National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Imaging

Techniques in

Medications

Development:

Preclinical and

Clinical Aspects

U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health

138
Imaging Techniques in

Medications Development:

Preclinical and Clinical

Aspects

Editors:

Heinz Sorer, Ph.D.

Rao S. Rapaka, Ph.D.

NIDA Research Monograph 138

1994

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGEMENT

This monograph is based on the papers from a technical review on

“Imaging Techniques in Medications Development: Preclinical and

Clinical Aspects” held in 1992. The review meeting was sponsored by

the National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse 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 the National

Institute on Drug Abuse 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.

National Institute on Drug Abuse

NIH Publication No. 94-3728

Printed 1994

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.

ii
Contents

Page

PET, SPECT, and Probes in Drug Design and Development . . . . . . 1

Henry N. Wagner, Jr.

Positron Emission Tomography in Studies of Drug Abuse . . . . . . 15

Edythe D. London

Imaging Brain Function in Animals To Understand

Drugs of Abuse and Potential Pharmacotherapies . . . . . . . . . . . 25

M.F. Piercey

Brain-Imaging Studies of the Combined Use of Cocaine

and Alcohol and of the Pharmacokinetics of Cocaethylene . . . . . . 41

Nora D. Volkow and Joanna S. Fowler

Positron Emission Tomography of Cocaine Binding Sites

on the Dopamine Transporter . . . . . . . . . . . . . . . . . . . . . . . . 57

Bertha K. Madras, David R. Elmaleh, Peter C. Meltzer,

Anna Y. Liang, Gordon L. Brownell,

and Anna-Liisa Brownell

Metabolic Mapping Methods for the Identification of the Neural

Substrates of the Effects of Novel Tropane Analogs . . . . . . . . . . . 71

Linda J. Porrinno, Lisa Williams-Hemby, and Huw M.L. Davies

Noninvasive Evaluation of the Sympathetic Nervous System

of the Heart by Nuclear Imaging Procedures . . . . . . . . . . . . . . . . 85

Markus Schwaiger, James C. Sisson, and Donald Wieland

iii
Development of PET/SPECT Ligands for the Serotonin

Transporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Ursula Scheffel, Robert F. Dannals, Makiko Suehiro,

George A. Ricaurte, F. Ivy Carroll, Michael J. Kuhar,

and Henry N. Wagner, Jr.

SPECT Imaging of Dopamine and Serotonin Transporters

in Nonhuman Primate Brain . . . . . . . . . . . . . . . . . . 131

Marc Laruelle, Ronald M. Baldwin, and Robert B. Innis

Cerebra Blood Flow Changes With Acute Cocaine Intoxication:

Clinical Correlations With SPECT, CT, and MRI . . . . . . . . . 161

I. Mena, R.J. Giornbetti, B.L. Miller, K. Garrett,

J. Villanueva-Meyer, C. Mody, and M.A. Goldberg

An Introduction to Magnetic Resonance Methods

for Clinical Research . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Gerald G. Blackwell

iv
PET, SPECT, and Probes in Drug

Design and Development

Henry N. Wagner, Jr.

INTRODUCTION

The technology of nuclear medicine has the potential of bringing

together three approaches to drug design and development: genetic,

biochemical, and pharmacologic (Burns et al. 1993; Bice et al. 1986;

Jefferies et al., submitted; Swann 1988). For example, using

polymerase chain reaction, one can synthesize large quantities of

complementary RNA related to the production of a specific

biochemical such as D2-dopamine receptors. The structure of the

receptor can be determined by means of x-ray crystallography. The

essential features of the structure of the receptor that relate to the

binding and activation of D2-dopamine receptors can be determined.

Using positron-emitting tracers, one can determine the site, quantity,

and degree of occupancy of the receptors and examine the reuptake of

dopamine into presynaptic dopaminergic neurons through the dopamine

transporter (Gamett et al. 1983; Wagner et al. 1983). Thus, the status

of both the presynaptic and postsynaptic neurons can be examined. If

one can successfully label oligonucleotides such as the antisense

oligonucleotide related to dopamine receptor synthesis, factors affecting

the synthesis of D2-dopamine receptors could be examined. In essence,

nuclear medicine makes it possible to relate genotypes, chemotypes,

and phenotypes. The essence of modem nuclear medicine is in vivo

biochemistry of living human beings.

At the annual meeting of the Society of Nuclear Medicine held in Los

Angeles in June 1992, Dewanjee and colleagues (1992) from the

University of Miami reported that they had labeled an antisense

oligonucleotide with a diethylenetriaminepenta-acetic acid (DTPA)

chelate of 111In. When injected intravenously, the tracer bound to

leukocytes. Many problems such as nonspecific binding remain to be

solved, but, if the oligonucleotide binds successfully to messenger

RNA (mRNA), it could represent a major focus of radiotracer

1
development for in vivo studies. The primary message is that carried

by mRNA, which controls the synthesis of polypeptides or proteins.

By looking at the receptors themselves, it is possible to determine the

required shape, hydrophilicity, charge, and other factors that result in

binding by the receptor with a high affinity. It is possible to design

tracers as well as therapeutic agents to conform to the structural

requirements. The pharmacokinetics of these agents can be examined

and the relationship between structure and function assessed.

Even with complete knowledge of the genome, it would be necessary

to examine the biochemical expression of the genome. One important

genetic principle is pleotropism, which states that an abnormal gene

can result in many different diseases. A defective polypeptide can

result in failure of multiple systems, resulting in many different

phenotypic manifestations of disease. A second principle is genetic

heterogeneity, which states that many different gene abnormalities can

result in or produce the same disease manifestations. Thus, the

approaches to disease should be genetic, biochemical, and

pharmacologic.

USING PET AND SPECT TO TREAT DISEASE

With positron emission tomography (PET) and single photon emission

computed tomography (SPECT), researchers can begin to close the

circle between brain chemistry and behavior. Not only amine receptors

but also polypeptide receptors have been studied successfully.

Peptides are bound by specific receptors, are inexpensive to produce

by genetic engineering techniques, and are quickly cleared from the

blood. Most of the 50-odd peptides are amenable to radiotracer

development. One characteristic of peptides is that they do not cross

the blood-brain barrier. With the application of the ability to examine

various parts of neuroreceptor systems, including the dopamine and

opiate systems, it is now possible to examine a whole new approach to

drug therapy-the direct installation of amines or peptides into specific

regions of the brain. Examples of potential applications of the

approach are the treatment of Parkinson’s disease (PD) by the infusion

of amines or nerve growth factors, of epilepsy by local infusion of

drugs (Frost et al. 1985; Mazziotta and Engel 1984), or of intractable

2
pain by blocking pain receptors. This approach of direct infusion into

specific regions of the brain might be useful for the treatment of

persons suffering from substance abuse.

PET and SPECT technology could help develop increasingly specific

drugs by adding in vivo biochemistry to physiology in drug design and

development. The vasopressin story is a prototype. The vasoconstrictive effect of native vasopressin was removed from the

antidiuretic effect of vasopressin, which makes it more effective in the

treatment of diabetes insipidus. Genetic engineering produces an

effective drug that has only an antidiuretic effect. Biochemical tracing

and biochemical treatment are two sides of the same coin-molecular

medicine. More and more specific tracers directed toward particular

biochemical recognition sites have been developed.

From the beginning of the pioneering studies with [

18F]deoxyglucose,

PET was concerned primarily with bioenergetics (Phelps et al. 1986).

Today, both PET and SPECT make it possible to examine intercellular

communication in living human beings. Life is maintained because

atoms and molecules can recognize each other. More diseases are

being characterized as problems with intracellular communication. In

the absence of vasopressin receptors, a person develops congenital

diabetes insipidus because vasopressin cannot be recognized.

Another example of a disease involving neurotransmission is PD. In

the mid-1960s, it was discovered that patients with PD have a

deficiency of dopamine in the basal ganglia as a result of degeneration

of dopaminergic neurons in the substantia nigra. In 1983, it became

possible to image and quantify D2-dopamine receptors in the living

human brain with the tracer [

11C]N-methyl spiperone (NMSP). Since

that time, both PET and SPECT have been used to examine many

aspects of dopamine biochemistry in the human brain. For example,

dopamine synthesis can be examined with fluorine-labeled L-dopa.

The secretion of dopamine can be measured by its competitive

inhibition of tracers that bind to dopamine receptors. It is possible to

assess the availability of both presynaptic and postsynaptic receptors,

including the presynaptic dopamine transporter involved in the action

of cocaine.

3
Although the state of the art of PET and SPECT imaging has a spatial

resolution of about 5 mm, specific chemicals can be used to

differentiate the effects of drugs on structures that are only micrometers apart. For example, if one produces a unilateral lesion of

presynaptic dopaminergic neurons by injecting the neurotoxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into a single

internal carotid artery, the impairment of presynaptic neurons, but not

postsynaptic neurons, can be documented by quantitative imaging of

the presynaptic dopamine transporter with [

11C]-labeled WIN 45-428

and of the postsynaptic dopamine receptors with [

11C]NMSP.

The dopaminergic system has been studied extensively by PET, and

the serotonergic system also is becoming a major focus. For example,

there is now an effective tracer of the serotonin presynaptic transporter

available in both active and inactive isomeric forms that makes

possible subtraction images and facilitates quantification.

COMPARISONS BETWEEN PET AND SPECT

What about the relative merits of PET and SPECT in drug design and

development? The most fundamental difference lies in the radionuclides that each employs. PET tracers are chiefly those of [

11C] and

[

11F] and, to a lesser degree, 12N and 15O. SPECT primarily uses

iodine- 123 ([123I]) or technetium-99m (Tc-99m). The major problem

with PET is related to the short half-life of [

11C] (20 minutes) and [18F]

(2 hours). The half-life of Tc-99m is 6 hours and that of [

123I] is

13 hours, which greatly decreases the demands of the study. If a study

can be carried out successfully with SPECT, it should be used. Many

studies cannot be done with SPECT. The chief advantage of PET is

related to [

11C]. Chemistry will always be easier with [11C] than it will

be with [

123I] or Tc-99m, so many new tracers can be developed faster

with PET than with SPECT. However, PET successes are translated

into more widespread use by extending them to SPECT.

Using PET facilitated the study of D2-dopamine receptors and

transporters. First, [

11C]NMSP was developed; its success led to the

development of [

123I]-labeled benzamides for the study of D2

-dopamine

receptors by SPECT. The presynaptic transporter initially was

examined with a [

11C] tracer that is specific for the dopamine

4
transporter. The SPECT tracer-[123I] RTI-55-was then developed

and found to bind to both serotonin and dopamine presynaptic

transporters. The iodinated tracer also was found to have a higher

affinity for the transporter than the [

11C] compound.

Another advantage of the [

123I] tracers is related to their 13-hour halflife, which makes it possible to carry out studies for 24 or more hours

after injection of the tracer. By that time, nonspecific binding is less

than that observed soon after injection of the [

11C]-labeled tracer

because of its 20-minute half-life.

USING PET AND SPECT TO PLAN TREATMENT

The purpose of studies with these new tracers of recognition sites is to

characterize patients biochemically, as well as physiologically, and

then plan treatment for them on the basis of their chemical

abnormalities. Because the classification is biochemical, the treatment

often can be chemical, and the response to treatment can be assessed

chemically. PET and SPECT do not just provide new tests for “old”

diseases. They provide a whole new way of looking at disease-an

approach based on in vivo biochemistry (i.e., molecular medicine).

An example of an “old” disease is Alzheimer’s disease. Dementia is

the primary presenting problem; senile plaques and neurofibrillary

tangles in histopathological sections of the brain are the basis of

classification. Dementia is analogous to heart failure in representing a

heterogeneous population. It is naive to search for a single biological

marker that will characterize all patients who meet the phenotypical

criteria for schizophrenia as described in the Diagnostic and Statistical

Manual of Mental Disorders (American Psychiatric Association 1987).

Schizophrenics are also a very heterogeneous population of patients.

The status of specific neuronal systems, the perturbation of which may

help the patients, must be determined. In vivo chemistry can be used

to try to answer the five basic questions that describe the practice of

medicine:

What is wrong with the patient?

How did it happen?

What is going to happen?

5
What can be done about it?

Is the problem being solved?

The approach to movement disorders is a good example. In patients

with typical idiopathic PD, both D1- and D2-dopamine receptors are

intact, and patients benefit from being given L-dopa to increase

dopamine synthesis or bromocriptine to stimulate the intact D2-

receptors. Other patients with movement disorders involving

nigrostriatal degeneration have impaired D2-dopamine receptors and

will not be helped by either L-dopa or bromocriptine treatment.

The development of a SPECT tracer of D2-dopamine receptors called

[

123I]iodobenzamide extended the PET studies to larger numbers of

patients with movement disorders. A recent study (Dewanjee et al.

1992) included 60 patients with idiopathic PD, 17 controls, and 30

patients with Parkinson’s syndrome. Quantification with PET was

more accurate than with SPECT. Some investigators (e.g., Dewanjee

et al. 1992) have used simple indices such as the basal ganglia to

frontal cortex activity ratios in [

123I]iodobenzamide studies of D2

-

dopamine receptors. There is a dangerous tendency to oversimplify

when a method becomes simple. SPECT studies should be carried out

drawing on the lessons learned from decades of PET. Attenuation

correction, positioning, errors related to the limitations in spatial

resolution, and many other technical factors are as important for

SPECT as for PET.

ROLE OF SOMATOSTATIN RECEPTORS IN CANCER

DIAGNOSIS

An example of an advance made recently in SPECT is the study of

somatostatin receptors by Krenning and colleagues (1992a) from the

Netherlands. They used an 8-amino acid polypeptide analog of

somatostatin, a growth suppressor. The tracer, octreotide-111, was

labeled with an 111In DTPA chelate. Developed in Sandoz Research

Institute, the labeled octreotide was investigated initially at the

University of Rotterdam by Krenning and colleagues. To date, they

have studied more than 1,000 patients with various types of

neuroendocrine and other tumors. Among the findings was the fact

6
that 70 percent of 70 patients with carcinoma of the breast had

somatostatin receptors.

Somatostatin receptor imaging should not be considered a test for

breast cancer. It is a test for somatostatin receptors. If a tumor has

somatostatin receptors, the patient is a candidate for somatostatin

therapy. Somatostatin is a stable drug.

An important finding by Krenning and colleagues (1992b) was that

patients with hyperthyroidism and exophthalmus have large numbers of

somatostatin receptors in the T lymphocytes behind the eyes. The

degree of accumulation of octreotide-111 behind the eyes was related

to the degree of the malignant exophthalmus. This finding is

important in planning treatment. If the disease is active, the patient

can be treated with steroids or an immunosuppressive drug. If there is

no evidence of activity of the disease, the exophthalmus is treated

better by surgery to remove the fibrous tissue behind the eyes. Thus,

again, one sees the sequence: chemical characterization of the disease,

planning of treatment, and monitoring of treatment on the basis of

chemistry.

An example of how effectively PET and SPECT technology can be

used to study the action of drugs is the recent report of Dewey and

colleagues (1992) from Brookhaven National Laboratory, who studied

the effects of a suicide inhibitor of the enzyme that breaks down

aminobutyric acid (GABA), an inhibitory neurotransmitter. Inhibition

of the metabolizing enzyme resulted in decreased production of

GABA. This in turn disinhibited the dopaminergic neurons, so

synaptic dopamine secretion increased. The secretion of dopamine

competed with the binding of [

11C]raclopride by the D2

-dopamine

receptors, so more dopamine receptors were available for binding

[

11C]raclopride. Dopamine secretion inhibited the release of

acetylcholine by cholinergic neurons; this inhibition was reflected in

increased binding of [

11C]benzotropine because less acetylcholine was

bound to the acetylcholine receptor. Thus, Dewey and colleagues

(1992) were able to demonstrate serial neurochemical effects among

three neurotransmitters-GABA, dopamine, and acetylcholine. Their

studies were also a demonstration of how endogenous neurotransmitter

release can be measured by competitive inhibition of radioligand

binding.

7
SIMPLE PROBE SYSTEM USES

Some types of problems do not need the spatial resolution provided by

PET or SPECT. Much simpler dual-detector systems can be used to

look at either the entire brain or specific regions such as the frontal

lobes or cerebellum. The use of the simple probe systems shows that

it is not necessary to use expensive high-spatial-resolution PET or

SPECT imaging to solve problems in which spatial resolution does not

have to be high. Probes are simple and less expensive by a factor of

30 or 50. The radiation dose also is reduced by orders of magnitude

compared with high-resolution imaging, in which the statistical

limitations are related to the number of counts per picture element.

Particularly in the study of drug effects, it is often not necessary to

have a high degree of spatial resolution. For example, a highresolution system is not needed to measure the quantitative effect of

naltrexone or naloxone on opiate receptors. The antagonists affect all

the u-type opiate receptors regardless of location. Conversely, in a

problem such as temporal lobe epilepsy, in which the search is for a

focus in the temporal or frontal lobe, a high degree of spatial

resolution is required.

A probe device developed at The Johns Hopkins University has been

used to study mice, rats, dogs, baboons, and human beings. For

example, in living mice it was possible to obtain a dose/response curve

showing the blocking effect of haloperidol on the binding of

[

11C]NMSP. Similar dose/response curves were obtained in human

beings, and the results correlated well with PET studies of the same

persons. One study (Brust et al. 1992) examined the effect of

vasopressin on [

11C]methionine accumulation in the brain of living

dogs. When nonradioactive phenylalanine was given before the dose

of [

11C]methionine, the rate of transport of methionine decreased

because of competitive inhibition. The purpose of the vasopressin

study was to test the hypothesis that vasopressin’s effect on memory

might be through amino acid transport because vasopressin itself does

not cross the blood-brain barrier. Vasopressin administration did

inhibit [

11C]methionine transport,

Another study (Lee et al. 1988) addressed the question of how long a

50-mg dose of naltrexone blocks opiate receptors. When

8
[

11C]carfentanil was used at various times after the administration of an

oral 50-mg dose of naltrexone, it was found that the half-time of

blockade of the receptors was 72 hours. Thus, even though a 50-mg

dose blocks the p-opiate receptors for about a week, it is recommended

that the dose be given every day, primarily because of the rapid blood

clearance of the drug. By examining the state of the receptors

themselves, one can get closer to the site of action of the drug than

one can get by measuring only plasma concentrations of the drug.

This study exemplifies the marriage of nuclear medicine technology

and drug design and development.

Probe-based systems have the potential of opening up PET and SPECT

studies to basic neuroscientists. It is unlikely that human-oriented PET

or SPECT scanners can be provided to basic neuroscientists, but

simpler systems can be used to take advantage of the new tracers

rapidly being developed. Probe studies also can be carried out safely

and effectively in public health studies. In the author’s laboratory, for

example, the effects of lead toxicity on brain neurochemistry are being

investigated. At present, only laboratory animals are being studied, but

it is hoped that the studies soon will extend to human beings. It is

unlikely that PET imaging could be used in public health studies.

Another direction is the development of less expensive imaging

devices for the study of animals. Green and colleagues (1992) at the

National Institutes of Health have developed a SPECT scanner for

animals that costs less than $50,000.

CONCLUSION

The main engine of nuclear medicine technology is the cyclotron

because of its chemical capabilities, chiefly [

11C] and [18F]. PET

studies are beginning to be used in community hospitals and research

centers from centrally located cyclotrons, some under commercial

sponsorship. One commercial supplier of cyclotron-produced

radiotracers is in Los Angeles; another is in Phoenix, where PET

imaging is carried out in community hospitals and health centers using

tracers supplied from a central cyclotron. More research and patient

studies can be carried out with Tc-99m and [

123I] because of their

longer half-lives. The half-lives of these tracers will never

9
change—for [

11C], 20 minutes; for [18F], 2 hours; for Tc-99m, 6 hours;

and for [

123I], 13 hours. At the 1992 Society of Nuclear Medicine

meeting mentioned previously, 51 papers were based on the use of

[

11C] compounds. Forty papers (not all discussing [

11C]) involved the

dopaminergic system, and 125 papers involved [

18F]. Ninety papers

were based on [

18F]deoxyglucose to study bioenergetics. Half of the

nearly 900 papers presented at the meeting involved PET or SPECT.

The number of studies of neuroreceptors continues to increase every

year. Blood flow and metabolism also are important topics. A major

area of research is the study of drug effects. Someday, a psychiatrist

or other physician will be measuring brain chemistry with simple probe

detectors the way physicians now use the sphygmomanometer to

measure blood pressure.

A description of a patient who presented with a lesion of the brain that

was seen in a magnetic resonance imaging study illustrates the use of

various imaging techniques. Was it a tumor? Was it radiation

necrosis? A [

18F]deoxyglucose study showed that the lesion was

hypometabolic and therefore not a tumor. The pituitary was very

hypermetabolic, indicating a tumor. The next question was whether

the tumor had dopamine receptors on its membrane, as is the case with

many pituitary tumors. If a pituitary tumor manifests dopamine

receptors, dopamine receptor agonists are helpful because the

dopaminergic system is inhibitory of cell growth, acting through

prolactin-secreting cells. This tumor contained dopamine receptors, so

bromocriptine (a dopamine agonist) therapy was begun. By studying

the metabolism of either deoxyglucose or amino acids, in 1 day it was

determined whether the metabolic activity of the tumor had been

reduced. If the metabolism was not affected, there was no reason to

treat the patient for months, waiting for anatomical changes to occur.

Thus, in 1 day it was possible to characterize the tumor, plan the

treatment, and monitor the effect of the treatment (Di Chiro, personal

communication, 1992).

Is there a competition between PET and SPECT? Their synergism is

documented by that fact that, as noted above, the use of both has been

increasing to the point that they were discussed in 50 percent of the

papers presented at the 1992 Society of Nuclear Medicine meeting.

PET and SPECT are like two hands with which complicated problems

10
can be approached. To emphasize one without the other is like tying

one hand behind the back. If SPECT can solve a problem, it should

be used; but PET will always be able to do things that SPECT cannot

do because this is a world of carbon, and there never will be a good

SPECT tracer for carbon. That is physics, and that is forever. There

is a PET and SPECT continuum, in which both will flourish and in

which SPECT extends PET. Yet, advances often will be made initially

by SPECT because of the greater simplicity and greater numbers of

patients who can be studied. PET, however, is unique in the

possibility of labeling pharmacological compounds without changing

their pharmacological behavior. Evaluating the behavior of a drug in

vivo through time is now a PET domain.

REFERENCES

American Psychiatric Association. Diagnostic and Statistical Manual of

Mental Disorders 3rd ed. Washington, DC: American Psychiatric

Press, 1987.

Brust, P.; Shaya, E.K.; Jeffried, K.J.; Gjedde, A.; Dannals, R.F.;

Ravert, H.T.; Wilson, A.A.; Wagner, H.N., Jr.; Ermisch, A.; and

Wong, D.F. Effects of vasopressin on blood-brain transfer of

methionin in dogs. J Neurochem 59: 1421-1429, 1992.

Dewanjee, M.K.; Ghafouripour, A.K.; Kapadvanjwala, M.; Glenn, S.;

Serafini, A.N.; Sfakianakis, G.N. A new direct method of celllabeling in whole blood by administration of In-111 labeled antisense oligonucleotide. Abstract. J Nucl Med 33:980, 1992.

