense once the initial endothelial cell

231
FIGURE 1. The endocytic activity of the endothelium at the luminal front in

rodent and primate brains is demonstrable upon exposure to

the fluid-phase tracer HRP administered intravenously. Within

5 minutes postinjection, BBB endothelia exhibit a plethora of

peroxidase-labeled dense bodies, tubules, and vesicles (A,

arrows); endocytic vesicles harboring peroxidase reaction product

very likely are derived from pits or invaginations (B, arrow) in

the luminal plasmalemma coated with peroxidase reaction

product (B, arrowheads). Tubular profiles (C, arrow) labeled with

reaction product are not identified as establishing membrane

continuity with the luminal and abluminal plasma membranes

simultaneously (Balin et al. 1987); however, some tubules are

confluent with peroxidase-labeled dense bodies, and both types

of organelles stain positively for acid hydrolase activity (Balin et

al. 1987; Broadwell and Salcman 1987) indicating that they are

secondary lysosomes. Peroxidase reaction product is not evident

on the abluminal surface or within the perivascular clefts of

cerebral endothelia deep in the CNS removed from non-BBB sites

(e.g., pial surface, circumventricular organs) in animals injected

intravenously with the tracer.

232
barrier is breached (Balin et al. 1986; Broadwell 1989; Broadwell and Brightman

1976; Broadwell and Salcman 1981; Broadwell and Sofroniew. submitted for

publication). Potential phagocytic cells associated with the fluid-brain barriers

include perivascular pericytes, microglia, and macrophages; arachnoid

and subarachnoid macrophages; and macrophages lying on the surfaces of

ependymal and choroid plexus epithelia. The subarachnoid and perivascular

phagocytes in the rodent and primate CNS label with the blood-borne, fluidphase tracer HRP, suggesting that this probe molecule is successful in

circumventing the fluid-brain barriers by extracellular routes (see below).

The internalization of cell surface membrane and endocytosis are demonstrable

circumferentially in choroid plexus and median eminence epithelia as evidenced

by exposure of these epithelia to blood-borne and CSF-borne peroxidase and

sequestration of HRP reaction product within epithelial vesicles, endosomes.

tubular profiles, multivesicular bodies, and dense body lysosomes (Balin and

Broadwell 1988; Broadwell et al. 1987a). BBB endothelia do not exhibit a

demonstrable endocytic activity circumferentially. Organelles in BBB endothelia

identical to those in choroid epithelia label with blood-borne peroxidase, but

they fail to do so when the abluminal plasmalemma of BBB endothelia is

bathed for 5 minutes through 12 hours in peroxidase delivered into the CNS

by ventriculocisternal perfusion (Balin et al. 1986, 1987; Broadwell 1989;

Broadwell et al. 1983a). Pits or invaginations in the abluminal plasmalemma

of cerebral endothelia readily fill with CSF-borne peroxidase (figure 2) and

have been misinterpreted as endothelial vesicles engaged in the transcytosis

of protein from blood to brain and from brain to blood under normal and

experimental conditions (for references and an indepth discussion, see

Broadwell 1989). A comparison of membrane behavior at the luminal vs.

abluminal front of the BBB suggests that, unlike epithelia of the blood-CSF

barrier, the endothelia of the BBB are polarized with regard to the internalization

or recycling of cell surface membrane and endocytosis of macromolecules

(Broadwell 1989; Broadwell et al. 1983a; Villegas and Broadwell, in press).

This polarity in the BBB suggests further that transcytosis of macromolecules

through nonfenestrated cerebral endothelia, if indeed the process occurs

significantly, is vectorial, from blood to brain but not from brain to blood.

Characteristics of the mammalian fluid-brain barriers considered above are

summarized in the list below.

Cell types

-Blood-brain barrier: Nonfenestrated endothelia

-Circumferential belts of tight junctional complexes (Reese and

Karnovsky 1967)

233
FIGURE 2. Exposure of the abluminal surface (A, arrows) of cerebral

endothelia for 5 minutes to 12 hours to peroxidase-delivered

by ventriculocisternal perfusion yields no concentration of

peroxidase-labeled organelles comparable to those that label with

blood-borne HRP (figures 1A, 1C), This observation suggests

that the endocytic activity and internalization of abluminal surface

membrane are insignificant or minor at best. lnvaginations or pits

in the abluminal plasmalemma label with peroxidase reaction

product and may be misinterpreted as endocytic vesicles (B,

arrows). Abluminal surface pits also can fill with blood-borne

peroxidase that has circumvented the BBB extracellularly under

normal conditions (figure 6) or that has moved through the

experimentally manipulated barrier: abluminal pits labeled

in this fashion are misinterpreted as vesicles engaged in

transendothelial transport and exocytosis of the tracer from

blood to brain (Broadwell 1989; Broadwell et al. 1983a;

Broadwell and Sofronie w, submitted for publication).

234
-Secondary lysosomes containing acid hydrolases (Broadwell and

Salcman 1981; Balin et al. 1987)

-Extralysosomal hydrolytic enzymes (e.g., monoamine oxidase)

(Betiler et al. 1963, 1966)

-Blood-CSF barrier

-Choroid plexus epithelium

-Circumferential belts of tight junctional complexes (Brightman 1968)

-Secondary lysosomes containing acid hydrolases (Balin and

Broadwell 1988)

-Arachnoid mater

-Circumferential belts of tight junctional complexes (Balin et al.