Dewey, S.L.; Brodie, J.D.; Smith, G.; Logan, J.; MacGregor, R.R.;

King, P.T.; Fowler, J.S.; Wolf, A.P.; Volkow, R.A.; and Ferrieri,

R.A. GABAergic modulation of endogenous dopamine and

acetylcholine release measured in vivo with positron emission

tomography. Abstract. J Nucl Med 33:847, 1992.

Frost, J.J.; Wagner, H.N., Jr.; Dannals, R.F.; Ravert, H.T.; Links, J.M.;

Wilson, A.A.; Bums, D.H.; Wong, D.F.; McPherson, R.W.;

Rosenbaum, A.E.; Kuhar, M.J.; and Snyder, S.H. Imaging opiate

receptors in the human brain by positron tomography. J Comput

Assist Tomogr 9(2):231-236, 1985.

Gamett, E.S.; Firnau, G.; and Nahmias, C. Dopamine visualized in the

basal ganglia of living man. Nature 305:137-138, 1983.

11
Green, M.V.; Markowitz, A.; Tedder, T.E.; Andrich, M.P.; Owens,

ES.; and Neumann, R.D. SPECT imaging in small animals.

Abstract. J Nucl Med 33:852, 1992.

Krenning, E.P.; Bakker, W.H.; Kooij, P.P.M.; Breeman, W.A.P.; Oei,

H.Y.; de Jong, M.; Reubi, J.-C.; Visser, T.J.; Bums, C.;

Kwekkeboom, D.J.; Reijs, A.E.M.; van Hagen, P.M.; Koper, J.W.;

and Lamberts, S.W.J. Somatostatin receptor scintigraphy with [

111InDTPA-Phe1

] octreotide in man: Metabolism, dosimetry and

comparison with [

1231-Ty3

]-octreotide. J Nucl Med 33:652-658,

1992a.

Krenning, E.P.; Oei, H.Y.; Kooij, P.P.M.; Wijnngaarde, R.; Lamberts,

S.W.J.; Wiersinga, W.M.; Prummel, M.F.; and van Royer, R.M.L.

Thyroid and orbital radiolabelled somatostatin accumulation in

Graves’ disease and Graves’ ophthalmopathy. Abstract. J Nucl Med

33:894, 1992b.

Lee, M.C.; Wagner, H.N., Jr.; Tanada, S.; Frost, J.J.; Bite, A.N.; and

Dannals, R.F. Duration of occupancy of opiate receptors by

naltrexone. J Nucl Med 29:1207-1211, 1988.

Mazziotta, J.C., and Engel, J., Jr. Advanced neuro-imaging techniques

in the study of epilepsy: PET, SPECT and NMR-CT. In: Pedley, T.,

and Meldrum, B., eds. Recent Advances in Epilepsy. Vol. 2.

Edinburgh, UK: Churchill, Livingstone, 1984.

Phelps, M.E.; Mazziotta, J.C.; and Schelbert, H.R., eds. Positron

Emission Tomography and Autoradiography: Application in the

Brain and Heart. New York: Raven Press, 1986.

Swann, J.P. Academic Scientists and the Pharmaceutical Industry.

Baltimore: The Johns Hopkins University Press, 1988.

Wagner, H.N., Jr.; Bums, H.D.; Dannals, R.F.; Wong, D.F.;

Langstrom, B.; Duelfer, T.; Frost, J.J.; Ravert, H.T.; Links, J.M.;

Rosenbloom, S.B.; Lukas, SE.; Kramer, A.V.; and Kuhar, M.J.

Imaging dopamine receptors in the human brain by positron

tomography. Science 221:1264-1266, 1983.

12
AUTHOR

Henry N. Wagner, Jr., M.D.

Professor

Divisions of Nuclear and Radiation Health Sciences

The Johns Hopkins Medical Institutions

615 North Wolfe Street, Room 2001

Baltimore. MD 21205-1279

13

Positron Emission Tomography

in Studies of Drug Abuse

Edythe D. London

INTRODUCTION

Positron emission tomography (PET) is a nuclear medicine procedure

that uses radiopharmaceuticals that are labeled with positron-emitting

radionuclides such as [

15O], [13N], [11C], and [18F]. In human

volunteers, PET scanning can be used to map and quantitate

biochemical processes in the brain. Applications include the labeling

of receptors for drugs and neurotransmitters, or functional studies, such

as assessments of regional cerebral blood flow and glucose

metabolism. This chapter focuses on how studies of glucose

metabolism have been applied to understanding the euphoriant actions

of drugs of abuse.

USING PET TO MEASURE CEREBRAL GLUCOSE

METABOLISM

The metabolic rate for glucose is an index of brain function because

glucose is the major metabolic substrate of the adult brain (Sokoloff

1972; Siesjö 1978). Physiological perturbations that increase energy

metabolism of the brain also increase the metabolism of glucose,

although nonoxidative metabolism of glucose also can be stimulated

(Lear and Ackermann 1989). A method for measuring the regional

cerebral metabolic rate(s) for glucose (rCMRglc) in intact animals was

developed using ex vivo autoradiography (Sokoloff et al. 1977). The

procedure involves the intravenous injection of radiolabeled 2-deoxyD-glucose and the collection of timed arterial blood samples for

estimation of radioactivity and glucose in plasma. An operational

equation allows calculation of rCMRglc from these data and tissue

radioactivity, as determined by quantitative autoradiography of

individual brain regions. This method had been adapted for noninvasive use in human subjects with PET scanning and the use of

15
[

18F]fluorodeoxy-glucose (FDG) (Phelps et al. 1979; Reivich et al.

1979) or [

11C]deoxyglucose (Reivich et al. 1982).

Studies of glucose metabolism can provide important insights into

normal brain function as well as the pathophysiology of diseases

(Reivich et al. 1983). For example, in patients with Alzheimer’s

disease, there are rCMRglc deficits in the temporoparietal cortex

(Friedland et al. 1983) and correlations between asymmetries in

rCMRglc and neuropsychological performance (Grady et al. 1986).

The FDG method also has been used to study regional brain function

in schizophrenia (Farkas et al. 1980; Gur et al. 1987) and in

neurological diseases, including stroke, neurodegenerative disorders,

and epilepsy (Kuhl 1983; Engel et al. 1983).

Measurements of rCMRglc obtained by the FDG method also have

been useful in drug abuse research. Studies of the acute cerebral

metabolic responses to drugs of abuse have been performed using this

method in human volunteers. Although only some of these studies

were designed to elucidate the neurobiological determinants of druginduced euphoria, the results from all of them provide information

about the mechanisms by which drugs of abuse produce their effects

on mood and feeling state. Drugs that have been studied in this way

include morphine (London et al. 1990a), psychomotor stimulants

(Wolkin et al. 1987; London et al. 1990b), A

9

-tetrahydrocannabinol

(Volkow et al. 1991), benzodiazepines (Buchsbaum et al. 1987; Foster

et al. 1987; de Wit et al. 1991), nicotine (Stapleton et al. 1992), and

alcohol (de Wit et al. 1990; Volkow et al. 1990).

In PET scan studies of substance abuse at the Addiction Research

Center, most of the subjects were male polydrug abusers who resided

on a closed research ward. The subjects underwent screening

procedures, including psychological and medical assessments. After a

drug-free period of several weeks, the subjects participated in

simulation sessions designed to reduce the novelty of the procedures

and also to provide training in the completion of specific

questionnaires. Each subject received either an x-ray computed

tomography scan or a magnetic resonance imaging scan. These

structural images were used to exclude individuals with frank structural

brain abnormalities and to aid in localization of specific regions of

interest in the functional images obtained by PET.

16
The subjects participated in two PET sessions, with placebo or active

drug administered in random sequence. During the measurement of

rCMRglc, subjective self-reports of mood and feeling state were

obtained. Questionnaires that are sensitive to the effects of

psychoactive drugs were administered before and after the PET scan

measurements and, in some cases, during the radiotracer uptake period.

These included the Addiction Research Center Inventory, composed of

the following three subscales:

Morphine-Benzedrine Group subscale, which is sensitive to the

positive (euphoriant) properties of opioids and psychomotor

stimulants;

Pentobarbital Chlorpromazine Alcohol Group subscale, which is

sensitive to fatigue and low motivation; and

Lysergic Acid Diethylamide (LSD) subscale, which contains items

that reflect weird feelings, increased awareness of somatic

sensations, and depersonalization (Haertzen 1974).

The Cocaine-Sensitive Scale (Sherer 1988; Muntaner et al. 1989) was

administered during the radiotracer uptake period after the injection of

either cocaine or placebo.

The responses to 30 mg morphine, injected intramuscularly, included

elevations on the Morphine-Benzedrine and Pentobarbital

Chlorpromazine Alcohol Group subscales of the Addiction Research

Center Inventory, indicating the presence of euphoria and, to a lesser

extent, of sedation and disorientation (London et al. 1990a). At the

same time, global cerebral glucose metabolism was reduced (10

percent reduction from placebo values), with statistically significant

decrements in six of the cortical areas assayed when controlling for the

contribution of PaCO2 by partial correlation analysis. The major

conclusion from this study was that opioid euphoria involved a

reduction of cortical activity, as indicated by rCMRglc. It seems

reasonable that reduced cortical activity could be a necessary

component of the production of opioid euphoria or that, alternatively,

the reduced activity is a response to the positive affective state that is

produced.

17
When polydrug abusers with histories of intravenous use of cocaine

were tested similarly, self-report ratings on items of the CocaineSensitive Scale indicated intense “rush” immediately after the injection

of cocaine; the response declined to negligible values within 20 min

(London et al. 1990b). Similarly, subjects reported feeling high,

pleasant, and powerful. These subjective ratings were accompanied by

an increase in beta activity in the spontaneous electroencephalogram

(R. Herning, R.L. Phillips, B. Glover, and E.D. London, unpublished

observations). Simultaneous measures of cerebral glucose utilization

showed a global decrease (mean reduction of 14 percent), and most of

the regions of interest that were assayed showed statistically significant

decrements in rCMRglc (5 percent to 26 percent). Thus, the

intravenous dose of cocaine that was used produced a more widespread

decrease in rCMRglc than the morphine dose used in the previous

study.

It is intriguing that drugs as different as morphine and cocaine would

produce similar effects on cerebral metabolism. While morphine

produces a constellation of effects, including sedation and decreases in

body temperature, cocaine produces restlessness, cardiovascular

stimulation, and increases in body temperature. Nonetheless, these

drugs share the common property of inducing a positive affective state,

termed “euphoria.” It is now a common view that this euphoria is

linked to a common brain mechanism, involving systems that use

dopamine as a transmitter. A variety of evidence, obtained from

studies in animal models for human drug abuse, suggests that the

primary sites in the brain that mediate reinforcement due to opiates and

psychomotor stimulants are different (for reviews, see Koob and

Bloom 1988; Hubner and Koob 1993). Specifically, studies in rats

suggest that opiates produce reinforcement by primary actions in the

ventral tegmental area, which contains the cell bodies of mesolimbic

dopaminergic neurons (Broekkamp and Phillips 1979; Bozarth and

Wise 1980; Joyce et al. 1981). In contrast, psychomotor stimulants

such as amphetamine and cocaine appear to produce reward by

primary actions on the terminals of mesolimbic dopaminergic neurons

(Carr and White 1983; Goeders and Smith 1983). Nonetheless.

irrespective of the initial target sites, the effect of opioid or

psychomotor stimulant action would be the same in that both classes of

drugs would increase intrasynaptic dopamine in terminal fields of the

mesolimbic dopamine system. Thus, the overall reduction of cerebral

18
glucose metabolism, particularly in the cortex, could result from

enhanced dopaminergic activity.

If one extends an examination of the effects of drugs of abuse on

cerebral glucose metabolism beyond studies of acute morphine and

cocaine effects, the emergent finding is that almost all euphoriants that

are compulsively self-administered reduce cerebral glucose utilization

(table 1). This statement derives from observations that amphetamine

(Wolkin et al. 1987) benzodiazepines (Buchsbaum et al. 1987; Foster

et al. 1987; de Wit et al. 1991), and ethanol (de Wit et al. 1990;

Volkow et al. 1990) all reduce rCMRglc. Similarly, preliminary

studies at the Addiction Research Center also indicate that intravenous

nicotine (Stapleton et al. 1992) and buprenorphine (Walsh et al. 1992)

produce liking and other positive subjective self-reports while

decreasing rCMRglc in experienced users of tobacco products and

opioids, respectively. An exception is the finding that an intoxicating

dose of -tetrahydrocannabinol does not consistently reduce rCMRglc.

Instead the most robust finding with -tetrahydrocannabinol is a

stimulation of rCMRglc in the cerebellum (Volkow et al. 1991), an

area that shows a high density of cannabinoid receptors (Herkenham et

al. 1990). Additional studies with euphorigenic doses of -tetrahydrocannabinol may be warranted. In general, the aforementioned

findings indicate that a decrease in cerebral glucose metabolism may

be a fundamental component of drug-induced euphoria.

TABLE 1. Reduction of rCMRglc by drugs of abuse

Morphine (London et al. 1990a)

Cocaine (London et al. 1990b)

Amphetamine (Wolkin et al. 1987)

Nicotine (Stapleton et al. 1992)

Benzodiazepines (Buchsbaum et al. 1987; Foster et al. 1987; de Wit et

al. 1991)

Ethanol (de Wit et al. 1990; Volkow et al. 1990)

Barbiturates (Theodore et al. 1986)

19
CONCLUSION

PET scanning is a valuable approach to understanding the brain

mechanisms that support drug abuse in human subjects. In particular,

functional studies using the FDG method have revealed that drugs of

abuse generally reduce rCMRglc when the drugs are given at

euphorigenic doses. Future investigations of brain mechanisms in

substance abuse might use such methodology to study the

physiological processes in the brain that underlie persistent states such

as craving and physiological dependence that are induced by drugs of

abuse.

REFERENCES

Bozarth, M.A., and Wise, R.A. Intracranial self-administration of

morphine into the ventral tegmental area in rats. Life Sci 28:551-

555, 1980.

Broekkamp, C.L., and Phillips, A.G. Facilitation of self-stimulation

behavior following intracerebral microinjections of opioids into the

ventral tegmental area. Pharmacol Biochem Behav 11:289-295,

1979.

Buchsbaum, MS.; Wu, J.; Haier, R.; Hazlett, E.; Ball, R.; Katz, M.;

Sokolski, K.; Lagunas-Solar, M.; and Langer, D. Positron emission

tomography assessment of effects of benzodiazepines on regional

glucose metabolic rate in patients with anxiety disorder. Life Sci

40:2393-2400, 1987.

Carr, G.D., and White, N.M. Conditioned place preference from intraaccumbens but not intracaudate amphetamine injections. Life Sci

33:2551-2557, 1983.

de Wit, H.; Metz, J.; and Cooper, M. Effects of ethanol, diazepam and

amphetamines on cerebral metabolic rate: PET studies using FDG.

In: Harris, L., ed. Problems of Drug Dependence 1990: Proceeding

of the 52nd Annual Scientific Meeting. National Institute on Drug

Abuse Research Monograph 105. DHHS Pub. No. (ADM)91-1753.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1991. pp.

61-67.

de Wit, H.; Metz, J.; Wagner, N.; and Cooper, M. Behavioral and

subjective effects of ethanol: Relationship to cerebral metabolism

using PET. Alcohol Clin Exp Res 14:482-489, 1990.

20
Engel, J., Jr.; Kuhl, D.E.; Phelps, M.E.; Rausch, R.; and Nuwer, M.

Local cerebral metabolism during partial seizures. Neurology

33:400-413, 1983.

Farkas, T.; Reivich, M.; Alavi, A.; Greenberg, J.H.; Fowler, J.S.;

MacGregor, R.R.; Christman, D.R.; and Wolf, A.P. The application

of [

18F]2-deoxy-2-fluoro-D-glucose and positron emission

tomography in the study of psychiatric conditions. In: Passonneau,

J.V.; Hawkins, R.A.; Lust, W.D.; and Welsh, F.A., eds. Cerebral

Metabolism and Neural Function. Baltimore: Waverly Press, Inc.,

1980. pp. 403-408.

Foster, N.L.; VanDerSpek, A.F.L.; Aldrich, M.S.; Berent, S.; Hichwa,

R.H.; Sackellares, J.C.; Gilman, S.; and Agranoff, B.W. The effect

of diazepam sedation on cerebral glucose metabolism in

Alzheimer’s disease as measured using positron emission

tomography. J Cereb Blood Flow Metab 7:415-420, 1987.

Friedland, R.P.; Budinger, T.F.; Ganz, E.; Yano, Y.; Mathis, C.A.;

Koss, B.; Ober, B.A.; Huesman, R.H.; and Derenzo, SE. Regional

cerebral metabolic alterations in dementia of the Alzheimer type:

Positron emission tomography with [

18Flfluorodeoxyglucose. J

Comput Assist Tomogr 7:590-598, 1983.

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

cocaine reinforcement. Science 221:773-775, 1983.

Grady, CL.; Haxby, J.V.; Schlageter, N.L.; Berg, G.; and Rapoport,

S.I. Stability of metabolic and neuropsychological asymmetries in

dementia of the Alzheimer type. Neurology 36:1390-1392, 1986.

Gur, R.E.; Resnick, S.M.; Alavi, A.; Gur, R.C.; Caroff, S.; Dann, R.;

Silver, F.L.; Saykin, A.J.; Chawluk, J.B.; Kushner, M.; and Reivich,

M. Regional brain function in schizophrenia: I. A positron emission

tomography study. Arch Gen Psychiatry 44:119-125, 1987.

Haertzen, C.A. An Overview of Addiction Research Center Inventory

Scales (ARCI): An Appendix and Manual of Scales. National

Institute on Drug Abuse. U.S. Department of Health, Education, and

Welfare. DHEW Pub. No. (ADM)74-92. Rockville, MD: Supt. of

Docs., U.S. Govt. Print. Off., 1974.

Herkenham, M.; Lynn, A.B.; Little, M.B.; Johnson, R.M.; Melvin,

L.S.; de Costa, B.N.; and Rice, K.C. Cannabinoid receptor

localization in brain. Proc Natl Acad Sci U S A 87:1932-1936,

1990.

21
Hubner, C.B., and Koob, G.F. Neurobiological substrates mediating the

reinforcing effects of psychomotor stimulant and opiate drugs. In:

London, E.D., ed. Imaging Drug Action in the Brain. Boca Raton,

FL: CRC Press, 1993. pp. 1-48.

Joyce, E.M.; Koob, G.F.; Strecker, R.; Iversen, S.D.; and Bloom, F.E.

The behavioral effects of enkephalin analogues injected into the

ventral tegmental area and globus pallidus. Brain Res 221:359-370,

1981.

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

drug dependence. Science 242:715-723, 1988.

Kuhl, D.E. Mapping local cerebral glucose metabolism in stroke,

degenerative diseases, and epilepsy. In: Magistretti, P.L., ed.

Functional Rodionuclide Imaging of the Brain. New York: Raven

Press, 1983. pp. 257-267.

Lear, J.L., and Ackermann, R.F. Why the deoxyglucose method has

proven so useful in cerebral activation studies: The unappreciated

prevalence of stimulation-induced glycolysis. J Cereb Blood Flow

Metab 9:911-913, 1989.

London, E.D.; Broussolle, E.P.M.; Links, J.M.; Wong, D.F.; Cascella,

N.G.; Dannals, R.F.; Sano, M.; Heming, R.; Snyder, F.R.; Rippetoe,

L.R.; Toung, T.J.K.; Jaffe, J.H.; and Wagner, H.N., Jr. Morphineinduced metabolic changes in human brain: Studies with positron

emission tomography and [fluorine 18]fluorodeoxyglucose. Arch

Gen Psychiatry 47:73-81, 1990a.

London, E.D.; Cascella, N.G.; Wong, D.F.; Phillips, R.L.; Dannals,

R.F.; Links, J.M.; Heming, R.; Grayson, R.; Jaffe, J.H.; and

Wagner, H.N., Jr. Cocaine-induced reduction of glucose utilization

in human brain. A study using positron emission tomography and

[fluorine 18]fluorodeoxyglucose. Arch Gen Psychiatry 47:567-574,

1990b.

Muntaner, C.; Cascella, N.G.; Kumor, K.M.; Nagoshi, C.; Heming, R.;

and Jaffe, J. Placebo responses to cocaine administration in humans:

Effects of prior administration and verbal instructions.

Psychopharmacology 99:282-286, 1989.

Phelps, M.E.; Huang, S.C.; Hoffman, E.J.; Selin, C.; Sokoloff, L.; and

Kuhl, D.E. Tomographic measurement of local cerebral glucose

metabolic rate in humans with (F-18)2-fluoro-2-deoxy-D-glucose:

Validation of method. Ann Neurol 6:371-388, 1979.

22
Reivich, M.; Alavi, A.; Wolf, A.; Greenberg, J.H.; Fowler, J.;

Christman, D.; MacGregor, R.; Jones, S.C.; London, J.; Shiue, C.;

and Yonekura, Y. Use of 2-deoxy-D[1-11C]glucose for the

determination of local cerebral glucose metabolism in humans:

Variation within and between subjects. J Cereb Blood Flow Metab

2:307-319, 1982.

Reivich, M.; Kuhl, D.; Wolf, A.; Greenberg, J.; Phelps, M.; Ido, T.;

Casella, V.; Fowler, J.; Hoffman, E.; Alavi, A.; Som, P.; and

Sokoloff, L. The [

18F]fluorodeoxyglucose method for the

measurement of local cerebral glucose utilization in man. Circ Res

44:127-137, 1979.

Reivich, M.; Rosen, A.D.; Kushner, M.; Gur, R.C.; and Alavi, A.

Local cerebral glucose consumption in man in health and disease.

In: Magistretti, P.L., ed. Functional Radionuclide Imaging of the

Brain. New York: Raven Press, 1983. pp. 311-318.

Sherer, M.A. Intravenous cocaine: Psychiatric effects, biological

mechanisms. Biol Psychiatry 24:865-885, 1988.

Siesjö, B.K. Brain Energy Metabolism. New York: Wiley, 1978.

Sokoloff, L. Circulation and energy metabolism of the brain. In:

Siegel, G.J.; Albers, R.W.; Katzman, R.; and Agranoff, B.W., eds.

Basic Neurochemistry. Vol. 2. Boston: Little, Brown and Co., 1972.

pp. 338-413.

Sokoloff, L.; Reivich, M.; Kennedy, C.; Des Rosiers, M.H.; Patlack,

C.S.; Pettigrew, K.D.; Sakurada, O.; and Shinohara, M. The

[

14C]deoxyglucose method for the measurement of local cerebral

glucose utilization: Theory, procedure, and normal values in the

conscious and anesthetized albino rat. J Neurochem 28:897-916,

1977.

Stapleton, J.M.; Henningfield, J.E.; Wong, D.F.; Phillips, R.L.; Gilson,

SF.; Grayson, R.F.; Dannals, R.F.; and London, E.D. Effects of

nicotine on cerebral metabolism and subjective responses in human

volunteers. Soc Neurosci Abstr 18:1074, 1992.

Theodore, W.H.; DiChiro, G.; Margolin, R.; Fishbein, D.; Porter, R.J.;

and Brooks, R.A. Barbiturates reduce human cerebral glucose

metabolism. Neurology 36:60-64, 1986.

Volkow, N.D.; Gillespie, H.; Mullani, N.; Tancredi, L.; Grant, C.;

Ivanovic, M.; and Hollister, L. Cerebellar metabolic activation by

delta-9-tetrahydrocannabinol in human brain: A study with positron

emission tomography and 18F-2-fluoro-2-deoxyglucose. Psychiatry

Res 40:69-78, 1991.

23
Volkow, N.D.; Hitzemann, R.; Wolf, A.P.; Logan, J.; Fowler, J.S.;

Christman, D.; Dewey, S.L.; Schlyer, D.; Burr, G.; Vitkun, S.; and

Hirschowitz, J. Acute effects of ethanol on regional brain glucose

metabolism and transport. Psychiatry Res 35:39-48, 1990.

Walsh, S.L.; Stapleton, J.M.; Preston, K.L.; Sullivan, J.T.; Dannals,

R.F.; Grayson, R.; Phillips, R.L.; Bigelow, G.E.; Jasinski, D.R.; and

London, E.D. Acute effects of buprenorphine on cerebral glucose

utilization in human substance abusers. Soc Neurosci Abstr 18:371,

1992.

Wolkin, A.; Angrist, B.; Wolf, A.; Brodie, J.; Wolkin, B.; Jaeger, J.;

Cancro, R.; and Rotrosen, J. Effects of amphetamine on local

cerebral metabolism in normal and schizophrenic subjects as

determined by positron emission tomography. Psychopharmacology

92:241-246, 1987.

AUTHOR

Edythe D. London, Ph.D.

Chief

Neuroimaging and Drug Action Section

Neuroscience Branch

Addiction Research Center

National Institute on Drug Abuse

National Institutes of Health

Baltimore, MD 21224

and

Associate Professor of Radiology

Department of Radiology

School of Medicine

The Johns Hopkins University

Baltimore, MD 2 1204

and

Adjunct Associate Professor

Department of Pharmacology and Experimental Therapeutics

School of Medicine

University of Maryland

Baltimore, MD 21201

24
Imaging Brain Function in

Animals To Understand Drugs of

Abuse and Potential

Pharmacotherapies

M. F. Piercey

INTRODUCTION

Most central nervous system (CNS) drugs, including those involved in

drug abuse, act by enhancing or depressing activities in specific brain

pathways. Some of these pathways are involved in acute reinforcing

properties such as euphoric or psychostimulant phenomena. Others are

involved with alterations in sensory perception or autonomic drug

effects. Thus, to understand functional effects of drugs of abuse, as

well as to design potential pharmacotherapies to treat drug abusers, it

is critical to define which neuronal structures are altered and how they

are altered (i.e., stimulated or inhibited).

Evaluating specific brain structures is challenging because of the large

number of such structures, many of which are extremely small.

Indeed, drugs frequently affect parts of the same structure differently.

To solve this problem, animal neuroscientists have turned to

autoradiography to map the functional neuroanatomy of the brain. The

development of positron emission tomography (PET) for imaging the

functional neuroanatomy of the human brain has at the same time

opened opportunities for evaluating how specific neuronal circuits

altered in animals are affected by the same drugs in humans.

IMAGING TECHNIQUES

Receptor binding autoradiography, usually carried out in vitro, is used

to determine the distribution of a specific receptor subtype among the

myriad brain structures (Kuhar 1985). When used in vivo, the

anatomy of receptor occupancy can be determined at doses known to

25
have biological effects. Receptor binding autoradiography uses

specific high-affinity radioligands to identify receptor sites. When

ligands interact with more than one receptor site, other drugs can

sometimes be used to mask the complicating receptors. Additionally,

drugs without apparent high affinity for any one site can be evaluated

for their ability to displace high-affinity ligands for well-documented

sites.

Nonspecific binding of radioactive ligands represents a potential

contaminating influence in receptor binding autoradiography.

Nonspecific binding can be isolated by removal of specific binding

with a nonlabeled ligand competing for the same receptor. Since

receptor-specific binding is saturable, whereas nonspecific binding is

not, cold ligand can be used to mask specific binding of the identical

radioactive ligand. Using adjacent brain sections and computer

imaging techniques, nonspecific binding is removed pixel by pixel to

yield the specific receptor binding image. Receptor binding identifies

where a drug can potentially act, but it does not actually reveal

whether any meaningful effect occurs.

2-Deoxyglucose (2-DG) autoradiography (Sokoloff et al. 1977) can be

used to determine drug effects, both stimulatory and inhibitory, on

brain energy metabolism, an indirect measure of neuronal activity.

This technique relies upon the fact that brain energy metabolism is

totally dependent upon blood glucose, since the brain does not store

glycogen. A steady state exists whereby glucose metabolism is equal

to glucose transported into the neurons. Tracer amounts of C

14 2-DG

enter brain neurons by competing with glucose for the transporter.

Once inside neurons, 2-DG is phosphorylated but not metabolized

further. 2-DG phosphate cannot diffuse outside cells, so radioactivity

accumulates at a rate proportional to glucose metabolism. If one

knows 2-DG and glucose concentrations, one needs only their

transporter affinities to obtain absolute amounts of glucose

metabolized. In practice, this is done by measuring timed samples of

arterial plasma glucose and radioactivity, measuring total radioactivity

in each brain region, and plugging the values into the equation

developed by Sokoloff and colleagues (1977) to quantitatively measure

glucose metabolism.

26
2-DG autoradiography measures not only a drug’s direct effects at sites

where its receptors are located but also its indirect effects occurring

downstream from these receptor sites. For example, phencyclidine

(PCP, “angel dust”) receptors are located throughout the circuit of

Papez, except in the mammillary body (MB). Nonetheless, PCP

intensely excites the MB by recruitment through activation of the

entire Papez circuit. Interestingly, indirect activation of the MB may

be important for PCP’s psychotropic effects (Piercey and Ray 1988a,

1988b; Piercey and Hoffmann 1989).

A major advantage of 2-DG imaging over receptor binding

autoradiography is that one does not need to know the mechanistic

effects or the receptor identity of the drug under study. In addition,

this technique does not require the development of a new high-affinity

ligand since C

14 2-DG is readily available. However, because brain

energy metabolism occurs in all tissues, including those not responding

to a drug, 2-DG imaging patterns are superimposed over an active

background, which tends to “blur” the image. In contrast, receptor

binding imaging negates nonspecific binding of its ligands. Thus,

receptor binding images are presented over essentially uniform inactive

black backgrounds. For this reason, receptor binding gives “sharper”

images that pinpoint active sites more clearly than 2-DG imaging.

In addition to receptor binding and 2-DG autoradiography, other brain

imaging techniques also are used. In particular, radiolabeled genetic

probes for in situ hybridization of messenger ribonucleic acid (mRNA)

(Uhl 1986) are being used with increasing frequency, particularly for

those receptors for which high-affinity receptor ligands are not

available. However, it must be remembered that receptor mRNA is a

measure of receptor synthesis, which quantitatively can be quite

different from the actual distribution of receptor protein. Antibody

staining of receptors occasionally has been used with some success

(McVittie et al. 1991). In addition to autoradiography and PET,

magnetic resonance imaging has some potential for evaluating drug

effects on some measures of brain function (Eastwood et al. 1985).

27
SITES OF DRUG ACTION

Receptor binding autoradiography, although dependent upon some

knowledge of the drug receptors activated by a drug, is probably the

most accurate method for identification of specific brain sites directly

affected by a drug. PCP, for example, binds to specific high-affinity

binding sites throughout the limbic system, especially the circuit of

Papez and the dopaminergic system (Gundlach et al. 1986a).

Interestingly, this high-affinity PCP receptor is codistributed with the

NMDA receptor subtype for the excitatory amino acid neurotransmitter, glutamate (Maragos et al. 1988). Electrophysiology

experiments confirm that PCP can block activation of NMDA ion

channels (Anis et al. 1983). The nonopioid sigma receptor has been

suggested as another major receptor involved in PCP’s

pharmacological effects. This receptor is distributed more caudally

than the high-affinity PCP receptor, with particularly high receptor

concentrations in the brain stem (Gundlach et al. 1986b).

Receptor binding autoradiography identifies where a drug binds to its

receptors but not necessarily where a drug induces its major biological

effects. By means of 2-DG autoradiography, PCP was found to induce

very large increases in energy metabolism in the forebrain, particularly

in the cerebral cortex, the dopaminergic system, and the limbic system

(especially the circuit of Papez). These excitatory effects were not

observed in the caudal brain or brain stem, although inhibitory effects

were seen occasionally (e.g., in the inferior colliculus) (Piercey and

Ray 1988a, 1988b; Piercey and Hoffmann 1989). Comparison of

binding and 2-DG data demonstrated a positive correlation between

PCP’s excitatory effects and those sites where the high-affinity PCP

receptor (i.e., NMDA channel) was located. In contrast, there was a

negative correlation between these sites for PCP excitation and those

where high concentrations of sigma receptors were found. It was

concluded that PCP’s stimulant effects are evoked through the highaffinity PCP site (Piercey and Ray 1988a, 1988b).

Knowing where a drug acts often can give great insight into how a

drug could produce some of its specific effects. For example,

excitation in dopaminergic areas could be responsible for many of the

psychotomimetic effects of PCP (Luisada 1978) since hyperactivity in

dopaminergic systems is thought to represent a major neurobiological

28
basis of schizophrenia (Matthysse and Sugarman 1978). However,

some of the more bizarre aspects of PCP intoxication could involve

excitatory effects in Papez’ circuit. Papez himself thought the circuit

to be involved in the integration of emotional sensation and expression,

with the MB in particular being critical for emotional outbursts (Papez

1937).

Where a drug acts is often dependent upon dose. High-dose effects

sometimes can be used to get an overall view of the pattern of actions

of a drug. Such patterns are particularly useful when using brain

imaging to identify what pharmacological class a drug belongs to (see

below, “Identification of Pharmacological Class”). Conversely, lower

doses may more specifically activate neuronal pathways responsible for

the dominant behavioral effects of the drug. For example, Porrino and

colleagues (1988) found that, at an intravenous (IV) dose of 0.5 mg/kg,

cocaine excited only the medial prefrontal cortex and nucleus

accumbens. Higher cocaine doses led to broader stimulation of

dopamine-related structures, especially those in the extrapyramidal

regions, accompanied by increases in locomotor activity. Porrino and

colleagues (1988) concluded that the nucleus accumbens and prefrontal

cortex may be involved in the reinforcing effects of cocaine and that

the extrapyramidal areas are more involved in the general stimulant

effects of the drug. Many of these conclusions are consistent with the

dopaminergic theory of drug reinforcement (Wise and Rompre 1989).

Other factors affecting sites of drug action include route and

sometimes time of injection relative to injection of the 2-DG. For

example, using intraperitoneal injections, London and colleagues

(1986) found that cocaine did not excite the nucleus accumbens at all.

Hoffmann and colleagues (1991) also did not observe excitation of the

nucleus accumbens or many of the other major dopaminergic areas

when cocaine was injected intravenously 10 minutes prior to the

injection of the 2-DG label. By comparison, Porrino and colleagues

(1988) injected cocaine only 2 minutes prior to 2-DG. Subsequent

(unpublished) experiments in the author’s laboratory in which cocaine

was injected 2 minutes prior to 2-DG resulted in a pattern of

stimulation extremely similar to that reported by Porrino and

colleagues (1988). Such sensitivity to timing seems likely to be

related to the short duration of action for cocaine, particularly

regarding its reinforcing effects (Gawin 1991).

29
A rarely discussed additional issue is the fact that, while most imaging

scientists would probably agree that a certain section of the brain can

be identified as a given brain nucleus, the specific component of that

nucleus used in the evaluation of the image probably varies greatly

among laboratories. This represents a problem since drug receptors

and responses to drugs often are not uniform within a brain area. For

example, even when cocaine is injected 2 minutes prior to the 2-DG

injection, this author’s laboratory did not see excitatory effects in the

nucleus accumbens measured at two different levels. However, neither

of these levels were as anterior as the area where Porrino and

colleagues (1988) observed the high nucleus accumbens sensitivity

(Porrino, personal communication, 1992). Thus, it probably would be

useful for authors to publish the atlases to identify precisely where

they analyze each brain area. In some cases, standardization might be

useful. However, this needs to be weighed against the need to explore

more brain regions.

IDENTIFICATION OF PHARMACOLOGICAL CLASS

Drugs acting through common mechanisms can be expected to affect

similar or identical brain areas. MK-801, like PCP, is an NMDA

channel blocking agent (Woodruff et al. 1987). Like PCP, MK-801

excites dopaminergic structures, Papez’ circuit, and other limbic and

cortical structures (Piercey et al. 1988). However, other psychostimulants such as amphetamine and cocaine excite dopaminergic

structures but have relatively fewer effects than MK-801 and PCP in

limbic areas (Porrino et al. 1988; Piercey and Hoffmann 1989). It is

interesting that amphetamine and cocaine, both of which increase

synaptic dopamine (albeit by different receptor mechanisms), in general

excite similar structures, according to 2-DG autoradiography. It also is

interesting that all of the psychostimulants excite dopaminergic

structures. This observation is consistent with the hypothesis that a

dopamine reward system may be responsible for the reinforcing effects

of these and possibly other agents (Wise and Rompre 1989). Also, as

noted previously, the fact that all of these drugs stimulate dopaminergic structures may account for some of the psychotomimetic

effects common to this group of compounds.

30
Identification of pharmacological class is not limited to psychostimulants; it applies to other classes of drugs as well. For example,

the hypnotic zolpidem acts through benzodiazepine receptors, even

though it is chemically not a benzodiazepine (Langer et al. 1988). In

2-DG autoradiography experiments, it was demonstrated that zolpidem

and triazolam (a benzodiazepine hypnotic) depressed metabolism in

precisely the same regions of the brain (Piercey et al. 1991). Indeed, a

strong positive linear correlation was demonstrated to exist between the

percent of depression exerted by both drugs over all regions of the

brain, whether or not the level of depression was statistically

significant. Thus, the distribution of pharmacological effects is linked

inextricably to what receptor is affected and not to the precise

chemical nature of the ligand.

DRUG INTERACTIONS

The 2-DG autoradiography method allows evaluation of the

interactions between centrally acting drugs, Such interactions can

occur between drugs acting at the same receptor and those acting at

different receptors. Receptor binding autoradiography can be used to

establish whether interacting drugs interact at the same receptor since,

when this is true, both will displace the same radioactive ligand.

Numerous imaging studies have demonstrated interactions between

drugs acting at the same receptors. McCulloch and colleagues (1982)

showed that the dopamine receptor antagonist, haloperidol, completely

eliminated the effects of the dopamine receptor agonist, apomorphine,

in all brain regions where apomorphine affected brain energy

metabolism. Similarly, flumazenil, a benzodiazepine receptor

antagonist, eliminates the effects of the benzodiazepine agonist,

diazepam (Ableitner et al. 1985). The benzodiazepine receptor partial

agonist, U-78875, also antagonized the effects of the benzodiazepine

agonist, alprazolam (Piercey et al. 1990). However, in this case,

antagonism was observed only in those brain areas where the drug had

low efficacy of its own. Areas of high efficacy presumably have large

receptor reserves, allowing full agonist effects even with low intrinsic

activity.

31
Meaningful interactions also occur between drugs acting on similar

systems but through different receptors. Piercey and Ray (1988b )

demonstrated that haloperidol altered the effects of PCP, particularly in

areas where dopamine is thought to be a major neurotransmitter.

Hoffmann and colleagues (1991) showed that cocaine’s stimulant

effects could be altered by treatment with a stimulant dopamine

antagonist. In both these studies, the antagonist interacted directly

with dopamine receptors, but the agonists’ actions at these sites were

mediated indirectly through dopamine released from endogenous

stores.

Interactions also can be demonstrated between agonists acting at

different receptors. Ray and colleagues (1992), for example, found

that the dopamine D1 receptor agonist, SKF 38393, and the dopamine

D2 receptor agonist, quinpirole, have different actions and interactions,

depending upon brain site. In some areas, there are clear synergistic

interactions, whereas in others there are antagonistic interactions. Yet,

in other brain areas, one of the drugs produced significant alterations,

whereas the other did not affect metabolism either by itself or interact

with the active drug.

ANIMAL/HUMAN CORRELATIONS

One of the more exciting opportunities for brain imaging is the

possibility of comparing effects observed in animals to those

observable in humans. It has been demonstrated, for example, that

there is a positive correlation between rodent and human brains when

comparing energy metabolism among different brain regions in the two

species (Blinn et al. 1991). When comparing drug effects, it is

interesting that while the distribution of effects may, in general, be

similar between animals and man, the directions of these pharmacological effects are sometimes opposite. For example, whereas PCP,

amphetamine, and cocaine mostly increase energy metabolism in

rodents, they produce predominantly inhibitory effects in humans

(Wolkin et al. 1987; London et al. 1990).

32
IMAGING OF POTENTIAL PHARMACOTHERAPIES FOR

DRUG ADDICTION

It is hoped that through greater understanding of how drugs of abuse

act, it will be possible to design specific pharmacotherapies to treat

drug abuse as a clinical disorder.

Knowledge that stimulation of dopaminergic structures is a common

finding for euphoric psychostimulants suggests that dopamine

antagonists might be therapeutic in this condition. Unfortunately, in

addict populations, dopamine antagonists tend to promote the

anhedonias (inability to perceive pleasure) associated with drug craving

(Gawin 1991). Thus, some alternative approach is probably necessary.

Whatever the approach, imaging experiments can, along with a full

battery of pharmacological evaluations, be helpful in appreciating the

proposed mechanism of any pharmacotherapy being considered for

human testing. In addition, by comparing autoradiographic imaging in

animals to PET scans in humans, imaging experiments almost uniquely

can be used to determine if the critical neurobiological effects observed

in animals actually occur in man as well.

One possible mechanism for treating cocaine and other psychostimulant abuse is the use of a dopamine partial agonist. To date, no

imaging experiments involving dopamine partial agonists have been

published. However, 2-DG autoradiography potentially can be used to

demonstrate partial agonist and antagonist pharmacology, as has been

done with the benzodiazepine partial agonist U-78875 (Piercey et al.

1990). It is hoped that benzodiazepine partial agonists will lack the

drug dependence associated with full agonists.

Another drug class with potential for use in treating cocaine abuse is

the stimulant dopamine antagonists such as (±)-AJ 76. The mechanism

for (±)-AJ 76’s stimulant effects is not clearly delineated, but the

stimulant effects may be due to a preferential blockade of dopamine

autoreceptors (Svensson et al. 1986). Although it antagonizes

amphetamine and cocaine locomotor activity, (±)-AJ 76 weakly

increases locomotor activity like a weak psychostimulant (Svensson et

al. 1986; Piercey et al. 1992). (±)-AJ 76 partially generalizes to

cocaine’s subjective cue (Callahan et al. 1992) but antagonizes cocaine

self-administration (Richardson et al., in press). In 2-DG experiments,

33
(±)-AJ 76 by itself sometimes weakly excites dopaminergic structures,

but it antagonizes the excitatory effects of cocaine and amphetamine

(Hoffmann et al. 1988, 1991; table 1). Because it can weakly stimulate

dopaminergic structures, it is possible that (±)-AJ 76 will not produce

anhedonias like other dopamine antagonists (Gawin 1991).

TABLE 1. Antagonism of cocaine stimulation by (+)-AJ 761

Control Cocaine2

Cocaine +

(+)-AJ 763

Subthalamus 100 ± 10 138 ± 6*103 ± 2+

AV Thalamus 100 ± 12 132 ± 3*97 ± 7+

VL Thalamus 100 ± 9 138 ± 3*107 ± 6+

Lateral Septum 100 ± 6 147 ± 3*108 ± 8+

PAG 100 ± 5 138 ± 8*136 ± 8

+

Dorsal Horn 100 ± 12 125 ± 5*112±4 +

1Glucose utilization, μmole/100 g brain/min expressed as percent

control.

2Cocaine, 5 mg/kg IV injection, 10 min before 2-DG injection plus

vehicle IV injection at 5 min before 2-DG injection.

3Cocaine, 5 mg/kg IV injection, 10 min before 2-DG injection plus

(+)-AJ 76, 15 mg/kg IV injection, 5 min before 2-DG injection.

*

 p < 0.05 versus control, ANOVA.

+

 p < 0.05 versus cocaine alone, ANOVA.

Piercey and Ray (1988b) noted that haloperidol either significantly

reduced or destroyed PCP’s effects in dopaminergic areas. However,

the importance of this finding may be more related to treatment of

behavioral toxicity rather than the drug abuse itself.

Drug development begins with chemical synthesis and pharmacological

testing. Brain imaging experiments in animals represent only one

component of the pharmacological evaluation of a drug. Only one

compound of many thousands synthesized is generally found to have a

sufficiently delineated mechanism of action combined with good

pharmacokinetic and toxicological profiles to justify evaluation in

34
humans. New chemical entities tested in humans do not always

produce the desired effect. It is not always clear whether this is due to

differences in drug pharmacokinetics and distribution, to some altered

pharmacological properties in humans, or to a general failure of the

proposed mechanism for treatment. The complexity of the nervous

system has made it difficult for both animal and clinical scientists to

compare their results to answer these questions. However, with the

parallel development of brain imaging techniques in both animals and

humans, it is now possible to compare a drug’s effects across species.

It seems likely that brain imaging techniques will, over time, play a

larger role in the development of drugs to treat psychiatric conditions

such as drug abuse.

SUMMARY

Brain imaging studies can be used to enhance current understanding of

how CNS drugs act. Because the brain is in reality a collection of

independent, albeit interconnected, neural organs, identification of sites

of brain action are critical to an appraisal of how each drug exerts its

different behavioral and physiological effects. For new drugs not yet

tested in humans, identification of which neuronal structures are

affected by a drug can give clues as to what to expect from the drug if

it is eventually exposed to clinical testing.

Receptor binding autoradiography, more than any other technique,

pinpoints the distribution of those sites at which a given drug acts.

When combined with 2-DG autoradiography, the intensity and

direction of the action at each of these sites, as well as the impact of

these actions on additional sites, can be appreciated. In addition to

identification of sites of drug action, brain imaging studies can be used

to identify classes of drug action, interactions between drugs, and,

perhaps more than any other technique, enhance the appreciation of the

similarities and differences between the effects in animals and those in

humans.

When applied to drugs of abuse, brain imaging studies can be used to

help identify key physiological events underlying drug reinforcement

as well as those important for various physiological effects of each

drug. Armed with such information, brain imaging techniques can

35
play a unique role as one component of an effort to discover

pharmacotherapies for drug abuse.

REFERENCES

Ableitner, A.; Wuster, M.; and Herz, A. Specific changes in local

cerebral glucose utilization in the rat brain induced by acute and

chronic diazepam. Brain Res 359:49-56, 1985.

Anis, N.A.; Berry, S.C.; Burton, N.R.; and Lodge, D. The dissociative

anesthetics, ketamine and phencyclidine, selectively reduce

excitation of central mammalian neurones by N-methyl aspartate.

Br J Pharmacol 79:1565-575, 1983.

Blinn, J.; Ray, C.A.; Chase, T.C.; and Piercey, M.F. Regional cerebral

glucose metabolism compared in rodents and humans. Brain Res

568:215-222, 1991.

Callahan, P.M.; Piercey, M.F.; and Cunningham, K.A. Effects of the

putative dopamine autoreceptor antagonists AJ 76 and UH 232 on

the discriminative stimulus properties of cocaine. Psychopharmacology 107:73-77, 1992.

Eastwood, L.M.; Hutchinson, J.M.S.; and Besson, J.A.O. Nuclear

magnetic resonance (NMR): I. Imaging biochemical change. Br J

Psychiatry 146:26-35, 1985.