1986; Broadwell and Sofroniew, submitted for publication;

Nabeshima et al. 1975)

— Phagocytes

-Circumventricular organ/subarachnoid macrophages (Balin et al.

1986; Broadwell 1989; Broadwell and Sofroniew. submitted for

publication; Villegas and Broadwell, in press)

-Perivascular cells: pericytes, microglia, macrophages (Balin et al.

1986; Broadwell 1989; Broadwell and Brightman 1976; Broadwell and

Salcman 1981; Broadwell et al. 1988, 1989; Broadwell and Sofroniew,

submitted for publication; Villegas and Broadwell. in press)

-Supraependymal macrophages, Kolmer cells (Villegas and Broadwell,

in press)

Polarity of the BBB. The endothelium is polarized with regard to

demonstrable internalization or recycling of its cell surface membrane

and endocytosis of non-lipid-soluble macromolecules. These events

occur from the blood side but not from the brain side of the endothelium

(Broadwell 1989; Broadwell et al. 1983a, 1988; Villegas and Broadwell,

in press).

Blood-brain barrier vs. brain-blood barrier. BBB is not absolute, whereas its

counterpart-the brain-blood barrier-may be. Potential adsorptive and

235
receptor-mediated transcytoses of macromolecules through the barrier is

from blood to brain but not from brain to blood; hence, transcytosis through

the endothelium appears to be vectorial (Broadwell 1989; Broadwell et al.

1988; Villegas and Broadwell, in press).

Blood-CSF barrier is not polarized. Internalization of cell surface membrane

associated with fluid-phase and adsorptive endocytoses is circumferential

in epithelial cells of the choroid plexus and median eminence (Balin and

Broadwell 1988; Brightman 1968; Broadwell et al. 1987a; Villegas and

Broadwell, in press). Adsorptive transcytosis through these epithelial cells is

bidirectional (Balin and Broadwell 1988; Villegas and Broadwell, In press).

CIRCUMVENTING THE FLUID-BRAIN BARRIERS: INTRACELLULAR

PATHWAYS

Non-lipid-soluble micromolecules and macromolecules from the periphery

are capable of circumventing the fluid-brain barriers by intracellular routes

related to three separate and distinct endocytic processes. The three

endocytic processes from the least to the most specific are fluid- or bulk-phase

endocytosis, adsorptive endocytosis, and receptor-mediated endocytosis

(table 1). All three processes involve the internalization of the external

molecule in association with cell surface membrane. Fluid-phase endocytosis

has been discussed above. Adsorptive endocytosis concerns molecules

such as lectins (e.g., wheat germ agglutinin [WGA]) that bind to carbohydrate

moieties on the cell surface and positively charged (&ionized) molecules that

bind to negatively charged cell surface components. Receptor-mediated

endocytosis is identified with the binding of a ligand (e.g., insulin, transferrin)

to a cell surface receptor specific for that ligand; the binding then triggers the

internalization of the receptor-ligand complex (Dautry-Varsat and Lodish 1984).

The suspected intracellular pathways circumventing the fluid-brain barriers are

considered below and are listed below.

1.

2.

3.

Cerebral endothelium: adsorptive and receptor-mediated transcytoses of

specific blood-borne macromolecules (Broadwell 1989; Broadwell et al.

1988; Villegas and Broadwell, in press)

Choroid plexus epithelium: bidirectronal, adsorptive transcytosis of protein

between the blood and CSF (Balin and Broadwell 1988; Villegas and

Broadwell, in press)

Primary olfactory neurons: anterograde axoplasmic transport and

transsynaptic transfer (adsorptive transcytosis) of extracellular protein

(Broadwell and Balin 1985)

236
TABLE 1. Endocytic process

Types Examples Specificity Organelles

Fluid phase HRP, ferritin Nonspecific Vesicles, endosomes,

lysosomes

Adsorptive phase WGA, rein,

cationized

probes

Specific oligo- Vesicles, endosomes,

saccharides lysosomes. Golgi

complex

Receptor-mediated Insulin, Specific Vesicles, endosomes,

transferrin receptors lysosomes, Golgi

complex (?)

4. Neurosecretory cells, motor and autonomic neurons: retrograde

axoplasmic transport and transsynaptic transfer (adsorptive transcytosis)

of blood-borne protein (Broadwell and Brightman 1976, 1979: Fishman

and Carrigan 1988; Villegas and Broadwell 1989, in press) and virus

(Ugolinr et al. 1989)

Fluid-Phase Endocytosis

Native HRP, arguably the most well-known and utilized fluid-phase tracer, has

no difficulty in gaining entry to cells in general by fluid-phase endocytosis.

Peroxidase is a 40,000 molecular-weight glycoprotein that sticks but does not

bind to membranes. When administered intravenously, HRP is endocytosed

by axon terminals supplying circumventricular organs and peripheral tissues

(i.e., ganglia, muscle) possessing permeable blood vessels. Peroxidase taken

into the axon terminals first appears in vesicles the size of synaptic vesicles

and subsequently undergoes retrograde axoplasmic transport in vesicles,

vacuoles, and tubular profiles for sequestration in dendrites and perikarya.

Neuronal systems so labeled with blood-borne peroxidase Include cranial and

spinal cord motor and preganglionic autonomic neurons and hypothalamic

neurosecretory cells; the latter are afferent to the neurohypophysis and perhaps

other circumventricular organs (Broadwell and Brightman 1976, 1979, 1983).