Gawin, F.H. Cocaine addiction: Psychology and neurophysiology.

Science 251:1580-1586, 1991.

Gundlach, A.L.; Largent, B.L.; and Snyder, S.H. Phencyclidine (PCP)

receptors: Autoradiographic localization in brain with the selective

ligand, 3H-TCP. Brain Res 386:266-279, 1986a.

Gundlach, A.L.; Largent, B.L.; and Snyder, S.H. Autoradiographic

localization of sigma receptor binding sites in guinea pig and rat

central nervous system with (=)-3-H-3-(3-hydroxyphenyl)-N-(lpropyl)piperidine. J Neurosci 6:1757- 1770, 1986b.

Hoffmann, W.E.; Lum, J.T.; and Piercey, M.F. Electrophysiological

and metabolic effects of (+)-AJ 76, a selective antagonist of

dopamine autoreceptors. Neurosci Abstr 14:935, 1988.

Hoffmann, W.E.; Svensson, K.; Callahan, P.; Cunningham, K.; Mead,

B.; Lum, J.T.; and Piercey, M.F. Interactions between cocaine and

the presumed preferential dopamine autoreceptor antagonists, (+)-AJ

76 and (+)-UH 232. Neurosci Abstr 17:681, 1991.

36
Kuhar, M.J. Receptor localization with the microscope. In: Yamamuru,

H.I.; Enna, S.J.; and Kuhar, M.J., eds. Neurotransmitter Receptor

Binding. 2nd ed. New York: Raven Press, 1985. pp. 153-175.

Langer, S.Z.; Arbilla, S.; Scatton, B.; Niddam, R.; and Dubois, A.

Receptors involved in the mechanism of action of zolpidem. In:

Savvanet, J.P.; Langer, S.Z.; and Morselli, P.L., eds.

Imidazopyridines in Sleep Disorders. New York: Raven Press, 1988.

pp. 55-70.

London, E.D.; Cascella, N.G.; Wong, D.F.; Phillips, R.L.; Dannals,

R.F.; Links, J.M.; Herning, R.; Grayson, R.; Jaffe, J.H.; and

Wagner, H.N., Jr. Cocaine-induced reduction of glucose utilization

in human brain: A study using positron emission tomography and

fluorine18-fluorodeoxyglucose. Arch Gen Psychiatry 47:567-574,

1990.

London, E.D.; Wilkerson, G.; Goldberg, S.R.; and Risner, M.E. Effects

of l-cocaine on local cerebral glucose utilization in the rat. Neurosci

Lett 68:173-78, 1986.

Luisada, P.F. The phencyclidine psychosis: Phenomenology and

treatment. In: Petersen, R.C., and Stillman, R.C., eds. Phencyclidine

(PCP) Abuse: An Appraisal. National Institute on Drug Abuse

Research Monograph 21. DHHS Pub. No. (ADM)017-024-00785-4.

Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1978. pp.

19-43.

Maragos, W.F.; Penney, J.B.; and Young, A.B. Anatomic correlation

of NMDA and 3H-TCP-labeled receptors in rat brain. J Neurosci

8:493-501, 1988.

Matthysse, S., and Sugarman, J. Neurotransmitter theories of

schizophrenia. In: Iversen, L.L.; Iversen, S.D.; and Snyder, S.H.,

eds. Neuroleptics and Schizophrenia. New York: Plenum Press,

1978. pp. 221-242.

McCulloch, J.; Savaiki, H.E.; and Sokoloff, L. Distribution of effects

of haloperidol in energy metabolism in the rat brain. Brain Res

243:81-90, 1982.

McVittie, L.D.; Ariano, M.A.; and Sibley, D.R. Characterization of

anti-peptide antibodies for the localization of D2 dopamine

receptors in rat striatum. Proc Natl Acad Sci U S A 88:1441-1445,

1991.

Papez, J.W. A proposed mechanism of emotion. Arch Neurol

Psychiatry 38:724-743, 1937.

37
Piercey, M.F., and Hoffmann, W.E. Cerebral pathways activated by

PCP-like compounds: Relevance to neurotransmitters and their

receptors. In: Harris, L.S., ed. Problems of Drug Dependence.

National Institute on Drug Abuse Research Monograph 95. DHHS

Pub. No. (ADM)90-1663. Washington, DC: Supt. of Docs., U.S.

Govt. Print. Off., 1989. pp. 264-269.

Piercey, M.F.; Hoffmann, W.E.; and Cooper, M. The benzodiazepine

hypnotics triazolam and zolpidem have identical metabolic effects

throughout the brain: Implications for benzodiazepine receptor

subtypes. Brain Res 554:224-252, 1991.

Piercey, M.F.; Kasckovsky, P.; and Hoffmann, W.E. MK-801A, a

potential antistroke agent, stimulates PCP receptors in limbic and

cortical areas. Psychopharmacology 96:561-562, 1988.

Piercey, M.F.; Lum, J.T.; Hoffmann, W.E.; Carlsson, A.; Ljung, E.;

and Svensson, K. Antagonism of cocaine’s pharmacological effects

by the stimulant dopaminergic antagonists, (+)-AJ 76 and (+)-UH

232. Brain Res 588:217-222, 1992.

Piercey, M.F.; Nielsen, E.O.; Honore, T.; and Hoffmann, W.E.

U-78875 is a benzodiazepine agonist in some brain sites and an

antagonist in others: A 2-DG autoradiography study. Pharmacologist

32: 136, 1990.

Piercey, M.F., and Ray, C.A. Dramatic stimulation of limbic and

cortical structures via high-affinity PCP receptors. Life Sci 43:379-

385, 1988a.

Piercey, M.F., and Ray, CA. Evidence from 2-DG autoradiography

that phencyclidine’s functional effects are mediated by specific PCP

rather than sigma receptors. In: Domino, E.F., and Kamenka, J.-M.,

eds. Sigma Opioid Phencyclidine-like Compounds as Molecular

Probes in Biology. Ann Arbor: NPP Press, 19886. pp. 285-295.

Porrino, L.J.; Domer, F.R.; Crane, A.M.; and Sokoloff, L. Selective

alterations in cerebral metabolism within the mesocorticolimbic

dopaminergic system produced by acute cocaine administration in

rats. Neuropsychopharmacology 1:109-117, 1988.

Ray, C.A.; Strutt, M.; Camacho-Ochoa, M.; and Piercey, M.F.

Synergistic and antagonistic interactions of dopamine D1 and D2

agonists: A 2-deoxyglucose autoradiography study. Neurosci Abstr

18:995, 1992.

38
Richardson, N.R.; Piercey, M.F.; Svensson, K.; Collins, R.J.; Myers,

J.E.; and Roberts, D.C.S. An assessment of the reinforcing and anticocaine effects of the preferential dopamine autoreceptor antagonist,

(+)-AJ 76. Brain Res, in press.

Sokoloff, L.; Reivich, M.; Kennedy, C.; Des Rosiers, M.H.; Patlak,

C.S.; Pettigrew, K.D.; Sakurada, O.; and Shinohara, M. The

[

14C]deoxyglucose method for the measurement of local cerebra1

glucose utilization: Theory, procedure, and normal values in the

conscious and anesthetized albino rat. J Neurochem 28:897-916,

1977.

Svensson, K.; Johansson, A.M.; Magnusson, T.; and Carlsson, A.

(+)-AJ 76 and (+)-UH 232: Central stimulants acting as preferential

dopamine autoreceptor antagonists. Naunyn Schmiedeberg Arch

Pharmacol 334:234-245, 1986.

Uhl, G.R. In Situ Hybridization in Brain. New York: Plenum Press,

1986.

Wise, R.A., and Rompre, P.-P. Brain dopamine and reward. Annu Rev

Psychol 40:191-225, 1989.

Wolkin, A.; Angrist, B.; Wolf, A.; Brodie, J.; Wolkin, B.; Jaeger, J.;

Cancro, R.; and Rotrosen, J. Effects of amphetamine on local

cerebral metabolism in normal and schizophrenic subjects as

determined by positron emission tomography. Psychopharmacology

92:241-246, 1987.

Woodruff, G.N.; Foster, A.C.; Gill, R.; Kemp, J.A.; Wong, E.H.F.; and

Iversen, L.L. The interaction between MK-801 and receptors for

N-methyl-D-aspartate: Functional consequences. Neuropharmacology 26:903-909, 1987.

ACKNOWLEDGMENTS

Cathy Ray and Bill Hoffmann played major roles in the imaging

experiments carried out in The Upjohn Company laboratory. Brenda

Mead was instrumental in the (+)-AJ 76 experiments with cocaine.

39
AUTHOR

M.F. Piercey

CNS Research 725 1-209-4

The Upjohn Company

301 Henrietta Street

Kalamazoo, MI 49001

40
Brain-Imaging Studies of the

Combined Use of Cocaine and

Alcohol and of the

Pharmacokinetics of

Cocaethylene

Nora D. Volkow and Joanna S. Fowler

INTRODUCTION

The simultaneous use of cocaine and alcohol is one of the most

frequent patterns of combined drug use (Grant and Harford 1990;

Sands and Ciraulo 1992). Alcohol is combined with cocaine to reduce

the dysphoria experienced after a cocaine binge (crash) and also to

prolong the euphoria. Recent epidemiological studies have shown that

combined use of cocaine and alcohol may be synergistically toxic

(Boag and Havard 1985). It has been estimated that the combined use

of cocaine and alcohol results in an eighteenfold increase in the risk of

sudden death (Rose et al. 1990). Although the factors mediating the

increased risk associated with the combined use of cocaine and alcohol

are not fully understood, recent studies have suggested that cocaethylene, a metabolite of cocaine formed in the presence of alcohol,

may contribute to enhanced morbidity and mortality (Hear-n et al.

1991a). Preclinical neurochemical and behavioral data on cocaethylene

have shown that it has pharmacological properties similar to those of

cocaine (Hearn et al. 1991a). Cocaethylene, like cocaine, binds to the

dopamine transporter, inhibiting dopamine uptake and indirectly

increasing the presynaptic concentration of dopamine (Jatlow et al.

1991; Woodward et al. l991), and it has been shown to be more toxic

than cocaine (Hearn et al. 1991b; Katz et al. 1992).

Although cocaine and cocaethylene are similar in structure, differences

in physical properties such as lipophilicity, plasma protein binding, and

the ability to serve as substrates for cholinesterases in plasma might be

expected to result in a different uptake and residence time in target

41
organs. Differences in availability could result in higher drug-tissue

concentrations and longer drug duration, which could accentuate and

prolong the effects of cocaine.

However, other factors also could explain the enhanced behavioral and

toxic properties of cocaine when combined with alcohol. The

enhanced behavioral effects could relate to the combination of two

drugs that enhance dopamine concentrations in the nucleus accumbens

(Ritz et al. 1987; Seeman and Lee 1974; Di Chiara and Imperato

1988). Also, cocaine’s metabolism and pharmacokinetics could be

changed in the presence of ethanol, leading to higher and longer

lasting concentrations of cocaine or active metabolites. Such an effect

of alcohol in drug pharmacokinetics has been demonstrated for

amphetamine where coadministration with alcohol prolongs the blood

and brain levels of amphetamine (Jonsson and Lewander 1973) and for

cocaine where coadministration with alcohol results in higher blood

levels of cocaine (Perez-Reyes and Jeffcoat 1992). The direct actions

of cocaine and alcohol on target organs such as the heart also could

account for the synergistic toxicity (Masur et al. 1989).

Positron emission tomography (PET) is a nuclear-imaging technique

that allows the direct measurement of drug distribution and

pharmacokinetics in the living human brain and body (Fowler et al.

1990). Investigations on the uptake and pharmacokinetics of

[

11C]cocaine in the living human and nonhuman primate brain using

PET have been done (Fowler et al. 1989, 1992a). In the current study,

the authors used [

11C]-labeled cocaethylene to investigate the

pharmacokinetics of cocaethylene in the baboon brain, heart, and

plasma; the findings were compared with those for [

11C]cocaine

(Fowler et al. 1992b). To assess whether alcohol affects the

pharmacokinetics of cocaine, the authors have performed PET studies

with [

11C]cocaine in human subjects scanned without alcohol and

during alcohol intoxication.

42
MATERIALS AND METODS

Baboon Studies

The baboon studies were designed to investigate the properties of

[

11C]cocaethylene as they compare with those of [11C]cocaine.

Radiotracers synthesis occurred as follows: [N-11C-methyl]cocaine

was prepared by the methylation of norcocaine with [

11C]methyliodide

(Langstrom and Lundqvist 1976; Fowler et al. 1989).

[N-11C-methyl]cocaethylene was prepared by the methylation of

norcocaethylene (Fowler et al. 1992a). Cocaine, norcocaine, and

cocaethylene were supplied by the National Institute on Drug Abuse

(NIDA).

Five adult female baboons (Papio Anubis) were anesthetized,

catheterized, and prepared for the PET study (Dewey et al. 1990).

Scanning was carried out on a CTI 931-08 Computer Technologies,

Inc., tomograph (spatial resolution of 6x6x6.5 mm full width half

maximum). Each baboon was scanned twice, 2 hours apart, to

compare [

11C]cocaine with [11C]cocaethylene. Seven paired studies

were done, five with [

11C]cocaine preceding [11C]cocaethylene and two

scans with [

11C]cocaethylene preceding [11C]cocaine. For the studies,

[

11C]cocaethylene (5 to 8 mCi:3 to 10 μg) or cocaine (5 to 8 mCi:4 to

6 μg) were injected sequentially. Dynamic scans were started

immediately after injection of the tracer and were continued for 50

minutes.

Plasma analyses were done on arterial samples to measure total

radioactivity and unchanged labeled tracer. Also, procedures regarding

blood sampling and analysis were done (Fowler et al. 1992a). The

concentration of the unchanged labeled tracer in plasma was used for

the data analysis. The amount of [

11C]cocaine and [11C]cocaethylene

bound to plasma proteins also was determined for these studies

(Fowler et al. 1992a).

For the analysis of the PET images, regions of interest were drawn

directly on the images. An approximate value for whole brain uptake

was obtained by averaging the activity in the five central slices.

Regions for the striatum, cerebellum, and thalamus were obtained in

the various slices where the regions were identified (Dewey et al.

43
1990). The activity in these regions of interest was used to obtain the

time-activity curve for regional tissue concentration. The time-activity

curves for tissue concentration and for unchanged tracer in plasma

were used to calculate the transport constant between plasma and tissue

(K1) and to obtain the distribution volume for the various regions using

a graphical analysis technique (Logan et al. 1990). The distribution

volume is a measure of Bmax/Kd.

Human Studies

Seven normal male volunteers (ages 22 to 26 years) were studied with

[

11C]cocaine (Fowler et al. 1989, 1992b). Subjects were screened for

absence of medical, psychiatric, and neurological disease. Subjects

with a past or present history of alcohol or other drug abuse in

themselves or in first-degree relatives were excluded from the

investigation. Urine samples were taken prior to the PET scan to

ensure lack of psychoactive substance consumption. Each subject

received two scans with [

11C]cocaine. The first scan was done with no

pharmacological intervention, and the second scan was done 2 hours

later during alcohol intoxication. Alcohol was given orally as 1 g/kg

of ethanol during a 45-minute period, and scans were done 40 minutes

later. For three subjects, scans were obtained for their brains; for four

subjects, scans were obtained for their hearts.

Blood samples from a radial artery were obtained to quantitate total

radioactivity, unchanged [

11C]cocaine, and blood alcohol concentration.

A plasma sample drawn 10 minutes after injection of [

11C]cocaine was

subjected to high-pressure liquid chromatography analysis to quantitate

for labeled cocaethylene (Fowler et al. 1992b).

Regions of interest were obtained directly on the emission scans.

Regions for striatum, cerebellum, thalamus, and frontal cortex were

drawn on the various planes where they were identified, and a

“composite” value was obtained using their weighted average (Fowler

et al. 1989). Regions in the heart were obtained for the left ventricle,

septum, and left atrium (Volkow et al. 1992). The tissue and plasma

concentrations of tracer were used to calculate the distribution volume

as described for the baboon studies.

44
RESULTS

Baboon Studies

Figures 1 and 2 show brain images for [

11C]cocaine and for

[

11C]cocaethylene taken 3 to 6 minutes and 24 to 54 minutes after

tracer injection. The pattern of distribution for both tracers in the

brain was similar during the early scanning period (3 to 6 minutes).

Maximal uptake for both tracers was obtained in the basal ganglia.

Kinetic analysis of [

11C]cocaine and [11C]cocaethylene in the brain

revealed that peak uptake occurred for both tracers between 3 and 5

minutes for striatum and between 2 and 5 minutes for cortex, thalamus,

and cerebellum. The peak uptake for these two tracers (expressed as

percent dose/cm3 of tissue) was not significantly different for any of

the brain regions analyzed. In contrast to the similar pattern of uptake

for these two tracers, significant differences were observed for their

clearance; [

11C]cocaethylene cleared more slowly than [11C]cocaine

from the brain.

Figure 3 shows the time-activity curve for [

11C]cocaine and

[

11C]cocaethylene in the whole brain (global), basal ganglia, and

cerebellum and illustrates the slower clearance for [

11C]cocaethylene

(CE) relative to that of [

11C]cocaine (C). The averages of the

clearance half-time are shown in table 1.

Analyses of the rate of unchanged tracer in plasma revealed that there

was a significantly higher percentage of unchanged tracer for

[

11C]cocaethylene than for [11C]cocaine. Average percentages of

unchanged tracer in plasma at 10 minutes were C = 32±9.4 percent

and CE = 39.7±8.9 percent; at 30 minutes they were C=16.6±6.8

percent and CE = 22.3±4.1 percent.

The values for the distribution volume in basal ganglia were not

significantly different for [

11C]cocaine (7.4±1.9 mL/g) and

[

11C]cocaethylene (7.7±1.9 mL/g). However, they were significantly

larger for [

11C]cocaethylene than for [11C]cocaine in the whole brain,

basal ganglia, thalamus, and cerebellum. The distribution volume

values are shown in table 2.

45
IFIGURE 1. Brain images for [

11C]cocaine and [11C]cocaethylene obtained 3 to 6 minutes postinjection

(PI). Brain images correspond to sequential planes at the level where the basal ganglia are

located. Darker areas denote the brain regions with the highest uptake. For both tracers,

highest uptake occurs in basal ganglia and, secondly, in thalamus.
FIGURE 2. Brain images for [11C]cocaine and [11C]cocaethylene obtained 24 to 54 minutes PI. Brain

images correspond to the same planes as for figure 1. Notice the clearance of [

11C]cocaine

in basal ganglia, whereas there is still a high concentration of [

11C]cocaethylene.
FIGURE 3. Time-activity curves for [

11C]cocaine and

[

11C]cocaethylene (X) in whole brain (global), basal

ganglia, and cerebellum. Notice the slower clearance of

[

11C]cocaethylene than of [11C]cocaine.

48
TABLE 1. Averages of the clearance half-time in min

CLEARANCE

REGION HALF-TIME

AVERAGES

Whole Brain C = 14.3 ± 4.3

CE = 9.13 ± 4.7

Striatum C = 17.2 ± 4.5

CE = 22.8 ± 3.2

Thalamus C = 10.7 ± 3

CE = 315.4 ± 4

Cerebellum C = 7.9 ± 1.9

CE = 12.0 ± 2.4

KEY: C = cocaine; CE = cocaethylene

Incubation of samples of labeled cocaine and labeled cocaethylene with

baboon plasma for 40 minutes at room temperature, followed by thinlayer chromatography analysis, showed that cocaine is metabolized at a

faster rate than cocaethylene. After 40 minutes, hydrolysis of cocaine

to ecgonine methyl ester is 45-percent complete, whereas hydrolysis of

cocaethylene to ecgonine ethyl ester is 15-percent complete. The

plasma-free fraction for cocaine was 33.5 percent, and for cocaethylene

it was 30.5 percent (Fowler et al. 1992b).

Human Studies

Alcohol intoxication did not significantly affect [

11C]cocaine

pharmacokinetics as reflected by the rate of uptake and clearance of

cocaine from the brain and heart. Also, alcohol intoxication did not

affect the pattern of distribution of [

11C]cocaine nor its uptake in the

brain or heart, nor did it affect rate of metabolism of [

11C]cocaine

in plasma.

49
TABLE 2. Values for the distribution volume in mL/g

REGION DISTRIBUTION

VOLUME VALUES

Whole Brain C = 4.9 ± 1

CE = 5.5 ± 1.3

Basal Ganglia C = 7.4 ± 1.9

CE = 7.7 ± 1.9

Thalamus C = 5.8 ± 1.3

CE = 6.7 ± 1.7

Cerebellum C = 4.6 ± 1.1

CE = 5.4 ± 1.4

KEY: C = cocaine; CE = cocaethylene

NOTE: p < 0.01 paired t-test, two-tail

Distribution volumes before and during alcohol intoxication for the

brain regions were not significantly different from each other and were

within the variations previously reported for studies done on the same

subject without intervention (Logan et al. 1990). The 10-minute

plasma sample did not detect the presence of labeled cocaethylene

(Fowler et al. 1992b).

DISCUSSION

The similar rate of uptake of [

11C]cocaethylene in the brain to that of

[

11C]cocaine suggests that both drugs cross the blood-brain barrier to

the same extent. The distribution of [

11C]cocaethylene in the baboon

brain was also similar to that of [

11C]cocaine. For both tracers, the

highest uptake was basal ganglia > thalamus > cortex = cerebellum.

Both these drugs reached peak concentration in the brain at the same

time (average 2 to 5 minutes after injection). The high uptake of

[

11C]cocaethylene in the basal ganglia probably represents binding to

50
the dopamine transporter as shown for [

11C]cocaine by Fowler and

colleagues (1989). This is likely because pharmacological studies have

shown that the affinity of cocaethylene for the dopamine transporter is

similar to that of cocaine (Heam et al. 1991a). Thus, differences

between cocaine and cocaethylene do not seem to be due to either a

difference in (1) the rate or speed of brain uptake or (2) their pattern

of regional distribution. Interestingly, the rate of clearance of

[

11C]cocaethylene from the brain was slower than that of [11C]cocaine.

This slower clearance could prolong the pharmacological effects when

cocaine and alcohol are taken in combination. Similarly, the slower

clearance in plasma of cocaethylene than that of cocaine could lead to

more prolonged effects. However, the extent to which the differences

in pharmacokinetics and metabolism between the two drugs could

explain the enhanced behavioral and toxic effects of cocaine is not

clear because tolerance to cocaine develops rapidly, and decreased

cardiovascular and behavioral responses are observed even with steadystate plasma concentrations of cocaine (Ambre 1989).

The human studies were unable to document an effect of alcohol

intoxication on the pharmacokinetics and distribution of [

11C]cocaine.

This is different from what has been reported for alcohol and

amphetamines. Also, these studies were unable to document the

presence of labeled cocaethylene during the 10 minutes after

administration of [

11C]cocaine.

In interpreting these results, one has to realize that the studies differ

from real-life situations in the following manner: (1) a tracer dose of

[

11C]cocaine was used rather than a pharmacologically active dose; (2)

alcohol was administered prior to cocaine, and usually drug abusers

consume alcohol after they have been administering cocaine; (3) a

single administration of [

11C]cocaine was given as opposed to the

repeated “binge” administration of cocaine by cocaine abusers; and (4)

the study does not reproduce the conditions of the chronic drug abuser

in whom metabolism of cocaine or alcohol may be impaired by

long-term exposure to these drugs.