Retrogradely transported HRP is not secreted from the parent perikarya and

dendrites; peroxidase-labeled organelles undergoing retrograde transport

eventually fuse with perikaryal secondary lysosomes or become secondary

lysosomes after fusing with primary lysosomes derived from the inner saccule

of the Golgi complex (Broadwell 1980; Broadwell and Brightman 1979;

Broadwell et al. 1980).

237
The retrograde labeling of well-defined neuronal perikarya positioned behind

the BBB suggests that toxins, neurovirulent viruses, and other substances

can enter the same neuronal groups, as does blood-borne peroxidase, from

cerebral and extracerebral blood. First-order olfactory neurons in the nasal

mucosa exposed extracellularly to HRP likewise will endocytose the tracer

protein, which subsequently undergoes anterograde axoplasmic transport

in lysosomes to axon terminals innervating the glomeruli of the olfactory

bulb (Balin et al. 1986; Broadwell and Balin 1985); organelles transporting

peroxidase into the olfactory terminals do not secrete their contents. The

transcytosis of fluid-phase markers is not well documented for cells of the

fluid-brain barriers or for any cell type;’ however, a retrograde transneuronal

transfer of herpes virus (Ugolini et al. 1989) and the C fragment of tetanus toxin

(Fishman and Carrigan 1988) are reported from the periphery through lower

motoneurons into the cortex and brain stem. The transcytosis of virus and

toxins may be related more to adsorptive or receptor-mediated endocytosis

than to fluid-phase endocytosis.

Adsorptive Transcytosis

Potential transcytosis of lectins and cationized molecules is not specific

for cells of the fluid-brain barriers; the potentral exists for all cells exposed

to the molecules, those located within the CNS as well as those situated

peripherally. To date, WGA (MW 36,000) conjugated to HRP is the only

molecule documented morphologically to be associated with adsorptive

transcytosis through the fluid-brain barriers, specifically cerebral endothelia

and choroid epithelia (Balin and Broadwell 1988; Broadwell 1989; Broadwell

et al. 1988; Villegas and Broadwell, in press). Biochemical data advocate

cationized serum proteins as additional molecules for adsorptive transcytosis

through BBB endothelia (Pardridge et al. 1990; Triguero et al. 1989; Pardridge,

this volume). Lectins and cationized molecules, therefore, may represent

excellent vehicles for brain delivery of molecules normally excluded entry by

the fluid-brain barriers (see below).

Subsequent to the binding of WGA to n-acetylglucosamine and sialic acid

moieties on the plasmalemma of cells (Gonatas and Avrameas 1973) the

intracellular pathway that WGA-HRP follows through cells of the fluid-brain

barriers and neurons projecting outside the BBB initially is similar to that of

fluid-phase molecules. Organelles sequestering WGA-HRP early on include

endocytic 40 to 70 nm vesicles, tubular profiles, vacuoles that may represent

endosomes or a prelysosomal compartment, and dense bodies comparable

morphologically to secondary lysosomes. With the passage of time (e.g., 1 to

3 hours) and unlike with fluid-phase molecules, the innermost saccule of the

Golgi complex also labels with reaction product for WGA-HRP. This labeling

238
of the inner Golgi saccule coincides with transcytosis of the lectin conjugate

from blood to brain through BBB endothelia (Broadwell et al. 1988; Villegas

and Broadwell, in press), with transsynaptic transfer of WGA-HRP from

the periphery in the anterograde direction through primary olfactory neurons

afferent to the olfactory bulb (Broadwell and Balin 1985) and with transsynaptic

transfer in the retrograde direction through hypothalamic neurosecretory

neurons (Balin and Broadwell 1987; Villegas and Broadwell 1989, in press).

The signals for transcytosis of WGA-HRP through BBB endothelia are

(1) sequestration of reaction product for blood-borne WGA-HRP within the

inner Golgi saccule of cerebral endothelia; (2) reaction product filling the

perivascular clefts; (3) labeling of perivascular phagocytes throughout

the CNS, suggesting that adsorptive transcytosis through the BBB is

global; (4) WGA-HRP reaction product in the innermost Golgi saccule of

perivascular phagocytes; and (5) WGA-HRP occupying extracellular clefts

and processes in the neuropil beyond the basal lamina surrounding the

perivascular phagocytes and endothelta (figure 3) (Broadwell et al. 1988;

Villegas and Broadwell, in press).

Adsorptive transcytosis of WGA-HRP figures prominently with the

Goigi complex and, therefore, with the Palade (1975) scheme of the

cellular secretory process. The innermost Golgi saccule, which exhibits

acid phosphatase activity enzyme cytochemcally, gives rise to primary

lysosomes and additional vesicles/vacuoles involved in delivering membrane

and macromolecules, such as enzymes, to other organelles; this Golgi saccule

also is charged with packaging molecules for export and exocylosis at the

cell surface (for references, see Balin and Broadwell 1988; Broadwell and

Balin 1985; Broadwell and Oliver 1981, 1983). WGA-HRP labeling of the

Golgi complex may be a consequence of overwhelming the cellular endosomal

compartment by the internalization of cell surface membrane tagged with the

lectin conjugate.

The endosome compartment is the common denominator among fluid-phase,

adsorptive, and receptor-mediated endocytic processes; it represents a clearing

center and first intracellular stop in the endocytic pathway for internalized cell

surface membrane associated with lectin, the ligand-receptor complex, and

fluid-phase molecules (Dautry-Varsat and Lodish 1984; Gonatas et al. 1984;

Helenius et al. 1983; Steinman et al. 1983). The endosome serves to

dissociate internalized membrane from the attached lectin/ligand so that the

membrane can recycle to the cell surface, leaving the lectin/ligand within the

endosome. In fluid-phase endocytosis, internalized cell surface membrane

likewise recycles from endosomes to the plasmalemma after endocytic

vesicles have deposited their contents in endosomes. Broadwell and

239
FIGURE 3. The lectin WGA conjugated to HRP and administered

intravenously to rodents labels perivascular phagocytes (A,

arrows) throughout the CNS at 3 to 6 hours postinjection. Prior to

3 hours, reaction product is identified ultrastructurally on the

luminal surface membrane (B, arrowheads) and within the inner

saccule of the Golgi complex (B, arrow), vesicles, tubules, and

dense bodies (C, arrows) of BBB endothelia. Reaction product

occupies the perivascular clefts (C, arrowheads) and extracellular

spaces (D, arrows) in the neuropil beyond the endothelial basal

lamina at postinjection times of 3 hours and longer (Broadwell

1989; Broadwell et al. 1983a; Villegas and Broadwell, in press).

240
colleagues (Balin and Broadwell 1988; Broadwell and Balin 1985; Villegas

and Broadwell, in press) speculated that, when individual endosomes are

overloaded with endocytic membrane associated with WGA-HRP, the “normal”

intracellular endocytic pathway may be perturbed. As a consequence,

internalized membrane with attached lectin may be diverted to the inner Golgi

saccule wherein specific enzymes reside that contribute to the processing of

membrane macromolecules, such as the addition or replacement of sialic acid

(Bennett and O'Shaughnessy 1981; Bennett et al. 1981) to which WGA binds

on the cell surface. Membrane and WGA-HRP recycled through the inner Golgi

saccule would be packaged for export to other organelles (e.g., endosomes,

secondary lysosomes, and plasmalemma) as well as for exocytosis.

Although Broadwell and colleagues have yet to identify the transcytosis of

macromolecules from brain to blood through BBB endothelia (see below and

Broadwell 1989; Broadwell et al. 1983a; Villegas and Broadwell, in press),

transcytosis of WGA-HRP does occur bidirectionally through choroid plexus

epithelia and perhaps without necessary involvement of the Golgi complex.

WGA-HRP delivered into the lateral cerebral ventricle is endocytosed avidly by

choroid epithelia at the microvillus face of the cells and is transcytosed through

the epithelia within 10 minutes for binding to fenestrated endothelia located at

the opposite pole of the epithelia; this transcytosis occurs in advance of WGAHRP labeling of the innermost Golgi saccule and is speculated to utilize the

endosome compartment as an intermediary in the transcytotic pathway (Balin

and Broadwell 1988). If Broadwell and colleagues are correct, the transcytotic

pathway through choroid epithelia is similar to that reported for immunoglobulinG through intestinal epithelia (Rodewald and Kraehenbuhl 1984). The author

and his group also find that the transcytosis of blood-borne WGA-HRP through

choroid epithelia is not without difficulty. The event requires 17 to 24 hours to

be identified ultrastructurally and is minor at best, with the signal for transcytosis

of the lectin conjugate represented by WGA-HRP labeling of the microvillus

border and of phagocytic (Kolmer) cells residing on the surface of the microvilli

(Villegas and Broadwell, in press). The binding of blood-borne WGA-HRP to

fuminal and abluminal surfaces of fenestrated endothelia supplying the choroid

plexus is so prominent that extracellular availability of the lectin for adsorptive

endocytosis by choroid epithelia appears compromised (Balin and Broadwell

1988). Suspected membrane trafficking within choroid epithelia is diagramed in

figure 4.

Receptor-Mediated Transcytosis

Data advocating the receptor-mediated transfer across BBB endothelia

abound for ligands, specifically a host of peptides (e.g., insulin, transferrin,

and vasopressin); however, the data are more biochemical (Banks and Kastin

1985; Banks et al. 1987; Barrera et al. 1989; Duffy and Pardridge 1987;

241
CSF

Blood

FIGURE 4. Three potential intracellularpathways through epithelia of

the choroid plexus are proposed for the transcytosis of

CSF-borne and blood-borne proteins and peptides that bind

to the plasmalemma. Such macromolecules would enter the

choroid epithelium by adsorptive endocytosis (e.g., lectins) or

receptor-mediated endocytosis (e.g., ligands). The transcytotic

pathways are vesicular transport (1) bidirectionally between the

basolateral surface and the apical or microvillus surface, or

indirect (2) by way of endosomes (E), or by recruitment of the

Golgi complex (G), either directly from the cell surfaces or

indirectly from endosomes (3). The transmost or inner Golgi

saccule gives rise to a host of transporting vesicles; some of the

vesicles are destined to engage in exocytosis at the apical

surface (3a) or basolateral surface (36) of the choroid epithelium,

242
whereas others represent primary lysosomes (3c) that ferry acid

hydrolytic enzymes to endosomes (prelysosomes) and secondary

lysosomes (L). Blood-borne/CSF-borne macromolecules entering

the choroid epithelium in vesicles derived from the plasmalemma

by fluid-phase endocytosis (e.g., native HRP, ferritin), adsorptive

endocytosis (WGA, cationized probes), or receptor-mediated

endocytosis (e.g., ligands) are most likely directed to endosomes

or secondary lysosomes (pathway 4): internalized plasmalemma

possessing receptors freed from ligand/lectin deposited in

endosomes may recycle to the cell surface. The endoplasmic

reticulum (Et?) and tubular profiles (T) do not participate in

the transepithelial transport and exocytosis of extracellular

macromolecules (Balin and Broadwell 1987; Broadwell et al.