The inability to detect [

11C]cocaine and cocaethylene 10 minutes after

injection of [

11C]cocaine during alcohol consumption is consistent with

a recent study in humans showing that cocaethylene does not peak

until 90 to 120 minutes after coadministration of cocaine and alcohol.

51
The amount of cocaethylene seen at peak is relatively low, corresponding to one-sixth of that for cocaine (McCance-Katz et al. 1991).

Another study (Perez-Reyes and Jeffcoat 1992) reported a similar low

conversion, which is consistent with a recent study on the enzymatic

transesterification of cocaine in the presence of ethanol (Boyer and

Petersen 1992).

Although cocaethylene may play a role in the amount of toxicity seen

in cocaine and alcohol coadministration, other variables are probably as

important and need to be considered. In fact, it may be the direct

action of cocaine and alcohol, not their interactions, that is mainly

responsible for the enhanced toxicity when used in combination. For

example, the cardiovascular enhanced toxicity could relate to the fact

that both these drugs (1) are directly toxic to the myocardium

(Urbano-Marquez et al. 1989; Przywara and Dambach 1989), (2)

release catecholamines from adrenals (Pohorecky and Brick 1988;

Chiueh and Kopin 1978), and (3) induce vasoconstriction (Pohorecky

and Brick 1988). Similarly, the acute behavioral effects of cocaine and

alcohol could result from their effects on the same brain reward

systems (Seeman and Lee 1974; Di Chiara and Imperato 1988).

In explaining the enhanced behavioral and toxic effects seen with

coadministration of cocaine and alcohol and relating them to the

formation of cocaethylene, one has to be aware that these effects are

not specific to this particular drug combination. In fact, combination

of other stimulant drugs with alcohol or other sedative hypnotics, such

as benzodiazepines, has been shown to enhance behavioral and toxic

effects of the individual drug (Rech et al. 1976; Iverson et al. 1975;

Coleman and Evans 1975). The latter studies and the current findings

emphasize the need to consider the direct actions of alcohol and of

cocaine as one of the underlying mechanisms for their enhanced

toxicity when used in combination.

52
REFERENCES

Ambre, J.J. Cocaine kinetics in humans. In: Redda, K.K.; Walker,

CA.; and Bemett, G., eds. Cocaine, Marijuana, Designer Drugs:

Chemistry, Phamtacology, and Behavior. Boca Raton, FL: CRC

Press, 1989. pp. 53-70.

Boag, F., and Havard, C.W.H. Cardiac arrhythmia and myocardial

ischemia related to cocaine and alcohol consumption. Postgrad Med

J 61:997-999, 1985.

Boyer, C.S., and Petersen, D.R. Enzymatic basis for the transesterification of cocaine in the presence of ethanol: Evidence for the

participation of carboxylesterases. J Pharmacol Exp Ther

260:939-945, 1992.

Chiueh, C.C., and Kopin, I.J. Endogenous epinephrine and norepinephrine from the sympathoadrenal medullary system of

anesthetized rats. J Phamacol Exp Ther 205:148-154, 1978.

Coleman, J.H., and Evans, W.E. Drug interactions with alcohol. Med

Times 103:145-150, 1975.

Dewey, S.L.; MacGregor, R.R.; Brodie, J.D.; Bendriem, B.; King, P.T.;

Volkow, N.D.; Schlyer, D.J.; Fowler, J.S.; Wolf, A.P.; Gately, S.J.;

and Hitzemann, R. Mapping muscarinic receptors in human and

baboon brain using [N-11C-methyl]-benztropine. Synapse 5:213-223,

1990.

Di Chiara, G., and Imperato, A. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic

systems of freely moving rats. Proc Natl Acad Sci U S A

85:5274-5278, 1988.

Fowler, J.S.; Volkow, N.D.; Logan, J.; MacGregor, R.R.; Wang, G.-J.;

and Wolf, A.P. Alcohol intoxication does not change [

11C]cocaine

pharmacokinetics in human brain and heart. Synapse 12:228-235,

1992b.

Fowler, J.S.; Volkow, N.D.; MacGregor, R.R.; Logan, J.; Dewey, S.L.;

Gatley, S.J.; and Wolf, A.P. Comparative PET studies of the

kinetics and distribution of cocaine and cocaethylene in baboon

brain. Synapse 12:220-227, 1992a.

Fowler, J.S.; Volkow, N.D.; Wolf, A.P.; Dewey, S.L.; Schlyer, D.J.;

MacGregor, R.R.; Hitzemann, R.; Logan, J.; Bendriem, B.; Gatley,

S.J.; and Christman, D. Mapping cocaine binding sites in human

and baboon brain in vivo. Synapse 4:371-377, 1989.

53
Fowler, J.S.; Wolf, A.P.; and Volkow, N.D. New directions in positron

emission tomography-Part II. Annu Rep Med Chem 25:261-269,

1990.

Grant, B.F., and Harford, T.C. Concurrent and simultaneous use of

alcohol with cocaine: Results of national survey. Drug Alcohol

Depend 25:97-104, 1990.

Hearn, W.L.; Flynn, D.D.; Hime, G.W.; Rose, S.; Cofino, J.C.;

Mantero-Atienza, E.; Wetli, C.V.; and Mash, D.C. Cocaethylene: A

unique cocaine metabolite displays high affinity for the dopamine

transporter. J Neurochem 56:698-701, 1991a.

Heam, W.L.; Rose, S.; Wagner, J.; Ciarleglio, A.; and Mash, D.C.

Cocaethylene is more potent than cocaine in mediating lethality.

Pharmacol Biochem Behav 39:531-533, 1991b.

Iverson, F.; Coldwell, B.B.; Downie, R.H.; and Whitehouse, L.W.

Effect of ethanol on toxicity and metabolism of amphetamine in the

mouse. Experientia 31:679-680, 1975.

Jatlow, P.; Elsworth, J.D.; Bradberry, C.W.; Winger, G.; Taylor, J.R.;

Russell, R.; and Roth, R.H. Cocaethylene: A neuropharmacologically active metabolite associated with concurrent

cocaine-ethanol ingestion. Life Sci 48:1787-1794, 1991.

Jonsson, J., and Lewander, T. Effects of diethyldithiocarbamate and

ethanol on the in vivo metabolism and pharmacokinetics of

amphetamine in the rat. J Pharm Pharmacol 25:589-591, 1973.

Katz, J.L.; Terry, P.; and Witkin, J.M. Comparative behavioral

pharmacology and toxicology of cocaine and its ethanol-derived

metabolite, cocaine ethyl ester (cocaethylene). Life Sci 50:

1351-1361, 1992.

Langstrom, B., and Lundqvist, H. The preparation of “C-methyl iodide

and its use in the synthesis of 11C-methyl-L-methionine. Int J Appl

Radial Isotope 27:357-363, 1976.

Logan, J.; Fowler, J.S.; Volkow, N.D.; Wolf, A.P.; Dewey, S.L.;

Schlyer, D.; MacGregor, R.R.; Hitzemann, R.; Bendriem, B.;

Gatley, S.J.; and Christman, D.R. Graphical analysis of reversible

radioligand binding time-activity measurements applied to

[N-11C-methyl]-(-)-cocaine PET studies in human subjects.

J Neurochem 10:740-747, 1990.

Masur, J.; Souza-Formigoni, M.L.O.; and Pires, M.L.N. Increased

stimulatory effect by the combined administration of cocaine and

alcohol in mice. Alcohol 6:181-182, 1989.

54
McCance-Katz, E.F.; Price, L.H.; McDougle, C.J.; Marek, G.J.;

Kosten, T.R.; and Jatlow, P.I. Cocaethylene formation following

sequential administration of cocaine and ethanol to humans:

Pharmacological, physiological, and behavioral studies. Soc

Neurosci Abstr 349.1:890, 1991.

Perez-Reyes, M., and Jeffcoat, A.R. Ethanol/cocaine interaction:

Cocaine and cocaethylene plasma concentrations and their

relationship to subjective and cardiovascular effects. Life Sci

51:1553-563, 1992.

Pohorecky, L.A., and Brick, J. Pharmacology of ethanol. Pharmacol

Ther 36:1335-427, 1988.

Przywara, D.A., and Dambach, G.E. Direct action of cocaine on

cardiac cellular activity. Circ Res 65:185-192, 1989.

Rech, R.H.; Vomachka, M.K.; and Rickert, D. Interactions between

amphetamine and alcohol and their effect on rodent behavior. Ann

N Y Acad Sci 281:426-440, 1976.

Ritz, M.C.; Lamb, R.J.; Goldberg, S.R.; and Kuhar, M.J. Cocaine

receptors on dopamine transporters are related to self-administration

of cocaine. Science 237:1219-1223, 1987.

Rose, S.; Heam, W.L.; Hime, G.W.; Wetli, C.V.; Ruttenber, A.I.; and

Mash, D.C. Cocaine and cocaethylene concentrations in human post

mortem cerebral cortex. Soc Neurosci Abstr 11.6:14, 1990.

Sands, B.F., and Ciraulo, D.A. Cocaine drug-drug interactions. J Clin

Psychopharmacol 12:49-55, 1992.

Seeman, P., and Lee, T. The dopamine-releasing actions of

neuroleptics and ethanol. J Pharmcol Exp Ther 190:131-140, 1974.

Urbano-Marquez, A.; Estruch, R.; Navarro-Lopez, F.; Grau, J.M.;

Mont, L.; and Rubin, E. Effects of alcoholism on skeletal and

cardiac muscle. N Engl J Med 320:409-415, 1989.

Volkow, N.D.; Fowler, J.S.; Wolf, A.P.; Wang, G.-J.; Logan, J.;

MacGregor, R.R.; Dewey, S.L.; Schlyer, D.; and Hitzemann, R.

Distribution and kinetics of carbon- 11 -cocaine in human body

measured with PET. J Nucl Med 33:521-525, 1992.

Woodward, J.J.; Mansbach, R.; Carroll, F.I.; and Balster, R.L.

Cocaethylene inhibits dopamine uptake and produces cocaine-like

actions in drug discrimination studies. Eur J Pharmacol 197:

235-236, 1991.

55
ACKNOWLEDGMENTS

This research was supported by NIDA grants RID-A06891 and RIDA06278 and by the U.S. Department of Energy under contract

DE-AC02-76CH00016.

AUTHORS

Nora D. Volkow, M.D.

Scientist

Medical Department

Joanna S. Fowler, Ph.D.

Senior Chemist

Brookhaven National Laboratory

Building 490

30 Bell Avenue

Upton, NY 11973

56
Positron Emission Tomography

of Cocaine Binding Sites on the

Dopamine Transporter

Bertha K. Madras, David R. Elmaleh, Peter C. Meltzer,

Anna Y. Liung, Gordon L. Brownell,

and Anna-Liisa Brownell

INTRODUCTION

Abuse of the highly addictive drug cocaine results in adverse

psychological and medical consequences. The addictive properties of

the drug appear to be related, in part, to the rapid entry of the drug

into the brain, association with the brain’s dopamine systems, and short

duration of action. Swift metabolism leads to multiple and frequent

dosing with the drug, which is an added risk for getting Acquired

Immunodeficiency Syndrome (AIDS) by the intravenous route. These

sequelae have catalyzed efforts to develop drug therapeutic approaches

for cocaine addiction, particularly in view of the reported benefits of

methadone and nicotine replacement for heroin and tobacco addictions.

Brain imaging techniques can contribute to and expedite the

development of therapeutic agents, Positron emission tomography

(PET) imaging is a sensitive method for localizing drug receptors in

living brain tissue. The method can be applied to detect receptor

occupancy of candidate therapeutic drugs and to determine whether

occupancy is associated with therapeutic benefit. This chapter surveys

preliminary studies by the authors to apply PET imaging to medications development. As converging evidence suggests that brain

dopamine systems are principal targets of cocaine in the brain, the

authors’ studies focused on these systems.

57
PARAMETERS OF THE STUDIES

The Dopamine Transporter

The dopamine transporter is localized on dopamine neurons and

suspends the effects of dopamine by transporting the neurotransmitter

back into the terminals from which it was released. Although cocaine

is a relatively nonselective inhibitor of the dopamine, serotonin, and

norepinephrine transport, blockade of the dopamine transporter is

strongly implicated in mediating the reinforcing properties of the drug

(Ritz et al. 1987; Madras et al. 1989a; Bergman et al. 1989). Imaging

of the dopamine transporter in vivo may be useful for monitoring

behaviorally relevant cocaine recognition sites in the brain and for

monitoring occupancy of potential drug therapies.

Cocaine Analogs as Probes for the Dopamine Transporter

The affinity of cocaine for the dopamine transporter in primate brain is

relatively low (Madras et al. 1989a), its dissociation from these sites

occurs within seconds, and approximately 25 to 30 percent of the

[

3H]cocaine binding is associated with targets unrelated to the

dopamine transporter. In recent years, a number of dopamine transport

inhibitors structurally dissimilar to cocaine, including GBR 12935 and

nomifensine, were proposed as probes for the dopamine transporter

(Kuhar et al. 1990). Their suitability as probes for the dopamine

transporter, however, is limited by their association with sites in

addition to the dopamine transporter (Andersen et al. 1987, 1989;

Seeman and Niznik 1990). This limitation, combined with the

possibility that cocaine and its congeners may bind differently to the

dopamine transporter than compounds of other chemical classes (Fahey

et al. 1989; Rothman 1990), suggested the need for novel approaches

to this problem. In 1989, the high-affinity cocaine congener WIN

35,428 (Clarke et al. 1973) was proposed as a probe for the dopamine

transporter (Madras et al. 1989a).

[

3H]WIN 35,428, also designated [3H]CFT, offers several advantages

over [

3H]cocaine as a probe (Madras et al. 1989b, table 1). The sites

labeled by [3H]CFT are of similar density and have virtually the same

pharmacological specificity as [

3H]cocaine. Furthermore, the binding

isotherms of [

3H]CFT model to two affinity components, a property

58
TABLE 1. Comparison of the binding properties of [3H]cocaine and

[

3H]CFT ([3H]WIN 35,428, Madras et al. 1989a, 1989b)

SIMILARITIES

Site density

High/Low affinity

NaCl requirement

[

3H]Cocaine [3H]CFT

431 pmol/g 388 pmol/g

yes yes

yes yes

DIFFERENCES

[

3H]Cocaine [

3H]CFT

Affinity 283 nM 16 nM

Dissociation time seconds 23 min

Nonspecific binding 20 to 30 percent 3 to 9 percent

characteristic of [

3H]cocaine binding. However, [3H]CFT is superior to

[

3H]cocaine inasmuch as its affinity is higher and its dissociation from

these sites is slower (> 20 min versus seconds), which results in highly

reproducible binding data. Most significantly for brain imaging,

nonspecific binding of [

3H]CFT is less than 10 percent, and frequently

less than 5 percent, a value considerably lower than that of [

3H]cocaine

(20 to 30 percent). The markedly low level of nonspecific binding

observed in homogenates was the authors’ initial impetus to develop

radiolabeled CFT and similar congeners as imaging agents.

Brain Distribution of [

3H]CFT (WIN 35,428)

Subsequent experimental findings with [

3H]CFT supported the authors’

initial proposal and the progressive development of [

3H]CFT for this

application. [

3H]CFT distributed primarily to dopamine-rich regions of

primate brain (Canfield et al. 1990; Kaufman et al. 1991; Kaufman and

Madras 1992). [

3H]CFT binding sites in the caudate-putamen and

nucleus accumbus were relevant to the behavioral effects of cocaine

(Madras 1989c). The sites were localized presynaptically (Kaufman

and Madras 1991). In this regard, earlier studies reported a severe

depletion of [

3H]CFT binding in post-mortem human Parkinson’s

diseased caudate and putamen compared with controls (Madras et al.

59
1990; Kaufman and Madras 1991). These brain regions undergo a

marked loss of dopamine fibers in the course of Parkinson’s disease.

An additional advantage of [

3H]CFT was the high striatum:cerebellum

ratio (3.3:5.6) observed by ex vivo autoradiography between 30 and 90

min, a period of time corresponding to a serviceable timeframe for a

PET imaging procedure (Kaufman and Madras 1992, 1993). Finally,

whereas a number of cocaine congeners bind to both the dopamine and

serotonin transporter (Boja et al. 1992; Kaufman and Madras 1992;

Laruelle et al. 1993), CFT demonstrated the highest selectivity,

fifteenfold, for the dopamine compared with the serotonin transporter

of compounds available during this period (Meltzer et al. 1993).

[

11C]CFT as a PET Ligand

A project was initiated to evaluate [

11C]CFT as a PET imaging probe

for cocaine recognition sites associated with the dopamine transporter.

Adult male and female cynomolgus monkeys (Macacafascicularis)

were used for the imaging studies. PET imaging was conducted with a

high-resolution positron emission tomograph (PCR-1, Brownell et al.

1985). The resolution of PCR-1 for a point source at the center is 4.5

mm, and the sensitivity is 46,000 counts per s for a source 20 cm in

diameter with a concentration of 1 uCi/ml and a plane thickness of 1

cm. The plane thickness, 5 mm, used in these studies were obtained

by the use of cylindrical collimators, which limited the effective height

of the detectors. Each monkey was anesthetized, and PET imaging

was conducted as described previously (Hantraye et al. 1992).

Indwelling venous and arterial catheters were used for drug injections

and for monitoring blood levels of [

11C]CFT. Sequential dynamic

imaging for 90 min was carried out at earbar levels corresponding to

30 mm, 25 mm, 20 mm, 15 mm, 10 mm, -5 mm, and - 10 mm. Data

were corrected for sensitivity and attenuation using a mathematical

correction. For image reconstruction, a Hanning windowed filter

convolution backprojection was used. Calibration of the camera was

performed using a cylindrical plastic phantom (0 = 6 cm) and 18Fsolution, and cross calibration with the same solution was conducted

using a gamma counter. Regions of interest (ROI) were drawn over

the caudate, putamen, cortex, and cerebellum, and subsequently

striatal:cerebellar and cortical:cerebellar ratios were calculated.

60
RESULTS

[

11C]CFT Accumulation in the Striatum

The accumulation of [

11C]CFT in the brain was evident within 2.5 min,

and it was observed initially in the cortex, striatum, and additional

brain regions. Within 40 min, regions other than the striatum were

cleared of the radioligand, and dynamic images showed high uptake of

[

11C]CFT in the caudate and putamen, which increased as a function of

time for at least 100 min (figure 1). The ratio of specific to nonspecific binding was determined by comparing the uptake in the

caudate nucleus and putamen with the uptake in the cerebellum. At 90

min, the striatum:cerebellum ratio was 4.21±0.02 and 4.88±0.25 for the

caudate nucleus and putamen, respectively. The ratio for the putamen

was consistently 15 percent higher, a finding that corresponded with an

earlier report using [

3H]CFT (Kaufman et al. 1991).

Pharmacological Specificity of [

11C]CFT Binding Sites

In order to determine whether binding was principally to the dopamine

transporter, the effects of a number of selective and nonselective

dopamine transport inhibitors on [

11C]CFT accumulation was

determined. The dopamine transport inhibitors (-)-cocaine, GBR

12909, and mazindol, at doses that produce behavioral stimulation

(Spealman et al. 1989), attenuated the accumulation of [

11C]CFT

(Madras et al. 1991). In contrast, the selective and high-affinity

serotonin transport inhibitor citalopram did not appreciably alter the

accumulation of [

11C]CFT in striatum at low doses, but it did so at

higher doses.

The present results clearly indicate that [

11C]CFT is a promising ligand

for monitoring behaviorally relevant cocaine binding sites in striatum

and for monitoring dopamine terminals. Its striatum:cerebellum ratio is

higher than other PET ligands targeted to the dopamine transporter, its

pharmacokinetic properties are suitable for imaging, and its initial

metabolism is minimal (Kaufman and Madras 1992). Consequently,

PET imaging of the dopamine transporter may expedite research in

cocaine abuse. Equally important, it may serve as a powerful method

for measuring the onset time and length of occupancy of cocaine

therapeutic agents targeted to the dopamine transporter.

61
FIGURE 1. Accumulation of [

11C]CFT in monkey striatum as a function of time
The low level of nonspecific binding also suggested that radiolabeled

CFT may be a marker for Parkinson’s disease. This application for

both [

11H]CFT and [11C]CFT has been documented (Madras et al. 1990;

Kaufman and Madras 1991; Hantraye et al. 1992), and it demonstrates

an unanticipated benefit of cocaine research.

IMPLICATIONS

Applications of [

11C]CPT for Medications Development

The mounting evidence that dopamine contributes significantly to

mediating the effects of cocaine offers several avenues for medications

development. Among classes of drugs to be considered are compounds that target the dopamine transporter or dopamine receptors,

including potential cocaine antagonists (Kitayama et al. 1992). Drug

substitutes are the most frequently prescribed and effective medications

used in the treatment of drug abuse. Cocaine substitutes require a

careful consideration of the potential benefits of candidate compounds.

In addition to efficacy, factors to be considered are comparisons

between candidate compounds and cocaine with regard to abuse

liability; behavioral, physiological, neurochemical, and toxic sequelae;

and oral efficacy. Such information is needed to weigh carefully the

risks and benefits of such an approach. Although a number of

dopamine transport inhibitors are thought to exhibit less abuse liability

than cocaine, this assumption should be validated by comparing the

effects of candidate compounds when administered by the same routes

as cocaine.

Criteria for therapeutic agents that target the dopamine transporter have

not been clearly established. A candidate therapeutic agent that

demonstrates a longer onset time and prolonged duration of action may

offer some advantages. In this regard, the slow onset may result in

diminished euphoric effects compared with cocaine, and the long

duration of action may minimize frequent dosing, thereby reducing the

risk of acquiring AIDS by the intravenous route. One example of such

a drug is indantraline (Lu 19-005), which has a relatively slow onset

and long duration of action (> 2 d) in primates (Rosenzweig-Lipson et

al. 1992).

63
The authors administered indantraline (0.2 mg/kg) 24 h prior to onset

of a PET imaging procedure. At this low dose, equivalent to one-fifth

the dose that generated behavioral stimulation for longer than 2 d,

uptake of [

11C]CFT was reduced in two of three animals. At 60 min

into the PET imaging procedure, [

11C]CFT accumulation was reduced

by 31 percent in the caudate nucleus and by 25 percent in the

putamen. These results suggest that prolonged stimulation by

indantraline may be related directly to occupancy of the dopamine

transporter. Furthermore, the results demonstrate the feasibility of

using PET imaging to identify whether candidate compounds occupy

the dopamine transporter in the brain and to measure the occupancy

period.

The long duration of action of indantraline may be related to its high

affinity (0.081 pM) for the dopamine transporter in monkey brain and

possibly to its occupancy of the serotonin transporter in the periphery

(Madras et al. 1993). In this regard, another high-affinity serotonin

transport inhibitor, RTI-55 (Boja et al. 1992), was shown to accumulate in high concentrations in lung, a potential depot for drugs that are

potent inhibitors of serotonin transport (Kaufman and Madras 1992).