19836; Cataldo and Broadwell 1986). Because the choroid

epithelium is not polarized with regard to its endocytic activity

and internalization of cell surface membranes, a bidirectional

transcytosis of extracellular macromolecules through the

epithelium between blood and CSF is conceivable. N,

nucleus; TJ, tight junctional complex.

Fishman et al. 1987; Pardridge 1986) than morphological (Broadwell 1989;

Broadwell et al. 1983a; Villegas and Broadwell, in press), but are not without

controversy (Meisenberg and Simmons 1983; Ermisch et al. 1985). One

laboratory is of the belief that selected peptides may be involved in a

bidirectional transfer across the BBB (Banks et al. 1988, 1989). These data

should be interpreted with caution for reasons to be considered shortly.

The potential pathways for receptor-mediated transcytosls through and

membrane trafficking within BBB endothelia are diagramed in figure 5; the

pathways are (1) direct vesicular transport from the luminal to the abluminal

side, (2) indirect vesicular transport utilizing the endosome compartment as an

intermediary, and (3) vesicular transport by way of the Golgl complex. Similar

pathways would apply for receptor-mediated transcytosis through epithelia of

the blood-CSF barrier (figure 4).

Data of ferrotransferrin (f-TRF; MW 35,000) conjugated to peroxidase for

receptor-mediated transcytosis through the BBB are, at best, suggestive

and raise important questions regarding the process in BBB endothelia

(Broadwell 1989; R.D. Broadwell. A. Wolf, M. Tangoren, B.J. Baker, and

C. Oliver, unpublished data; Tangoren et al. 1988). lmmunohistochemistry

has demonstrated that endothelia possessing the receptor for f-TRF are

243
Brain

Blood

FIGURE 5. Potential transcytotic routes through endothelial cells (EC)

of the BBB and membrane events associated with fluid-phase,

adsorptive, and receptor-mediated endocytic processes

are represented in pathways 1-4. A direct transendothelial

vesicular transfer (7) or an indirect route (2) through the

endosome compartment (E) may apply for the receptor-mediated

transcytosis of blood-borne ligands (e.g., transferfin, insulin);

available data suggest the indirect route is the more plausible of

the two (see text). Macromolecules entering the endothelium by

adsorptive endocytosis are channeled to endosomes (2) and

either directly or indirectly to the inner saccule (3) of the Golgi

complex (G); this Golgi saccule is responsible for packaging

macromolecules for exocytosis at the abluminal (a) and luminal

(6) faces of the endothelium. Alternatively, some Golgi-derived

vesicles represent primary lysosomes charged with delivering

acid hydrolases to endosomes (E; pathway c) and secondary

lysosomes (L; pathway d). Fluid-phase macromolecules and

some macromolecules taken into the endothelium by receptormediated and adsorptive endocytic processes are directed to

endosomes and secondary lysosomes (4) for degradation. In

pathway 4, the internalized cell surface membrane may recycle to

the luminal plasmalemma as vesicles from the endosome

compartment. Reports that the endoplasmic reticulum (ER) of

the cerebral endothelium may be involved with the transcytosis

of blood-borne macromolecules (Mollgard and Saunders 1975,

1977) are not confirmed (Broadwell et a/. 19836; Cataldo and

Broadwell 1986). Endocytosis and retrieval of cell surface

membrane at the abluminal front are not conspicuous in

comparison to the same events at the luminal plasmalemma

(see text for discussion).

244
restricted to cerebral vessels (Jefferies at al. 1984). Receptor recognition

of f-TRF may promote the transport and delivery of transferrin and iron from

plasma into brain. The f-TRF-HRP used by Broadwell and colleagues is run

on gel electrophoresis and chromatographed to ensure that the conjugate

administered into the carotid artery of rats (6 mg in 0.5-1.0 cc saline) is not

contaminated with free HRP. At 1 hour postinjection, reaction product for

f-TRF-HRP is observed within endothelial endocytic vesicles, tubules,

spherical endosomes, and dense bodies and within the perivascular clefts and

perivascular phagocytes. Subarachnoid macrophages, the circumventricular

organs, and Golgi saccules in BBB endothelia are free of reaction product.*

Infrequently, reaction product fills a presumptive “exocytic vesicle” or pit

positioned at the abluminal plasmalemma. The data suggest a priori that

the entire f-TRF-HRP conjugate may have undergone receptor-mediated

transcytosis through the BBB; however, at this time this interpretation must be

viewed with caution. Because of the specificity of the endothelial transferrin

(TRF) receptor in brain, blood-borne TRF may be an ideal vehicle for ferrying

substances across the barrier.

If receptor-mediated transcytosis of f-TRF-HRP does occur through cerebral

endothelia, the treatment of f-TRF by this endothelium differs from that in other

cell types. In the hepatocyte, for example, the f-TRF associated with the cell

surface f-TRF receptor is internalized and directed to endosomes, wherein iron

is dissociated from the transferrin peptide and transferred to the iron-storing

protein ferritin; the iron-free apotransferrin remains bound to its membrane

receptor and is recycled with it to the cell surface (Dautry-Varsat and Lodish

1983). The iron-free apotransferrin is released to bind additional iron when the

receptor-apotransferrin complex encounters the neutral pH of the extracellular

medium.

Broadwell and colleagues hypothesize that the fate of f-TRF in the endosome

of BBB endothelia is dissociation of the ligand from its receptor, which IS

recycled to the luminal surface; f-TRF is anticipated to be transferred to a

vesicle of endosome origin for export and exocytosis at the abluminal front

(R.D. Broadwell, A. Wolf, M. Tangoren, B.J. Baker, and C. Oliver, unpublished

data; Broadwell 1989). Iron would dissociate from the TRF once exocytosis

into the perivascular space commences. Perivascular phagocytes are in

position to endocytose the free TRF. This scheme excludes consideration of

a direct transendothelial vesicular transport comparable to that diagramed in

figure 5. To date, direct transendothelial vesicular transport is only a suggestion

in the BBB literature without confirmation. lmmunocytochemrstry utilizing a

monoclonal antibody against a BBB protein (Sternberger and Sternberger 1987)

fails to support movement of luminal surface membrane to abluminal surface;

the antibody recognizes a luminal membrane antigen that is internalized but is

245
not directed to the abluminal surface (N. Sternberger and C. Shear, personal

communication, November 1990).

THE ENIGMA OF TRANSCYTOSIS THROUGH THE BBB

The event of transcytosis through the BBB implies that the membrane

of exporting vesicles engaged in exocytosis at the abluminal front of the

endothelium fuses with the plasmalemma. In so doing, exocytic vesicle

membrane is added necessarily to abluminal surface membrane. The

Palade (1975) scheme of the cellular secretory process states that exocytosis

and endocytosis are complementary event-vents well recognized in bona

fide secretory cells such as the neuron and those of endocrine and exocrine

glands. With the addition of secretory vesicle or granule membrane to the

plasmalemma for exocytosis to commence, the endocytic process signals the

retrieval of cell surface membrane as a compensatory response to exocytosis,

thereby ensuring that the overall surface area of cell membrane remains static

with each exocytic event. The endocytic activity at the luminal front of the

BBB is demonstrable upon even brief exposure to blood-borne tracer

(figure 1) but not so at the abluminal front exposed for 5 minutes through 24

hours to native peroxidase or WGA-HRP filling the perivascular clefts following

ventriculocisternal perfusion of the proteins (Balin et al. 1986; Broadwell

1989; Broadwell et al. 1983a; Villegas and Broadwell, in press). In the latter

preparation, peroxidase-labeled endosomes and dense bodies are exceedingly

rare within the endothelia, and labeled tubular profiles and Golgi saccules are

nonexistent (figure 2). Presumptive exocytic and endocytic vesicles at the

abluminal face are impossible to discern morphologically and unequivocally

from static pits studding the abluminal plasmalemma. Abluminal surface

pits are appreciated readily in preparations incubated to reveal the alkaline

phosphatase activity inherent to the plasma membranes and fill with peroxidase

that has gained access to the perivascular clefts from the blood through patent

extracellular routes (considered below), from ventriculocisternal perfusion

(figure 2), or from experimental manipulation of the BBB (Broadwell 1989).

The enigma of a significant transcytosis of blood-borne proteins and peptides

through the BBB concerns how this endothelial barrier compensates for the

absence of a demonstrable endocytic activity subsequent to exocytosis at its

abluminal plasma membrane. Such a discrepancy in the cellular secretory

process applied to the BBB requires clarification for defining transcytosis

through the BBB. The problem is not encountered with the bidirectional

transcytosis through the blood-choroid epithelial barrier, because the choroid

epithelium demonstrates endocytic activity circumferentially. The apparent

absence of macromolecular transcytosis through the cerebral endothelium

246
from brain to blood suggests that the CNS possesses a brain-blood barrier

that indeed may be absolute, whereas its counterpart-the BBB-is not.

EXTRACELLULAR PATHWAYS

Extracellular pathways circumventing the fluid-brain barriers (listed below)

are comparable in the CNS of rodents and a subhuman primate (Balin et al.

1986; Broadwell 1989; Broadwell and Sofroniew, submitted for publication).

The most highly documented extracellular route is through the circumventricular

organs (e.g., median eminence, organum vasculosum of the lamina terminalis,

subfornical organ, and area postrema), all of which contain fenestrated

capillaries and, therefore, Iie outside the BBB. Blood-borne macromolecules,

specifically fluid-phase molecules, escaping fenestrated vessels supplying the

circumventricular organs move extracellularly into adjacent brain areas located

behind the BBB (for references, see Broadwell and Brightman 1976; Broadwell

et al. 1987a; Gross 1987).

Permeable blood vessels

—Circumventricular organs (e.g., median eminence, area postrema)

(Broadwell and Brightman 1976; Broadwell et al. 1983a, 1987a, 1987b,

1989).3

— Pial surface (Balin et al. 1986; Broadwell 1989; Broadwell et al. 1991;

Broadwell and Sofroniew, submitted for publication).3

Patent intercellular junctional complexes

—Nasal epithelium; absence of a nose-brain barrier (Balin et al. 1986).