PET Imaging of the D1 Dopamine Receptor

Because they are the indirect targets of cocaine, dopamine receptors

represent another potential target for therapeutic agents. Although two

families of dopamine receptors are found in the brain, D1 and D2, the

majority of drugs currently available are of the D2 type. D2 agonists

and antagonists have received attention as possible therapeutic agents

(Preston et al. 1992). The adverse side effects of agonists

(dyskinesias) and antagonists (extrapyramidal signs) has focused

interest on antagonists with reduced extrapyramidal signs. Recently,

researchers have focused more attention on drugs that target the D1

dopamine receptor. The authors have initiated studies to determine

whether D1 agonists occupy D1 dopamine receptors in vivo. The D1

receptor was imaged with [

11C]SCH 39166, a selective D1

 receptor

antagonist that accumulates in high concentrations in the striatum. A

preliminary study using an MPTP-treated monkey has demonstrated D1

receptor occupancy by the selective D1 agonist, SKF 81297 (Madras et

al. 1992).

64
CONCLUSION

These preliminary studies demonstrate the potential utility of PET as a

technique for imaging the dopamine transporter, a principal target of

cocaine in the brain. Furthermore, they illustrate a potential application of PET imaging in medications development. PET imaging

appears to be useful for identifying the receptor targets of candidate

compounds. PET also may be useful for assessing whether a compound, which produces prolonged stimulant effects, occupies the

dopamine transporter during the timeframe that behavioral effects are

measurable. Thus, combined with other strategies, PET imaging

studies can accelerate preclinical evaluation of potential cocaine

therapeutic agents.

REFERENCES

Andersen, P.H. The dopamine uptake inhibitor GBR 12909: Selectivity

and molecular mechanism of action. Eur J Pharmacol 166:493-504,

1989.

Andersen, P.H.; Jansen, J.A.; and Nielsen, E.B. [

3H]GBR 12935

binding in vivo in mouse brain: Labelling of a piperazine acceptor

site. Eur J Pharmacol 144:1-6, 1987.

Bergman, J.; Madras, B.K.; Johnson, S.E.; and Spealman, R.D. Effects

of cocaine and related drugs in nonhuman primates: III. Selfadministration by squirrel monkeys. J Pharmacol Exp Ther 251:150-

155, 1989.

Boja, J.W.; Mitchell, W.M.; Patel, A.; Kopajtic, T.A.; Carroll, F.I.;

Lewin, A.H.; Abraham, P.; and Kuhar, M.J. High-affinity binding

of [

125I]RTI-55 to dopamine and serotonin transporters in rat brain.

Synapse 12:27-36, 1992.

Brownell, G.L.; Bumham, C.A.; and Chesler, D.A. High resolution

tomograph using analog coding. In: Greitz, T.; Ingvar, D.H.; and

Widen, L., eds. The Metabolism of the Human Brain Studied With

Positron Emission Tomography. New York: Raven Press, 1985.

Canfield, D.R.; Spealman, R.D.; Kaufman, M.J.; and Madras, B.K.

Autoradiographic localization of cocaine receptors by [

3H]CFT in

monkey brain. Synapse 5:189-195, 1990.

65
Clarke, R.L.; Daum, S.J.; Gambino, A.J.; Aceto, M.D.; Pearl, J.;

Levitt, M.; Cumisky, W.R.; and Bogado, E.F. Compounds affecting

the central nervous system: IV. 2-ß-phenyltropane-2-carboxylic

esters and analogs. J Med Chem 16:1260-1267, 1973.

Fahey, M.A.; Canfield, D.R.; Spealman, R.D.; and Madras, B.K.

Comparison of [

3H]GBR 12935 and [3H]cocaine binding sites in

monkey brain. Soc Neurosci Abst 15:1090, 1989.

Hantraye, P.; Brownell, A.-L.; Elmaleh, D.; Spealman, R.D.; Wullner,

U.; Brownell, G.L.; Madras, B.K.; and Isacson, O. Dopamine fiber

detection by 11C-CFT and PET in a primate model of Parkinsonism.

Neuroreport 3:265-268, 1992.

Kaufman, M.J., and Madras, B.K. Severe depletion of cocaine

recognition sites associated with the dopamine transporter in

Parkinson’s diseased striatum. Synapse 9:43-49, 1991.

Kaufman, M.J., and Madras, B.K. Cocaine recognition sites labeled by

[

3H]CFT and [125I]RTI-55 in monkey brain: II. Ex vivo autoradiographic distribution. Synapse 12:99-111, 1992.

Kaufman, M.J., and Madras, B.K. [

3H]CFT ([3H]WIN 35,428)

accumulation in dopamine regions of monkey brain: Comparison of

a mature and an aged monkey. Brain Res 611:322-325, 1993.

Kaufman, M.J.; Spealman, R.D.; and Madras, B.K. Distribution of

cocaine recognition sites in monkey brain: I. In vitro autoradiography with [

3H]CFT. Synapse 9:177-187, 1991.

Kitayama, S.; Shimada, S.; Xu, H.; Markham, L.; Donovan, D.M.; and

Uhl, G.R. Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc Natl Acad

Sci U S A 89:7782-7785, 1992.

Kuhar, M.J.; Sanchez-Roa, P.M.; Wong, D.F.; Dannals, R.F.;

Grigoriadis, D.E.; Lew, R.; and Milberger, M. Dopamine

transporter: Biochemistry, pharmacology, and imaging. Eur Neurol

30:15-20, 1990.

Laruelle, M.; Baldwin, R.M.; Malison, R.T.; Zea-Ponce, Y.; Zoghbi,

S.S.; Al-Tikriti, M.S.; Sybirska, E.H.; Zimmermann, R.C.;

Wisniewski, G.; Milius, R.A.; Wang, S.; Smith, E.O.; Roth, R.H.;

Chamey, D.S.; Hoffer, P.B.; and Innis, R.B. SPECT imaging of

dopamine and serotonin transporters with [

125I]ß-CIT: Pharmacological characterization of brain uptake in nonhuman primates.

Synapse 13:295-309, 1993.

66
Madras, B.K. “Cocaine Recognition Sites in Primate Nucleus

Accumbens and Medial Prefrontal Cortex: Relationship to the

Behavioral Effects of Cocaine-like Drugs.” Paper presented at the

annual meeting of the American College of Neuropsychopharmacology, Maui, Hawaii, 1989c.

Madras, B.K.; Elmaleh, D.; Meltzer, P.; Lee-Parritz, D.E.; Hantraye,

P.; Isacson, O.; Brownell, G.L.; and Brownell, A.-L. Parkinsonian

signs in MPTP-treated monkeys are alleviated by the high efficacy

D1 dopamine receptor agonist SKF 81297. Soc Neuosci Abstr

18:935, 1992.

Madras, B.K.; Fahey, M.A.; Bergman, J.; Canfield, D.R.; and

Spealman, R.D. Effects of cocaine and related drugs in nonhuman

primates: I. [

3H]Cocaine binding sites in caudate-putamen.

J Pharmacol Exp Ther 251:131-141, 1989a.

Madras, B.K.; Fahey, M.A.; and Kaufman, M.J. [

3H]CFT and [3H]Lu

19-005: Markers for cocaine receptors/dopamine nerve terminals in

Parkinson’s disease. Soc Neurosci Abstr 16:14, 1990.

Madras, B.K.; Fahey, M.A.; Kaufman, M.J.; Spealman, R.D.;

Schumacher, J.; Isacson, O.; Brownell, A.-L.; Brownell, G.L.; and

Elmaleh, D.R. Cocaine receptor probes in nonhuman and human

brain: In vitro characterization and in vivo imaging. Soc Neurosci

Abstr 17:190, 1991.

Madras, B.K.; Spealman, R.D.; Fahey, M.A.; Neumeyer, J.L.; Saha,

J.K.; and Milius, R.A. Cocaine receptors labeled by [

3H]2ßcarbomethoxy-3ß-(4-fluorophenyl)tropane. Mol Pharmacol 36:518-

524, 1989b.

Meltzer, P.C.; Liang, A.Y.; Brownell, A.-L.; Elmaleh, D.R.; and

Madras, B.K. Substituted 3-phenyltropane analogs of cocaine:

Synthesis, inhibition of binding at cocaine recognition sites, and

positron emission tomography. J Med Chem 36:855-862, 1993.

Preston, K.L.; Sullivan, J.T.; Strain, E.C.; and Bigelow, G.E. Effects of

cocaine alone and in combination with bromocriptine in human

cocaine abusers. J Pharmacol Exp Ther 262:279-291, 1992.

Ritz, M.C.; Lamb, R.J.; Goldberg, S.R.; and Kuhar, M.J. Cocaine

receptors on dopamine transporters are related to self-administration

of cocaine. Science 237:1219-1223, 1987.

Rosenzweig-Lipson, S.J.; Bergman, J.; Spealman, R.D.; and Madras,

B.K. Stereoselective behavioral effects of Lu 19-005 in monkeys:

Relation to binding at cocaine recognition sites. Psychopharmacology 107:186-194, 1992.

67
Rothman, R.B. High affinity dopamine reuptake inhibitors as potential

cocaine antagonists: A strategy for drug development. Life Sci

46:PL-17-PL-21, 1990.

Seeman, P., and Niznik, H.B. Dopamine receptors and transporters in

Parkinson’s disease and schizophrenia. FASEB J 4:2737-2744,

1990.

Spealman, R.D.; Madras, B.K.; and Bergman J. Effects of cocaine and

related drugs in nonhuman primates: II. Stimulant effects on

schedule-controlled behavior. J Pharmacol Exp Ther 251:142-149,

1989.

ACKNOWLEDGMENTS

This research has been supported by United States Public Health

Service grants DA06303 and RR00168. Dr. D. Lee-Parritz for advice

on anesthesia, and Dr. J. Amt (Lundbeck A/S) for indantraline. The

animals were maintained in accordance with the guidelines of the

Committee on Animals of the Harvard Medical School and according

to DHHS publication No. (NIH)85-23.

AUTHOR

Bertha K. Madras, Ph.D.

Associate Professor

Harvard Medical School

New England Regional Primate Research Center

One Pine Hill Drive

Southborough, MA 01772

David R. Elmaleh, Ph.D.

Associate Professor

Gordon L. Brownell, Ph.D.

Professor

68
Anna-Liisa Brownell, Ph.D.

Nuclear Physicist

Department of Radiology

Massachusetts General Hospital

Boston, MA 02115

Peter C. Meltzer, Ph.D.

Chemist

Anna Y. Liang, Ph.D.

Chemist

Organix, Inc.

Wobum, MA 01801

69

Metabolic Mapping Methods for

the Identification of the Neural

Substrates of the Effects of

Novel Tropane Analogs

Linda J. Porrino, Lisa Williams-Hemby, and Huw M.L. Davies

INTRODUCTION

The administration of any psychoactive drug to an organism produces

a variety of pharmacological actions that may include both central and

peripheral effects. These physiological and behavioral responses are

unlikely to result from a single action in a single brain region; they are

more likely to be the product of multiple processes at a number of

distinct anatomical sites. In addition, most drugs, particularly when

administered over a broad range of doses, may not act on only one

neurotransmitter system or receptor subtype but on some combination

thereof. These issues are particularly pertinent when new compounds

are to be investigated. Such compounds may have actions in vivo not

necessarily predictable from in vitro characterizations.

In order, then, to determine the neural substrates of the actions of a

drug, especially when the actions of the drug are not completely

specified (as is the case when drugs are in development), it is

necessary to identify neural events in circuits and pathways throughout

the brain, not just at one location. This requires either that researchers

investigate small portions of the brain one at a time, as with electrophysiological or in vivo microdialysis methods, or that they use

methods capable of surveying the entire brain simultaneously. The

development of the 2-[14C]deoxyglucose (2-DG) method by Sokoloff

and colleagues (1977) has provided the means to accomplish the latter

approach. The 2-DG method is a means with which functional events

in the brain related to various physiological, pharmacological, and

behavioral states can be investigated. It has been used extensively in

neuropharmacology to identify the neural circuits that mediate the

effects of a wide variety of pharmacological agents (for reviews cf.

71
McCulloch 1982; Porrino and Pontieri 1993). This chapter will

describe the 2-DG method and its application to the study of a series

of novel tropane analogs recently synthesized by Davies and colleagues

(1991, 1993).

MEASUREMENT OF RATES OF GLUCOSE UTILIZATION

The 2-DG method measures rates of local cerebral glucose utilization

(LCGU) throughout all portions of the central nervous system (CNS).

It is a biochemical method that measures a biological processglucose utilization or the rate at which energy is consumed in neuroanatomically defined regions in the CNS of conscious animals. The

value of the measurement of rates of glucose utilization stems from the

fact that in the brain, as in other tissues that do physicochemical work,

the amount of energy used is correlated with the amount of work done

in that tissue. Glucose utilization in the brain is a measure of energy

use under normal physiological conditions because glucose is virtually

the exclusive substrate for energy metabolism. The basic rationale of

the 2-DG method is that functional activity in any given brain region is

related directly to energy metabolism in that region. It is possible,

therefore, through the measurement of changes in rates of glucose

utilization to identify brain regions in which functional activity is

altered during various experimental manipulations.

Although there are a number of energy-requiring processes in the brain

that contribute to basal rates of glucose utilization, transmitter synthesis, release and reuptake, and protein synthesis, the largest portion of

energy in the CNS, however, is used to maintain and restore ionic

gradients. It has been estimated that 80 percent of the energy generated in the brain is used for this purpose (Kurumaji et al. 1993). It is

important to appreciate that the changes in rates of glucose utilization

that are evoked by an experimental manipulation are thought to result

mainly from increases or decreases in electrical activity or synaptic

activity in the CNS. These changes in electrical activity in turn

produce corresponding increases or decreases in the activity of Na+,

K

+

,ATPase, the energy consuming enzyme involved in the restoration

of neuronal ionic gradients to their resting state. Experi-ments with 2-

DG have shown that the coupling of glucose utilization to functional

activity is dependent on Na+, K

+

-ATPase activity in that ouabain, an

72
ATPase inhibitor, can block the increases in glucose utilization that

accompany electrical stimulation (Mata et al. 1980).

The measurement of rates of glucose utilization follows the basic

principles for the measurement of rates of any reaction with radioactive

tracers. The amount of product formed over an interval of time is

determined, related to the integrated specific activity (i.e., the ratio of

the labeled precursor to the total precursor pool integrated over the

time of measurement), and corrected for kinetic differences between

the labeled and unlabeled compounds. Rates of LCGU are measured

with a radioactively labeled analog of glucose, 2-DG, that, like

glucose, is transported into cerebral tissue by the same carrier and

phosphorylated by hexokinase but, unlike glucose, is not metabolized

further and is, therefore, trapped within cells. It is this sequestration

within cells that allows quantitative autoradiography to be used to

measure actual rates of glucose utilization in individual brain regions.

The details of the derivation of the 2-DG method and an extensive

discussion of its theoretical basis are beyond the scope of this chapter,

but for further discussion see Sokoloff and colleagues (1977, 1982)

and Sokoloff and Porrino (1986). The techniques used for the

application of the 2-DG method have also been described in detail

elsewhere (Porrino and Crane 1990).

ADVANTAGES AND DISADVANTAGES OF THE 2-DG

METHOD

Advantages

One of the principal advantages of the 2-DG method is the use of

autoradiography, which permits the visualization of individual structures within the brain as well as anatomical subdivisions within a given

structure. The resolution of the 2-DG method is approximately 100μ.

The limiting factors are the energy of the isotope and the diffusion and

migration of the labeled compound in the tissue during freezing and

cutting of the brain. Even in the rat, though, it is possible to distinguish subdivisions of a structure as small as the lateral habenula. For

example, glucose utilization in the lateral portion of the lateral

habenula is decreased significantly following the administration of

73
dopaminergic agonists (McCulloch et al. 1982; Wechsler et al. 1979;

Porrino et al. 1984, 1988). With many other neurochemical methods,

such as in vivo microdialysis or neurotransmitter turnover determinations, it is not possible to ascribe effects to particular divisions or

subdivisions of brain regions because the areas sampled are by

necessity rather large. In 2-DG studies, the use of quantitative

autoradiography makes it possible to evaluate changes in functional

activity in highly circumscribed brain regions.

Another advantage is that a broad survey of effects at all levels of the

brain is possible. This means that investigations are not limited to

those sites in which effects are expected, a particularly important factor

when a drug has not been characterized completely. If the whole brain

is examined, effects of a pharmacological agent may be seen that

otherwise would have gone unnoticed. In a study of the effects of

lesions of the substantia nigra of primates induced by the infusion of

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, alterations in cerebral

glucose utilization rates were evident not just within the basal ganglia

as expected but also in circuits mediating eye movements (Porrino et

al. 1987). Using this kind of an approach, it is possible to survey

effects in a number of systems without using different sets of animals.

Although earlier experiments with the 2-DG method were conducted in

restrained or partially restrained animals, methods have been developed

that permit the use of the 2-DG method in freely moving, conscious

animals (Crane and Porrino 1989). This approach allows the application of the method to the study of behavior. In pharmacological

studies, behavioral effects produced by drug administration can be

related directly to changes in functional activity in specific brain

regions.

One problem with the 2-DG method is that the uptake of 2-deoxyglucose is dependent on variables such as blood pressure, cardiac

output, and plasma glucose levels. Overall differences in uptake based

on these variables could obscure differences between groups that are

the result of changes in functional activity, particularly if non- or semiquantitative methods are used. Because the calculation of absolute

rates of glucose utilization with the 2-DG method takes these variables

into account, however, valid comparisons can be made between

groups, allowing both the identification of brain areas with altered

74
functional activity and the magnitude of these changes. This is clearly

an advantage in pharmacological studies in which the physiological

status of the animal frequently is altered by drug administration.

Disadvantages

Despite its advantages, the 2-DG method has a number of limitations.

Foremost among these is the inability to differentiate between direct

and indirect effects of a given stimulus. An entire pathway or circuit

may be activated metabolically, even though the direct action of the

stimulus may occur only at the origin of or at some point along the

pathway. The 2-DG method, therefore, cannot identify the sites at

which a drug binds or the sites at which a drug initiates its actions.

This is illustrated by the results of a study in which the effects of

diazepam on cerebral metabolism were measured (Ableitner et al.

1985). Little overlap was evident between regions in which significant

changes in rates of metabolism were found and those areas identified

as having high benzodiazepine binding. More specifically, glucose

utilization was increased in the lateral thalamus, an area low in binding

sites, and not altered in areas such as the ventromedial hypothalamus

and medial amygdala, both rich in binding sites. It is important to

consider that glucose utilization changes reflect only a corresponding

change in afferent input to that region and not a change in the functional activity of cell bodies in that structure. An entire pathway may

be activated, even though the direct action of the drug occurred only at

the origin of the circuit. Although this lack of specificity may be a

limitation in some instances, as in the case of determining the primary

site of action of a drug, it is an advantage when the goal is to identify

the neural circuits or pathways that mediate a specific behavioral

response.

Other disadvantages include the fact that it is not possible to identify

the specific neurotransmitters, neuromodulators, or receptor types that

are responsible for changes in functional activity and the inability to

distinguish between inhibitory and excitatory processes on the basis of

increases or decreases in 2-DG uptake. Both excitation and inhibition

at the neuronal level involve similar metabolic processes in that

maintenance and restoration of ionic gradients have similar energy

requirements, regardless of whether excitatory or inhibitory neurotransmitters are secreted at synaptic terminals.

75
The 2-DG method also has poor time resolution. The rather long

experimental time period can be a significant limitation in drug studies

because steady state or constant conditions need to be maintained for

accurate measurement of rates of glucose utilization. Physiological and

behavioral responses to drug administration can vary significantly

across the 45 minutes required for the application of the 2-DG method.

For example, although the total experimental time is 45 minutes, the

uptake and phosphorylation of 2-DG in the brain reflect predominantly

its metabolism in the first 5 to 10 minutes of this period. This is

because the tissue concentrations of free 2-DG available for metabolism are highest during the first 5 to 10 minutes after its injection

(Sokoloff et al. 1977). Correct choice of the optimal time for drug

administration in relation to the time of injection of 2-DG is, then,

important to ensure maximal sensitivity of the method as well as to

ensure that data will be comparable across treatments. Drug administration should be timed so that the maximum drug response coincides

with the time of maximum incorporation of 2-DG.

A final disadvantage that should be noted is that repeated measurements are not possible in the same animal. Groups of separate animals

must be used to determine the effects of each experimental condition,

which increases variability as well as the number of animals necessary.

Despite these constraints, the 2-DG method has been applied effectively in many neuropharmacological studies. The method can provide

novel information about the identity of brain regions functionally

involved in mediating the effects of a given drug. It can be particularly useful when the actions of the drug in question have not been

described completely. An example of such an application is discussed

in the next section.

NOVEL TROPANE ANALOGS

One approach to the study of the neurobiological actions of cocaine

has been through the use of cocaine analogs. Because many of the

important behavioral and physiological effects of cocaine, in particular

its euphorogenic properties, have been attributed to its actions at

dopaminergic synapses, tropane analogs that mimic or are more potent

than cocaine at these sites can be useful tools. A variety of tropane

76
analogs have been synthesized by a number of investigators to date

(Abraham et al. 1992; Lewin et al. 1992; Carroll et al. 1992a; 1992b;

Davies et al. 1991; Kozikowski et al. 1991).

One such series of novel tropane analogs was reported recently by

Davies and colleagues (1991, 1993). These analogs are synthesized

with a novel strategy utilizing vinylcarbenoid precursors instead of

more traditional methods in which cocaine is used as the starting

material. Characterization of these compounds in vitro has shown that

several of the compounds are as much as 900 to 1300 times more

potent than cocaine in displacing [

125I]RTI-55 binding in rat striatal

membrane preparations (Davies et al. 1993). In addition to being more

potent than cocaine at dopamine transporters, some analogs in this

series also were significantly more potent than cocaine in binding to

serotonin transporters in rat frontal cortex. As with cocaine, many of

the analogs tended to be comparatively nonselective with respect to

dopamine and serotonin (Childers, Bennett, Sexton, Saikali, and

Davies, unpublished observations). Several of the analogs, however,

demonstrated a relative selectivity for dopamine, including 2ßpropanoyl-3ß-(4-toluyl) tropane (PTT), which displayed at least a

twentyfold selectivity for dopamine in binding assays. Results from

uptake studies have confirmed both the potencies and specificities of

these tropanes as well (Bennett, Hyde, Saikali, and Davies, unpublished observations).

To date, PTT has been the only analog that has undergone a thorough

evaluation in vivo. These studies have verified that, like cocaine, PTT

acts as a stimulant in many respects (Porrino et al., submitted). These

studies, however, also show that PTT differs from cocaine qualitatively

in other aspects, as might be expected from an analog with a different

specificity than cocaine. For example, PTT produced significant dosedependent behavioral activating effects when administered to rats

intraperitoneally. Within the range of doses tested in these comparison

studies, PTT was approximately 10 to 30 times more potent than

cocaine in stimulating locomotor activity (Porrino et al., submitted).

This corresponds closely to the relative potency of this novel tropane

analog at binding to the dopamine transporter as well as its potency at

inhibiting dopamine uptake (Davies et al. 1993). There is, then, good

agreement between the relative potencies of PTT and cocaine in

in vitro and in vivo behavioral studies,

77
The behavioral effects of PTT, however, did not always coincide with

those of cocaine when examined more closely (Porrino et al., submitted). Animals treated with low doses of PTT, for example,

exhibited little movement. These rats remained awake and alert but

relatively motionless for long periods. In contrast, at moderate doses

PTT produced cocaine-like behavioral effects in the form of increases

in exploratory behaviors such as sniffing, rearing, and forward locomotion, and the administration of higher doses of PTT were accompanied by intense patterns of stereotypic head movements. PTT

elicited a far greater degree of stereotypic movements than cocaine,

and the character of these behaviors also was quite different. Even at

the highest dose tested in this assay, cocaine treatment was associated

mainly with forward locomotion and rearing with few stereotyped

behaviors evident, whereas PTT administration produced focused,

circular head bobbing restricted to one comer of the test chamber. The

differences in the behavioral effects elicited by cocaine and PTT support the view that they are qualitatively different in vivo.