— Ependymal lining of median eminence and area postrema (Broadwell

et al. 1983a, 1987a; Gotow and Hashimoto 1979, 1981; Richards

1978).

lntracerebral transplants

—Blood vessels supplying solid tissue grafts of peripheral origin are leaky

and are indigenous to the grafted tissue; the grafts are deficient in a

BBB and a brain-blood barrier (Broadwell et al. 1991).

—Blood vessels supplying cell suspension grafts of peripheral origin are

leaky and are of host origin; the vessels have forfeited their BBB and

brain-blood barriercharacteristics (Broadwell et al. 1991).

247
Additional extracellular avenues into the CNS are believed to be associated

with sites possessing patent intercellular junctional complexes: the nasal

mucosa, the epithelial linings of the median eminence and area postrema, and

possibly the endothelia of large vessels on the pial surface and/or occupying

the Virchow-Robin spaces. The degeneration-regeneration of cells in the nasal

mucosa allows the intercellular clefts of this epithelium to be patent to fluidphase macromolecules instilled in the nares (i.e., air borne, applied topically)

or delivered to the epithelium from the blood through leaky capillaries. The

extracellular route continues along the olfactory nerve into the subarachnoid

space at the level of the olfactory bulb (Balin et al. 1986). The absence of

a nose-brain barrier is of importance when the nose is considered a site

for delivery of drugs, viruses, and environmental toxins associated with

neurological disease (Barthold 1988; Langston 1985; Talamo et al. 1989).

Junctional complexes among ependymal cells lining the area postrema

and median eminence are suspected of being discontinuous and not

circumferentially tight, unlike the tight junctional complexes among BBB

endothelia and epithelia of the choroid plexus; the apparent absence of

a blood-CSF barrier at these two circumventricular organs heralds the

bidirectional exchange of micromolecules and macromolecules between

the blood and CSF (Broadwell et al. 1983a, 1987a; Gotow and Hashimoto

1979, 1981; Richards 1978). Even if the junctional complexes among

ependymal cells in the median eminence and area postrema were

circumferentially, tight, blood-borne, fluid-phase substances leaking from

vessels in the circumventricular organs could move extracellularly around

the tight junctions and enter the CSF through gap junctions among ependymal

cells adjacent to the median eminence and area postrema.

The absence of a BBB to blood-borne macromolecules at the pial surface of

the brain was reported initially in mice injected intravenously with the fluidphase tracer HRP (Balin et al. 1986) and recently confirmed in rat and monkey

(Broadwell 1989; Broadwell and Sofroniew, submitted for publication). The

latter studies suspect that the “leak” lies at the level of larger vessels on the pial

surface and/or within the Virchow-Robin spaces. Blood-borne peroxidase that

has entered the subarachnoid space by this route easily moves extracellularly

through the pia mater and glial limitans into the extracellular clefts of the

underlying neuropil and through the Virchow-Robin spaces for widespread

distribution within the perivascular clefts (figure 6). The circumventricular

organs also contribute to the dissemination of blood-borne HRP through the

perivascular clefts. Subarachnoid macrophages followed by perivascular

phagocytes lying superficially and deep within the brain parenchyma label with

blood-borne HRP in that order. Fluid-phase markers may be propelled through

the perivascular clefts in vivo by the pulsatile activity of arterioles (Rennels et al.

248
FIGURE 6. Blood-borne HRP enters the rodent and primate brain

extracellularly through permeable vessels supplying the pial

surface (A and B, small arrows) and circumventricular organs,

such as the median eminence (A, large arrow). Once on the pial

surface (B and D. arrows), blood-borne peroxidase can enter

the perivascular clefts (B, arrowheads) and move deeper into

the CNS; peroxidase is endocytosed by phagocytic cells on the

pial surface (D, large arrow) and in the per/vascular spaces (C,

arrows). Blood-borne peroxidase on the pial surface is able to

pass extracellularly among cells of the pia mater and glial limitans

to enter the subpial neuropil (D, arrowheads).

1985). The patent extracellular pathways serve to explain how perivascular

phagocytes throughout the rodent and monkey CNS become exposed to and

label with blood-borne peroxidase in less than 1 hour postinjection (Balin et

al. 1986; Broadwell 1989; Broadwell and Sofroniew, submitted for publication).

CNS sites leaky to fluid-phase molecules circumventing the BBB are not

249
insignificant; they allow the ingress of blood-borne, fluid-phase, endogenous

molecules the size of IgM (MW 165,000) (Broadwell and Sofroniew, submitted

for publication) and represent nonimmunologically privileged sites in the

CNS (Broadwell 1989; Broadwell and Sofroniew, submitted for publication;

Sentos and Valdimarsson 1982). Absence of a BBB in the circumventricular

organs and subarachnoid space is compensated for partially by populations

of microglia, macrophages, and class II cells of the major histocompatibility

complex (MHC) occupying these sites (R.D. Broadwell, B.J. Baker, A. Wolf,

and J.S. Villegas, unpublished data).

WGA-HRP as an adsorptive-phase tracer also may follow the extracellular

avenues into brain from the blood despite avid binding of the WGA molecule

to the plasma membrane of cells (Balin and Broadwell 1988; Broadwell and

Balin 1985; Villegas and Broadwell, in press). Whether blood-borne, receptormediated phase molecules not binding to specific endothelial receptors gain

access to extracellular routes circumventing the BBB remains to be determined.