METABOLIC MAPPING OF PTT

To investigate the differences between PTT and cocaine further and to

determine the neuroanatomical substrates of these differences, metabolic mapping with the quantitative autoradiographic 2-DG method

was utilized (Porrino, unpublished observations). The purpose of this

study was to identify the effects of PTT on functional activity and to

compare these effects to those of cocaine. The effects of the acute

intravenous (IV) administration of low (0.1 mg/kg), moderate (0.5

mg/kg), and high (1.0 mg/kg) doses of PTT on rates of LCGU were

measured in portions of the dopaminergic mesocorticolimbic and

nigrostriatal systems as well as in limbic, cortical, and thalamic

regions.

At moderate and high doses, widespread increases in LCGU were seen

in all portions of the nigrostriatal motor system (see figure 1 for

example) and throughout the thalamus and cortex. PTT at these doses

also elevated rates of cerebral metabolism in all portions of the mesocorticolimbic system with the largest increases in medial prefrontal

cortex, olfactory tubercle, and nucleus accumbens (see figure 2 for

example). This distribution of LCGU changes closely parallels the

78
dose-dependent metabolic changes observed following cocaine administration (Porrino et al. 1988). The similarities between PTT and other

psychostimulants are depicted in figures 3 and 4 in which the effects

of high, medium, and low doses of PTT, cocaine, and methamphetamine on rates of cerebral metabolism in portions of the nigrostriatal

and mesocorticolimbic systems are compared. The similarities are

consistent with increased activity that accompany treatment with these

drugs.

In contrast, at low doses, PTT had few effects on LCGU in most brain

regions but significantly decreased metabolism in the mesocorticolimbic system (figure 2). These metabolic changes may reflect the

behavioral suppression seen at low doses in the activity studies

previously described. The depression of metabolic activity in the

nucleus accumbens, olfactory tubercle, and medial prefrontal cortex

following PTT administration clearly is different from the pattern of

changes produced by cocaine, which at low doses produces selective

increases in metabolism in these same brain regions (Porrino et al.

1988). Figure 4 compares the effects of high, medium, and low doses

of PTT, cocaine, and methamphetamine on cerebral metabolism in the

olfactory tubercle and nucleus accumbens. The decreases in metabolism evident following PTT administration are not seen following

acute treatment with other psychostimulants or other dopaminergic

drugs, regardless of the dose range tested. PTT, then, is unique in its

effects at low doses, although at higher doses its effects strongly

resemble those of other psychostimulants.

CONCLUSIONS

The 2-DG method has provided a comprehensive view of the functional involvement of various brain areas in response to the administration of a novel tropane analog and has furnished information about the

specific neural substrates responsible for the behavioral and physiological effects of PTT. As demonstrated here, this method can reveal

unexpected actions of a drug not immediately obvious with either

behavioral testing or in vitro pharmacological assays. The significance

of the 2-DG method for medication development, then, is that it can

provide a powerful tool for investigating the nature, specificity,

79
anatomical substrates, and potential neurotoxicity of medications

developed for treatment of substance abuse or other diseases.

FIGURE 1. Effects of various doses of PTT on rates of LCGU in the

dorsomedial caudate (left) and dorsolateral caudate (right).

Values represent means and standard errors of rates of

glucose utilization (μmol/100 g/min), n = 4 in each group.

FIGURE 2. Effects of various doses of PTT on rates of LCGU in the

nucleus accumbens (left) and medial prefrontal cortex

(right). Values represent means and standard errors of

rates of glucose utilization (μmol/100 g/min), n = 4 in each

group.

80
FIGURE 3. Comparison of the alterations in LCGU accompanying the

administration of PTT and other psychostimulants in the

globus pallidus (left) and the entopeduncular nucleus

(right). Values are percent change from control for low,

medium, and high doses of PTT = 2ß-propanoyl-3ß-(4-

toluyl)-tropane (0.1, 0.5, and 1.0 mg/kg, IV; Porrino et al.

in preparation); Cocaine (0.25, 1.0, and 5.0 mg/kg, IV,

Porrino et al. 1988); and Meth = methamphetamine (0.5,

1.0, and 2.5 mg/kg, IV; Pontieri et al. 1990). Note the

similarities in the pattern of changes across the three drugs.

81
FIGURE 4. Comparison of the alterations in LCGU accompanying the

administration of PTT and other psychostimulants in the

olfactory tubercle (left) and the nucleus accumbens (right).

Values are percent change from control for low, medium,

and high doses of PTT = 2ß-propanoyl-3ß-(4-toluyl)-

tropane (0.1, 0.5, and 1.0 mg/kg, IV; Porrino et al., in

preparation); Cocaine = (0.25, 1.0, and 5.0 mg/kg, IV;

Porrino et al. 1988); and Meth = methamphetamine (0.5,

1.0, and 2.5 mg/kg, IV; Pontieri et al. 1990). Note the

differences in the patterns of glucose utilization changes

between PTT and cocaine and methamphetamine. Low

doses of PTT produce depressions in glucose utilization in

these structures not seen with cocaine or methamphetamine.

82
REFERENCES

Ableitner, A.; Wuster, M.; and Herz, A. Specific changes in local

cerebral glucose utilization in the rat brain induced by acute and

chronic diazepam. Brain Res 359:49-56, 1985.

Abraham, P.; Pitner, J.B.; Lewin, A.H.; Boja, J.W.; Kuhar, M.J.; and

Carroll, F.I. N-Modified analogues of cocaine. Synthesis and

inhibition of binding to the cocaine receptor. J Med Chem 35:141,

1992.

Carroll, F.I.; Gao, Y.; Abraham, P.; Lewin, A.H.; Parham, K.A.; Boja,

J.W., and Kuhar, M.J. Isopropyl and phenyl esters of 3ß-(4-substituted phenyl)tropane-2ß-carboxylic acids. Potent and selective

compounds for the dopamine transporter. J Med Chem 35:1813,

1992b.

Carroll, F.I.; Lewin, A.H.; Boja, J.W.; and Kuhar, M.J. Cocaine

receptor: Biochemical characterization and structure-activity

relationships of cocaine analogues at the dopamine transporter.

J Med Chem 35:969, 1992a.

Crane, A.M., and Porrino, L.J. Adaptation of the quantitative 2-

[

14C]deoxyglucose method for use in freely-moving rats. Brain Res

499:87-92, 1989.

Davies, H.M.L.; Saikali, E.; and Sexton, T.; Childers, S.R. Novel

2-substituted cocaine analogs: Binding properties at dopamine

transport sites in rat striatum. Eur J Pharmacol 244:93-97, 1993.

Davies, H.M.L.; Saikali, E.; and Young, W.B. Synthesis of

(±)-ferruginine and (±)-anhydroecgonine methyl ester by a tandem

cyclopropanation/Cope rearrangement. J Org Chem 56:5696, 1991.

Kozikowski, A.P.; Xiang, L.; Tanaka, J.; Bergmann, J.S.; and Johnson,

K.M. Use of nitrile oxide cycloaddition (NOC) chemistry in the

synthesis of cocaine analogs; mazindol binding and dopamine

uptake studies. Med Chem Res 1:312, 1991.

Kurumaji, A.; Dewar, D.; and McCulloch, J. Metabolic mapping with

deoxyglucose autoradiography as an approach for assessing drug

action in the central nervous system. In: London, E.D., ed. Imaging

Drug Action in the Brain. Boca Raton, FL: CRC Press, 1993. pp.

219-245.

Lewin, A.H.; Gao, Y.; Abraham, J.W.; Boja, J.W.; Kuhar, M.J.; and

Carroll, F.I. 2ß-Substituted analogues of cocaine. Synthesis and

inhibition of binding to the cocaine receptor. J Med Chem 35:135,

1992.

83
Mata, M.; Fink, D.J.; Gainer, H.; Smith, C.B.; Davidsen, L.; Savaki,

H.E.; Schwartz, W.J.; and Sokoloff, L. Activity-dependent energy

metabolism in rat posterior pituitary primarily reflects sodium

pump activity. J Neurochem 34:213-215, 1980.

McCulloch, J. Mapping functional alterations in the CNS with

[

14C]deoxyglucose. In: Iverson, L.; Iverson, S.; and Snyder, S., eds.

Handbook of Psychopharmacology. Vol. 15. New York: Plenum,

1982. pp. 321-410.

McCulloch, J.; Savaki, H.E.; and Sokoloff, L. The distribution of

alterations in energy metabolism in the rat brain produced by

apomorphine. Brain Res 243:67-80, 1982.

Pontieri, F.E.; Crane, A.M.; Seiden, L.S.; Kleven, M.S.; and Porrino,

L.J. Metabolic mapping of the effects of intravenous methamphetamine administration in freely-moving rats. Psychopharmacology 102:175- 182, 1990.

Porrino, L.J.; Bums, R.S.; Crane, A.M.; Palombo, E.; Kopin, I.J.; and

Sokoloff, L. Changes in local cerebral glucose utilization associated with Parkinson’s syndrome induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in the primate. Life Sci 40:

1657-1664, 1987.

Porrino, L.J., and Crane, A.M. Metabolic mapping of the effects of

drugs of abuse with the 2-[14C]deoxyglucose method. In: Adler,

M.W., and Cowan, A., eds. Modem Methods in Pharmacology:

Testing and Evaluation of Drugs of Abuse. New York: Alan R.

Liss, 1990. pp. 147-164.

Porrino, L.J.; Domer, F.R.; Crane, A.M.; and Sokoloff, L. Selective

alterations in cerebral metabolism within the mesocorticolimbic

dopaminergic system produced by acute cocaine administration in

rats. Neuropsychopharmacology 1:109-118, 1988.

Porrino, L.J.; Lucignani, G.; Dow-Edwards, D.L.; and Sokoloff, L.

Correlation of dose-dependent effects of acute amphetamine

administration on behavior and local cerebral metabolism in rats.

Brain Res 307:311-320, 1984.

Porrino, L.J.; Migliarese, K.; Davies, H.M.L.; Saikali, E.; and Childers,

S.R. Behavioral effects of the novel tropane analog, 2ß-propanoyl3ß-(4-toluyl)-tropane (PTT). Neuropharmacology. Submitted for

publication.

84
Porrino, L.J., and Pontieri, F.E. Metabolic mapping of the effects of

psychomotor stimulants in rats. In: London, E.D., ed. Imaging

Drug Action in the Brain. Boca Raton, FL: CRC Press, 1993. pp.

247-263.

Sokoloff, L. The radioactive deoxyglucose method: Theory, procedure,

and applications for the measurement of local glucose utilization in

the central nervous system. In: Agranoff, B.W., and Aprison, M.H.,

eds. Advances in Neurochemistry. Vol. 4. New York: Plenum,

1982. pp. 7-36.

Sokoloff, L., and Porrino, L.J. Some fundamental considerations in the

application of the deoxyglucose method to pharmacological studies.

In: Kriegelstein, I. ed. Pharmacology of Cerebral Ischemia.

Amsterdam: Elsevier, 1986. pp. 65-76.

Sokoloff, L.; Reivich, M.; Kennedy, C.; DesRosiers, M.H.; Patlak,

C.S.; Pettigrew, K.D.; Sakurada, O.; and Shinohara, M. The

[

14C]deoxyglucose method for the measurement of local cerebral

glucose utilization: theory, procedure and normal values in the

conscious and anesthetized albino rat. J Neurochem 28:897-916,

1977.

Wechsler, L.R.; Savaki, H.E.; and Sokoloff, L. Effects of d- and

l-amphetamine on local cerebral glucose utilization in the

conscious rat. J Neurochem 32:15, 1979.

ACKNOWLEDGMENTS

This work was supported by United States Public Health Service grants

P50 DA06634, DA07522 (LJP), and DA06301 (HMLD) and Training

Grant DA07246 (LWH) from NIDA.

AUTHORS

Linda J. Porrino, Ph.D.

Associate Professor

Lisa Williams-Hemby, B.S.

Graduate Student

85
Department of Physiology and Pharmacology

Bowman Gray School of Medicine

Medical Center Boulevard

Winston-Salem, NC 27157-1083

Huw M.L. Davies, Ph.D.

Associate Professor

Department of Chemistry

Wake Forest University

Winston-Salem, NC 27 104

86
Noninvasive Evaluation of the

Sympathetic Nervous System of

the Heart by Nuclear Imaging

Procedures

Markus Schwaiger, James C. Sisson, and Donald Wieland

INTRODUCTION

The autonomic nervous system plays an important role in the

regulation of cardiovascular performance under physiologic and

pathophysiologic conditions. The sympathetic and parasympathetic

innervations of the heart facilitate the electrophysiologic and hemodynamic adaptations to changing cardiovascular demands. Both

sympathetic and parasympathetic tones control the rate of electrophysiologic stimulation, whereas the contractile performance of the

heart is modulated primarily by sympathetic neurotransmission. The

evaluation of the sympathetic nervous system has been limited in the

past to catheterization of the coronary sinus vein and measurement of

plasma levels of norepinephrine (NE) and its metabolites in arterial

and venous blood across the heart (Goldstein et al. 1988). These

procedures are invasive and do not provide regional assessment of

neuronal integrity within the heart. More recently, radiolabeled

pharmaceuticals have been introduced for the noninvasive assessment

of the sympathetic nervous system using nuclear medicine imaging

approaches (Wieland et al. 1983). Improvement in radiochemistry and

imaging technology allows the specific visualization of sympathetic

nerve terminals and adrenergic receptors of the living heart. Iodinated

compounds have been used with planar and tomographic gamma

camera imaging for the delineation of sympathetic nerve terminals.

Recently, positron emission tomography (PET) has been employed to

assess more specifically uptake as well as synthesis of neurotransmitters in the heart. This new imaging modality, in combination with

short-lived isotopes, permits the quantitative definition of regional

myocardial tracer distribution, which may be useful for the quantification of regional neuronal function in the living human heart.

87
This chapter addresses the emerging role of nuclear imaging procedures in the noninvasive characterization of the cardiac sympathetic

nervous system. It addresses the physiology of the sympathetic

nervous system and discusses radiochemical approaches to tracing

sympathetic neurotransmission. Finally, it describes initial experimental and clinical experience with these radiopharmaceuticals in the

animal model and in human patients with various cardiac diseases.

PHYSIOLOGY OF THE SYMPATHETIC NERVOUS SYSTEM

Presynaptic Nerve Terminal

The postganglionic sympathetic fibers travel along vascular structures

on the surface of the heart. On entering the myocardial wall, the fibers

branch into multiple sympathetic nerve terminals. These terminals

include vesicles, which are the storage pools for neurotransmitters,

enzymes, and other proteins. NE, the dominant neurotransmitter in

cardiac sympathetic nerve terminals, is synthesized from the amino

acid tyrosine by several enzymatic steps (figure 1). Synthesis of dopa

from tyrosine is the rate-limiting step in the biosynthesis of catecholamines. Dopamine, converted from dopa, is transported into

storage vesicles by an active transport mechanism. Within the storage

vesicles, NE is synthesized, stored, and released on nerve stimulation

by exocytosis (Levitt et al. 1965). The regulation of neuronal NE

release is complex. Alpha-2-receptors have been postulated to exist on

the membrane of the presynaptic nerve terminal. Activation of these

receptors by NE leads to a negative feedback of the exocytotic process.

In addition to the presynaptic -2-receptors, the presence of other

inhibitory presynaptic receptors suggests that multiple control mechanisms modulate the NE release from sympathetic nerve terminals

(Francis 1988).

The reuptake of released NE by the nerve terminal from the synaptic

cleft is an important and efficient mechanism to maintain the stores of

neurotransmitters. As much as 80 percent of the NE that is released

from the nerve terminal is taken up through this mechanism (uptake-I

mechanism). Following reuptake, NE recycles into the storage vesicles

by active transport (Goldstein et al. 1988).

88
FIGURE 1. Schematic display of the sympathetic nerve terminal. NE is

synthesized from dopamine by the dopamine-betahydroxylase and stored in vesicles. Released NE undergoes

reuptake by the nerve terminal (uptake I). Only a small

amount of NE binds to the adrenergic receptors or is taken

up by nonneuronal tissue (uptake II).

A fraction of the released NE diffuses back into the vascular space

where it can be measured as NE spillover in the coronary sinus venous

blood (Goldstein 1981). Only a small amount of NE in the synaptic

cleft is actually available to activate receptors on the surface of the

myocyte or to enter the cardiac cell directly (uptake-II mechanism).

89
NE undergoes rapid metabolism in neuronal and extraneuronal tissue.

Metabolism of the neurotransmitter in the neuronal cytosol is by

monoamine oxidase (MAO), whereas extraneuronal NE is metabolized

by the catechol-o-methyltransferase (COMT) enzyme system (Axelrod

1960; Hertting et al. 1961).

Adrenergic Receptors

General classes of postsynaptic adrenergic receptors are designated as

or ß-receptors. This distinction is based on pharmacological studies

describing specific functional responses of each receptor system

(Lefkowitz and Caron 1988). A positive inotropic response to catecholamine stimulation is mediated primarily by ß-receptors. In the

normal left and right ventricular myocardium, the subset of receptors,

ß1, makes up about 80 percent of the total pool of ß-receptors (Stiles

et al. 1983). It has been speculated that ß2-receptors are most prevalent in the atria and that ß2-selective agonists elicit a more chronotropic response than do ß1-selective drugs (Carlsson et al. 1977).

Recent investigations indicate that functional ß-receptors of the heart

exist on the surface of cell membranes, but other nonfunctional ßreceptors are within the cells. Internalization of the receptors may be

in response to homologous desensitization (a method of regulation) of

ß-receptors under various physiologic and pathophysiologic conditions

(Lefkowitz and Caron 1988; Bristow et al. 1982).

RADIOPHARMACEUTICALS

The complex neurophysiology of cardiac innervation makes it apparent

that the noninvasive characterization of neuronal function by a single

tracer is difficult. Physiologic function of the sympathetic nerve

terminal includes the synthesis and release of NE as well as reuptake

and metabolism of neurotransmitters. The characterization of this

complex physiologic system by radiopharmaceuticals requires detailed

understanding of their physiologic behavior. Several approaches of

tracer design that can be pursued for the evaluation of the sympathetic

nervous system include the use of analogs, neurotransmitter precursors,

and receptor antagonists. Table 1 lists radiopharmaceuticals currently

used for the characterization of the sympathetic nervous system by

nuclear medicine procedures.

90
TABLE 1. Currently available tracers for the noninvasive

characterization of presynaptic and postsynaptic

sympathetic nervous system

NE Analogs

Radiopharmaceutical research efforts have focused initially on the

design of radiotracers that serve as markers for the presynaptic neuron.

An obvious tracer for the delineation of adrenergic nerve terminal is

NE. The first in vivo image of the heart based on adrenergic nerve

accumulation of a radiotracer was accomplished, appropriately, with

[

11C]NE in 1975 (Fowler et al. 1975). In the early 1980s, MIBG was

developed at the University of Michigan for selective mapping of the

heart’s sympathetic nerve endings using conventional nuclear medicine

procedures (figure 2) (Wieland et al. 1981a).

Recently, radiopharmaceuticals have been developed at the University

of Michigan for the PET characterization of the autonomic nervous

system. Development of these radiopharmaceuticals centered around

the false neurotransmitter metaraminol, which is a close structural

analog of NE (figure 2). Metaraminol has been used extensively in

animal models as a tool to study the transport characteristic of the

neuronal uptake (Anton and Berk 1977). This tracer has high affinity

for the catecholamine uptake mechanism, and its retention in tissue is

not complicated by neuronal metabolism. The metabolic refractoriness

91
FIGURE 2. Chemical structure of NE, MIBG, and [11C]HED

of metaraminol relative to NE is caused by the presence of the

methyl group that effectively blocks the action of MAO and the

absence of a catechol group that confers resistance to COMT.

Following the initial validation of titrated metaraminol as a suitable

presynaptic tracer of the heart, [

18F]metaraminol was synthesized using

the electrophilic fluorination method (Wieland et al. 1990). However,

the specific activity of [

18F]metaraminol achievable with this synthetic

method was not high enough to avoid pharmacological effects if

injected intravenously.

Another analog of metaraminol subsequently has been synthesized at

the authors’ laboratory (Rosenspire et al. 1990). HED is structurally

very similar to metaraminol, but it can be labeled with [

11C] to yield a

radiopharmaceutical with very high specific activity (figure 2). This

radiopharmaceutical behaves very similarly to [

11F]metaraminol, but,

because of a short physical half-life, it can be administered in large

doses and in combination with other PET tracers such as flow and

metabolic markers.

NE Precursors

A biochemical parameter that can be used to assess sympathetic function is the rate of endogenous NE synthesis. [

18F]Fluorodopamine

allows imaging of sympathetic nerve terminals as demonstrated in

animal experiments (Eisenhofer et al. 1989; Goldstein et al. 1990).

Because this tracer is converted to [

18F]NE within the sympathetic

92
nerve terminals, synthesis and release can be studied using this radiopharmaceutical. However, the tracer kinetics are expected to be complicated, and sophisticated tracer kinetic modeling will be necessary to

deconvolute synthesis and neuronal release of this radiotracer in the

living heart. Because secreted NE is taken up by the neuron terminal,

pharmacological interventions to inhibit the uptake-I pathway are

needed to quantify the neuronal release rate of NE using

[

18F]fluorodopamine.

Adrenergic-Receptor Ligands

A number of receptor sites or uptake carriers in the adrenergic

neuron could be targeted with labeled ligands. Numerous reports

exist on the subject of radiolabeled adrenergic-receptor antagonists.

[

11C]Propanolol and [11C]practolol have been synthesized and evaluated

for PET imaging (Syrota 1990). [

123I]ICYP has been proposed as a

marker for the assessment of myocardial ß-receptor density using

camera imaging techniques. However, these tracers are lipophilic and

exhibit considerable binding to lung tissue, limiting the accurate

quantification of regional receptor binding by imaging approaches.

Recently, a hydrophilic ß-receptor antagonist (CGP12177) was labeled

with [

11C] for use in combination with PET (Syrota 1990). Initial

results in the animal model are promising for the noninvasive quantification of myocardial ß-receptor density. However, such radiopharmaceuticals are still considered experimental.

EXPERIMENTAL AND CLINICAL IMAGING OF THE

SYMPATHETIC NERVOUS SYSTEM

Radiolabeled MIBG

Labeled with [

131I], MIBG concentrates in adrenergic tumors, and the

radiopharmaceutical has become popular to portray scintigraphically

the locations of pheochromocytomas (Shapiro et al. 1985; Sisson et al.

1981) and neuroblastomas (Geatti et al. 1985; Hoefnagel et al. 1987).

MIBG also can be labeled with [

123I], a radionuclide that, relative to

[

131I], is safer in larger doses and exhibits more favorable imaging

characteristics. Thus, [

123I]MIBG was selected to depict the adrenergic

neurons in the heart (Kline et al. 1981).

93
Studies in Cell Cultures and Animals. The pharmacology of

MIBG largely has been delineated. The agent enters adrenergic cells,

including neuron terminals, through the uptake-I pathway (Jaques et al.