INTRACEREBRAL TRANSPLANTS AND THE BLOOD-BRAIN BARRIER

lmmunohistochemistry with antibodies directed against MHC class I antigen

on the luminal surface of endothelia has demonstrated that blood vessels

inherent to intracerebrally placed grafts of CNS or peripheral origin are

sustained and anastomose with host cerebral vessels (Broadwell et al.

1991). Consequently, solid brain tissue grafted intracerebrally presents a

BBB to blood-borne macromolecules (Broadwell 1988; Broadwell et al.

1987b, 1989, 1991). Cell suspensions of astrocytes or neurons injected

intracerebrally are supplied with host CNS vessels and a BBB to circulating

protein (Broadwell et al. 1991). Solid tissue of the anterior pituitary gland

(Broadwell et al. 1987b, 1991) adrenal medullary gland (Rosenstein 1987)

muscle, skin, and superior cervical ganglia (Rosenstein and Brightman

1986; Wakai et al. 1986) grafted intracerebrally do not exhibit a BBB nor

do intracerebral cell suspensions of peripheral origin (e.g., fibroblasts, PC12

cells). The latter are supplied exclusively with host BBB vessels that lose their

BBB properties and become fenestrated and/or have patent interendothelial

junctional complexes (Broadwell et al. 1991). Not only will peripheral tissue/

cell suspension grafts create a “window” in the BBB for entry of blood-borne

substances, such as chemotherapeutics, to the brain, but also the absence

of a BBB in these grafts necessarily means the absence of a brain-blood

barrier as well. This fact introduces a significant problem to the intracerebral

application of peripheral tissue/cell suspension grafts for production and release

of a neurotransmitter or peptide in clinical treatment for neurodegenerative

disorders (e.g., Parkinson’s disease). Any neurotransmitter or peptide

anticipated to be secreted from intracerebral, peripheral tissue/cell suspension

250
grafts would likely fail to enter the surrounding host neuropil in large enough

concentration to effect a significant clinical improvement; rather, the secreted

product most likely would enter the non-BBB vessels supplying the graft and

be removed within the general circulation. Broadwell and coworkers have

confirmed that the presence or absence of blood-brain and brain-blood barriers

within intracerebrally positioned grafts is dictated by the grafted tissue/cells and

not by the surrounding host tissue contributing vessels to the graft (Broadwell et

al. 1991).

SUMMARY AND CONCLUSIONS

The potential intracellular and extracellular pathways that blood-borne

substances may follow for circumventing the fluid-brain barriers and entry

to the CNS are numerous. The extracellular avenues, patent to blood-borne

protein the size of IgM, and movement of blood-borne macromolecules

through perivascular clefts deep into the CNS complicate the interpretation

and identification of bona fide transcytosis through the BBB. The often-stated

belief in literature reviews of the BBB that nonfenestrated cerebral endothelia

fail to engage in endocytosis and possess few vesicles under normal conditions

is invalid. Endocytic vesicle formation and vesicular traffic among constituents

of the endomembrane system are no different in BBB endothelia than in

other cell types. Available biochemical and morphological data advocate

the transcytosis of blood-borne protein and peptides through nonfenestrated

cerebral endothelia. However, absence of demonstrable endocytic activity

at the abluminal front compared with a very prominent endocytic activity at the

luminal surface of BBB endothelia argues against bidirectional membrane

trafficking through the BBB and supports the concept of a brain-blood barrier.

The latter is no less significant functionally than the BBB and may be more so

in deterring transendothelial transfer of peptides and proteins bidirectionally

through the nonfenestrated cerebral endothelium. The difficulty in interpreting

transcytosis through BBB endothelia is not encountered for epithelia of the

blood-CSF barrier at the level of the choroid plexus. Choroid epithelia engage

in endocytosis circumferentially; hence, the potential for transcytosis and

circumvention of the blood-CSF barrier through an intraepithelial route exists

bidirectionally in the choroid plexus.

NOTES

1. The potential for native HRP to undergo transcellular transport following

fluid-phase endocytosis exists in the epithelium of the seminal vesicle

(Mata and David-Ferriera 1973) and in somatotrophs of the anterior

pituitary gland (Broadwell and Oliver 1983); HRP introduced extracellularly

to both cell types in vivo becomes sequestered within the innermost Golgi

251
saccule of the cells for packaging, export, and possible exocytosis. In

these cell types, native HRP may behave as a membrane-bound marker

(comparable with a lectin or ligand) rather than as a soluble or fluid-phase

marker. The binding may involve the carbohydrate moieties of the

peroxidase molecule. Available data suggest that HRP (Straus 1981)

may bind to mannose-6-phosphate receptors on the cell surface (Sly et

al. 1981).

2. In vitro studies suggest that the transferrin receptor in some cell types may

recycle through the Golgi complex (Fishman and Fine 1987; Snider and

Rogers 1985; Woods et al. 1986).

3. These sites lie outside the barrier and, therefore, are not immunologically

privileged sites within the CNS.

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ACKNOWLEDGMENT

This research was supported in part by U.S. Public Health Service grant NS18030 from the Stroke and Trauma Program, National Institute of Neurological

Disorders and Stroke, National Institutes of Health.

AUTHOR

Richard D. Broadwell, D.Phil.

Professor of Neurological Surgery and Director of Laboratories of Neurological

Surgery

Division of Neurological Surgery

Department of Surgery

257
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118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. Carl G.

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119 PROBLEMS OF DRUG DEPENDENCE 1991: 53RD ANNUAL

SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

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DHHS Publication No. (ADM) 92-1910

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1992