1984). Some MIBG may enter the adrenergic tissues by diffusion

(Jaques et al. 1984), but, under ordinary conditions, it seems likely that

this route carries only a small fraction of the radiopharmaceutical.

Within an adrenergic cell, MIBG is retained in specialized storage

vesicles (Gasnier et al. 1986; Wieland et al. 1981b). These characteristics enable the uptake and retention of MIBG to represent the

integrity of adrenergic neurons, particularly those in the heart. Pretreatment with the neurotoxin 6-hydroxydopamine (De Champlain and

Nadeau 1971) caused a generalized decrease in the MIBG uptake

retention in the hearts of rats, indicating the specificity of this tracer

for nerve terminals (Sisson et al. 1987a). Regional denervation can be

produced by the application of phenol to localized areas of pericardium

(Barber et al. 1984). This type of treatment produced, within the

region of neuronal injury, decreases in MIBG concentrations that were

easily detectable by scintigraphic imaging procedures (Dae et al. 1989;

Minardo et al. 1988; Sisson et al. 1988).

Thus, images of the heart made with [

123I]MIBG are capable of demonstrating neuronal injuries (i.e., denervation) that are either global or

regional in extent. Minardo and coworkers (1988) employed this

approach for the delineation of denervated, viable myocardium following an ischemic injury. Regions of infarction can be identified by

flow tracers such as thallium-201(201TI) and subtracted from the defect

appearing on the [

123I]MIBG image, which includes both denervated

and infarcted myocardium. However, the effects of reduced blood

flow on the delivery of the MIBG to the peri-infarct myocardium must

be taken into account (Sisson et al. 1990a). Large regions of periinfarct denervation can be visualized readily (Minardo et al. 1988), but

the identification of smaller regions and the quantification of denervation will require new and sophisticated approaches to the analysis of

neuron integrity and regional myocardial perfusion.

In images of the heart made soon after injection of MIBG, the nonneuronal uptake of the radiopharmaceutical appeared to be as much as

20 percent. However, after several hours, the vast majority of MIBG

probably resides within neurons (Sisson et al. 1987a). The physiologic

function of adrenergic neurons leads to secretion of NE by exocytosis,

94
and MIBG exits the neuron terminals by this same mechanism (Jaques

and Tobes 1985). The rate of loss of NE from the neurons in an organ

such as the heart appears to be proportional to the functional activity

of these nerves (Hasking et al. 1986); the NE “spills over” into the

circulation. The rate of loss of MIBG from the rat heart followed a

pattern similar to that of NE, but the rate for MIBG was more rapid

(Sisson et al. 1987a). MIBG possibly exits more rapidly from neurons

because a greater fraction of MIBG than of NE is extravesicular, where

leakage from a neuron may be independent of functional activity in the

nerve. Nevertheless, changes in neuron activity are reflected in

changes in the rates of loss of MIBG from the heart. This was true

when nerve traffic was increased by eating (Sisson et al. 1987b ) or

stimulated by the pharmacologic action of yohimbine (Sisson et al.

1990b) and when the nerve function was decreased by inhibition with

clonidine (Sisson et al. 1990b).

The animal studies indicate that scintigraphic portrayal of uptake and

retention (over hours) of [

123I]MIBG will identify healthy neurons in

the heart, and injured or destroyed neurons will be depicted by

diminished or absent uptake and retention. In addition, quantified

changes in rates of loss of the radiopharmaceutical may provide an

index of changes in nerve activity within that organ.

Studies in Human Subjects. As in dog hearts, [

123I]MIBG in the

normal human myocardium appears to be homogeneously distributed

throughout the walls of the left ventricle (Sisson et al. 1987b) (figure

3); the apical segments contain lesser quantities because there is less

myocardium and diminished innervation (Pierpont et al. 1985). The

nonneuronal component of MIBG in the human heart probably is

reduced to low levels by 2 hours after injection (Sisson et al. 1987b).

Transplanted hearts are denervated, at least for many months, and,

hence, represent a model in which neuronal function is absent. In such

hearts, the uptake of MIBG, even at 1 hour, was virtually undetectable

(Glowniak et al. 1989). Accordingly, generalized adrenergic neuropathies affecting the heart are associated with diminished concentrations of MIBG throughout most of the heart. However, the neuronal

injuries may not be uniform (Sisson et al. 1987b). Uneven patterns of

MIBG conceivably may define the heterogenous nature of neuronal

injury and detect patterns that predict serious arrhythmias (Kahn et al.

95
FIGURE 3. Tomographic images of a normal volunteer 20 hours after

injection of [123I] MIBG. Left: short axis (oblique) slice

through midheart; the left ventricle wall is most prominent,

but activity can be seen in the right ventricle (to the left).

Activity below is in the liver. Mid-oblique coronal slice

showing left ventricle walls and apex; right ventricle is

again seen. Right: oblique sagittal slice showing only left

ventricle.

1988). The rapid early egress of MIBG from the hearts of patients

with autonomic neuropathy (Nakajo et al. 1985) may be the consequence of a greater proportion of the more transient, nonneuronal

MIBG, or from neurons with a lesser capacity to retain MIBG, or both.

During heart failure, the adrenergic nervous system becomes dysfunctional, a state associated with diminished levels of NE in the myocardium (Chidsey et al. 1966; DeQuattro et al. 1973). However, the

increase (Hasking et al. 1986) and decrease (DeQuattro et al. 1973) of

the synthesis of NE in failing myocardium suggest that the identified

abnormalities of neurons vary and possibly change during the course of

the disease. Reports of cardiac uptake and retention of [

123I]MIBG in

patients with heart failure caused by congestive cardiomyopathy have

recorded differing results. When the concen-trations of MIBG were

measured between 15 and 85 minutes after injection, the hearts of

normal subjects showed no change, but those of patients with cardiomyopathy exhibited a significant loss of 25 percent (Henderson et al.

1988). In contrast, other investigators observed, by visual assessment

of images, that patients with subnormal ejection fractions had low or

absent levels of cardiac MIBG at 2 hours, but there was no appreciable

96
change over the next 4 hours (Schofer et al. 1988). Another group

found, by quantifying the radiopharmaceutical in the heart, a sustained

decline in concentrations of MIBG over a time period of from 1 to 16

hours and a more rapid loss in the patients with cardiomyopathy

(Glowniak et al. 1989). Summarizing these reports indicates the

probability that the rates of loss of MIBG from the cardiomyopathic

heart are increased. This more rapid exit of MIBG possibly signifies

an increased rate of loss of NE as well, but the concentrations of NE

are low in these hearts (Schofer et al. 1988); thus, the total adrenergic

nerve function, measured as the total NE secretion, may not be elevated. Moreover, it is possible that some of the effects occur because

of neuron injury (perhaps ischemia), and the increase in rate of escape

of MIBG and of NE loss occurs through a pathway unrelated to the

physiologic activity of the nerves.

It is important to remember that generalized decreases in the uptake

and retention of MIBG may be caused by a number of factors, some

of which are not related directly to heart and nerve function. For

example, elevated levels of catecholamines (Nakajo et al. 1983) and a

variety of drugs (Sisson et al. 1987b; Khafagi et al. 1989) will

diminish the uptake of MIBG by the heart.

[

18F]Metaraminol and [11C]HED

Animal Validation. Extensive testing with [

18F]metaraminol and

[

11C]HED has been carried out in animal models. To determine

whether [

18F]metaraminol or [11C]HED is actually stored in the vesicles

of sympathetic nerve terminals, the retention of both tracers was

assessed in the myocardial tissue of a second group of animals that had

been pretreated with reserpine to inhibit vesicular storage. Reset-pine

reduced the retention of [

18F]metaraminol and [11C]HED by more than

70 percent, suggesting that the majority of the tracer is retained in the

vesicles of the sympathetic nerve terminals. Furthermore, the tracer

retention was reduced following treatment with 6-hydroxydopamine,

which destroys sympathetic neurons in the heart (Wieland et al. 1990).

The results of these pharmacological interventions confirmed that the

nonneuronal binding of both tracers in myocardial tissue is very low.

An animal model with a short period of regional ischemia and

subsequent reperfusion was used to demonstrate the sensitivity

97
of sympathetic neurons to ischemia (Schwaiger et al. 1990a).

[

18F]Metaraminol was injected into an open-chest dog model, following a 30-minute occlusion of the left anterior descending artery,

and tissue activity was determined 30 minutes after intravenous injection. Despite restoration of blood flow, [

18F]metaraminol retention in

the reperfused myocardium was reduced significantly as compared with

the control myocardium. These data confirmed previous observations

that neuronal dysfunction can occur without myocardial necrosis.

Tracers such as [

18F]metaraminol and [11C]HED can be used to delineate the integrity of the neuronal uptake mechanism of catecholamines.

Because NE is stored in the vesicles with a high concentration

gradient, it is expected that this storage mechanism is sensitive to

deprivation of oxygen and high-energy phosphates. Further longitudinal studies are needed to define the possible recovery of neuronal

function following an ischemic injury.

Clinical Application. The only PET radiopharmaceutical available for

the evaluation of the sympathetic nervous system in humans at the

present time is [

11C]HED (Schwaiger et al. 1990b). Figure 4 shows

[

11C]HED images obtained in a normal volunteer following intravenous

injection of the tracer. These cross-sectional images through the left

ventricle were obtained 40 minutes after injection and demonstrate the

high image quality achievable with this tracer. Although [

11C]HED

clears rapidly from blood, levels of the agent remain stable in the

myocardium. The heart-to-blood ratio increases rapidly and averages

at 5 minutes after injection more than 5:l. The retention of [

11C]HED

is homogeneous throughout the left ventricle, reflecting the distribution

of sympathetic nerve terminals in this organ. The findings in normal

volunteers were compared with those obtained in patients with cardiac

transplantation, which provides a unique clinical model of sympathetic

denervation. [

11C]HED uptake in the left ventricle was reduced

markedly in the transplant patients (figure 5). The relative retention of

[

11C]HED was reduced by 76 percent in five patients with recent

cardiac transplantation as compared with the control population. This

indicates a relatively high specificity of [

11C]HED for the sympathetic

nervous system in the human heart.

It is interesting to note that regional [

11C]HED uptake in patients with

remote cardiac transplant showed a heterogeneous pattern. In particular, the basal aspects of the left ventricle appeared to extract [

11C]HED

98
FIGURE 4. Cross-sectional PET images obtained in normal volunteer

40 minutes following injection of [

11C]HED (above). Note

the high contrast between myocardial and blood pool

activity. For comparison, blood flow images with (82Rb) are

displayed below.

to a greater extent than early after surgery. These data offered the first

direct evidence of sympathetic reinnervation of the human cardiac

transplant. Such reinnervation has been documented extensively in the

animal model, but there is ongoing controversy about reinnervation in

the human transplant (Schwaiger et al. 1990c).

Figure 6 shows a [

11C]HED study obtained in a patient with acute

anterior myocardial infarction. This patient underwent thrombolytic

therapy, and the [

11C]HED PET study was obtained within 3 days after

the therapy. The blood flow images (

82Rb) indicate reduced blood

flow in the anteroapical regions of the left ventricle. The [

11C]HED

study clearly indicates a larger defect of tracer retention. The

combined tracer approach using a blood flow tracer in [

11C]HED

99
FIGURE 5. [

11C]HED PET images obtained in a patient with dilated

cardiomyopathy. Short-axis images from base (left upper

row) to apex (right lower row) are displayed. The tracer

distribution is heterogenous with an uptake gradient from

base to apex of the left ventricle indicating nonuniform

neuronal damage in patients with dilated cardiomyopathy.

allows accurate delineation of neuronal dysfunction in relation to the

infarct territory. Thus, [

11C]HED may be useful in the evaluation of

neuronal integrity in patients with ischemic heart disease. Since

heterogeneity of sympathetic tone has been linked to dysrhythmias in

patients with ischemic heart disease, such an imaging approach may

provide prognostic information for the identification of high-risk

patients.

100
FIGURE 6. [

11C]HED (right) and 82Rb (left) images obtained in a

patient with recent anterior myocardial infraction. The

arrows indicate the flow and neuronal defect within the

anterior wall. Please note the larger [11C]HED defect

suggesting neuronal dysfunction beyond the infarct area

depicted by the flow tracer.

As discussed above, alterations of the sympathetic system have been

observed in patients with heart failure. Reduced cardiac-pump function leads to increased sympathetic tone involving high plasma NE

levels and increased central stimulation of the cardiac sympathetic

nerves. Because the reuptake and storage of NE in the presynaptic

nerve terminals are important mechanisms to regulate NE content, the

dysfunction of the nerve terminals may be an important pathophysiologic factor of the overexposure of the postsynaptic receptor sites to

catecholamines. [

11C]HED allows assessment of the integrity of

101
presynaptic nerve terminals in these patients. Myocardial retention of

[

11C]HED was quantified in patients with dilated cardiomyopathy.

Figure 6 displays the [

11C]HED images in a patient with dilated cardiomyopathy. [

11C]HED retention is heterogeneously reduced with

gradient from base to apex of left ventricle. The neuronal catecholamine storage capacity as assessed by quantitation of [

11C]HED

tissue retention was reduced markedly in patients with cardiomyopathy,

approaching values found in the denervated transplanted heart

(Schwaiger et al. 1990d). The relatively large standard deviation of

the measurements in patients with cardiomyopathy indicates a large

variability of findings. These quantitative data obtainable by PET

indicate that the presynaptic uptake of catecholamines is altered

severely in patients with cardiomyopathy; this may affect the reuptake

of extraneuronal NE by sympathetic nerve terminals and, hence, result

in an overexposure of myocytes to NE. This hypothesis is supported

by recent findings that indicate a beneficial effect of ß-receptor

blockade in patients with congestive heart failure (Waagstein et al.

1989). Thus, the quantitative delineation of the sympathetic nervous

function in such patients by PET may aid in the selection of individuals for therapy with ß-receptor blockers and enhance understanding of the cardiac sympathetic nervous system in patients with

dilated cardiomyopathy.

The Effect of Cocaine on [

11C]HED Kinetics

Cocaine has well-documented cardiotoxicity. Acute exposure to this

drug results in coronary vasoconstriction, which together with

increased oxygen demand may lead to myocardial ischemia. Although

the pharmacology of cocaine is well described, little is known about its

direct effect on cardiac neurons. Imaging approaches using [

11C]HED

allow the investigation of the pharmacokinetics of cocaine on the

uptake of catecholamines in the sympathetic nerve terminal. Preliminary results by Melon and colleagues from the authors’ institution

demonstrate the marked reduction of [

11C]HED uptake in the canine

heart following intravenous injection. The effect of one injection is

sustained for several hours (Melon et al. 1992). These studies suggest

that PET may be uniquely suited to define the time course and intensity of drug effects on cardiac sympathetic neurons. Future studies in

human drug abuse may define possible alterations of sympathetic

innervation following chronic drug abuse.

102
REFERENCES

Anton, A., and Berk, A. Distribution of metaraminol and its relation to

norepinephrine. Eur J Pharmacol 44:161-167, 1977.

Axelrod, J. The fate of adrenaline and noradrenaline. In: Vane, J.;

Wolstenholme, G.; and O’Connor, M., eds. Adrenergic Mechanisms.

Boston: Little, Brown, 1960. pp. 28-39.

Barber, M.; Mueller, T.; Davies, B.; and Zipes, D. Phenol topically

applied to canine left ventricular epicardium interrupts sympathetic

but not vagal afferents. Circ Res 55:532-544, 1984.

Bristow, M.; Ginsburg, R.; Minobe, W.; Cubicciotti, R.S.; Sageman,

W.S.; Lurie, K.; Billingham, M.E.; Harrison, D.C.; and Stinson,

E.B. Decreased catecholamine sensitivity and beta-adrenergic

receptor density in failing human hearts. N Engl J Med 307:

205-211, 1982.

Carlsson, E.; Dahof, C.; and Hedberg, A. Differentiation of cardiac

chronotropic and inotropic effects of beta-adreno-ceptor angonists.

Naunyn Schmiedebergs Arch Pharmacol 300:101-106, 1977.

Chidsey, C.; Sonnenblick, E.; Morrow, A.; and Braunwald, E.

Norepinephrine stores and contractile force of papillary muscle from

failing human heart. Circulation 33:43-51, 1966.

Dae, M.; O’Connell, J.; Botvinick, E.; Aheam, T.; Yee, E.; Huberty,

J.; Mori, H.; Chin, M.; Hattner, R.; Herre, J.; and Munoz, L.

Scintigraphic assessment of regional cardiac adrenergic innervation.

Circulation 79:634-644, 1989.

De Champlain, J., and Nadeau, R. 6-Hydroxydopamine, 6-hydroxydopa, and degeneration of adrenergic nerves. Fed Proc 30:877-885,

1971.

DeQuattro, V.; Nagatsu, T.; Mendez, A.; and Verska, J. Determinants

of cardiac noradrenaline depletion in human congestive failure.

Cardiovasc Res 7:344-350, 1973.

Eisenhofer, G.; Hovevey-Sion, D.; Kopin, I.; Miletich, R.; Kirk, K.;

Finn, R.; and Goldstein, D. Neuronal uptake and metabolism of 2-

and 6-fluorodopamine: False neurotransmitters for positron emission

tomographic imaging of sympathetically innervated tissues.

J Pharmacol Exp Ther 248:419-427, 1989.

103
Fowler, J.; Wolf, A.; Cristman, R.; MacGregor, R.; Ansari, A.; and

Atkins, H. Carrier-free 11C-labeled catecholamines. In:

Subramanian, G.; Rhodes, B.; Cooper, J.; and Sodd, V., eds.

Radiopharmaceuticals. New York: Society of Nuclear Medicine,

1975. pp. 196-204.

Francis, G. Modulation of peripheral sympathetic nerve transmission.

J Am Coll Cardiol 12:250-254, 1988.

Gasnier, B.; Roisin, M.; Scherman, D.; Coornaert, S.; Desplanches, G.;

and Henry, J. Uptake of metaiodobenzylguanidine by bovin

chromaffin granule memberances. Mol Pharmacol 29:275-280,

1986.

Geatti, O.; Shapiro, B.; Sisson, J.; Hutchinson, R.; Mallette, S.; Eyre,

P.; and Beierwaltes, W. 131-Metaiodobenzylguanidine (131-IMIBG) scintigraphy for the location of neuroblastoma: Preliminary

experience in 10 cases. J Nucl Med 26:736-742, 1985.

Glowniak, J.; Turner, F.; Gray, L.; Palac, R.; Lagunas-Solar, M.; and

Woodward, W. Iodine- 123 metaiodobenzylguanidine imaging of the

heart in idiopathic congestive cardiomyopathy and cardiac

transplants. J Nucl Med 30:1182-1191, 1989.

Goldstein, D. Plasma norepinephrine as an indicator of sympathetic

neural activity in clinical cardiology. Am J Cardiol 48:1147, 1981.

Goldstein, D.; Brush, J., Jr.; Eisenhofer, G.; Stull, R.; and Esler, M. In

vivo measurement of neuronal uptake of norepinephrine in the

human heart. Circulation 78:41-48, 1988.

Goldstein, D.; Chang, P.; Eisenhofer, G.; Miletich, R.; Finn, R.;

Bather, J.; Kirk, K.; Bacharach, S.; and Kopin, I. Positron emission

tomographic imaging of cardiac sympathetic innervation and

function. Circulation 81:1606-1621, 1990.

Hasking, G.; Esler, M.; Jennings, G.; Burton, D.; and Korner, P.

Norepinephrine spillover to plasma in patients with congestive heart

failure: Evidence of increased overall and cardiorenal sympathetic

nervous activity. Circulation 73:615-621, 1986.

Henderson, E.; Kahn, J.; Corbett, J.; Jansen, D.; Pippin, J.; Kulkami,

P.; Ugolini, V.; Akers, M.; Hansen, C.; Buja, L.; Parkey, R.; and

Willerson, J. Abnormal I-123 metaiodobenzylguanidine myocardial

washout and distribution may reflect myocardial adrenergic

derangement in patients with congestive cardiomyopathy.

Circulation 78:1192-1199, 1988.

104
Hertting, G.; Axelrod, J.; Kopin, J.; and Whitby, L. Effect of drugs on

the uptake and metabolism of H3-norepinephrine. J Pharmacol Exp

Ther 134:146-153, 1961.

Hoefnagel, C.; Voute, P.; de Kraker, J.; and Marcuse, H. Radionuclide

diagnosis and therapy of neural crest tumors using iodine-131

metaiodobenzylguanidine. J Nucl Med 28:308-314, 1987.

Jaques, S., Jr., and Tobes, M. Comparison of the secretory mechanisms

of metaiodobenzylguanidine (MIBG) and norepinephrine (NE) from

cultured bovin adrenomedullary cells. J Nucl Med 26:17, 1985.

Jaques, S., Jr.; Tobes, M.; Sisson, J.; Baker, J.; and Wieland, D.

Comparison of the sodium dependency of uptake of metaiodobenzylguanidine and norepinephrine into cultured bovin

adrenomedullary cells. Mol Pharmacol 26:539-546, 1984.

Kahn, J.; Sisson, J.; and Vinik, A. Prediction of sudden cardiac death

in diabetic autonomic neuropathy. J Nucl Med 29:1605-1606, 1988.

Khafagi, F.; Shapiro, B.; Fig, L.; Mallette, S.; and Sisson, J. Labetalol

reduces iodine-131 MIBG uptake by pheochromocytoma and normal

tissues. J Nucl Med 30:481-489, 1989.

Kline, R.; Swanson, D.; and Wieland, D. Myocardial imaging in man

with I- 123 meta-iodobenzylguanidine. J Nucl Med 22:129-132,

1981.

Lefkowitz, R., and Caron, M. Adrenergic receptors. J Biol Chem

263:4993-4996, 1988.

Levitt, M.; Spector, S.; Sjoerdsma, A.; and Udenfriend, S. Elucidation

of the rate-limiting step in norepinephrine biosynthesis in the

perfused guinea-pig heart. J Pharmacol Exp Ther 148:1, 1965.

Melon, P.; DeGrado, T.; Nguyen, N.; Tooronigian, S.; Hutchins, G.;

and Schwaiger, M. Noninvasive assessment of regional myocardial

sympathetic neuronal function following intravenous cocaine

injection in dogs. J Nucl Med 33:994, 1992.

Minardo, J.; Tuli, M.; Mock, B.; Weiner, R.; Pride, H.; Wellman, H.;

and Zipes, D. Scintigraphic and electrophysiological evidence of

canine myocardial sympathetic denervation and reinnervation

produced by myocardial infarction or phenol application. Circulation

78:1008-1019, 1988.

Nakajo, M.; Shapiro, B.; and Glowniak, J. Inverse relationship between

cardiac accumulation of meta-[131I]-iodobenzylguanidine (I-131-

MIBG) and circulating catecholamines in suspected pheochromocytoma. J Nucl Med 24:1127-1134, 1983.

105
Nakajo, M.; Shimabukuro, K.; Miyaji, N.; Shimada, J.; Shirono, K.;

Sakata, H.; Yoshimura, H.; Yonekura, R.; and Shinohara, S. Rapid

clearance of iodine- 13 1 MIBG from the heart and liver of patients

with adrenergic dysfunction and pheochromocytoma. J Nucl Med

26:357-365, 1985.

Pierpont, G.; DeMaster, E.; Reynolds, S.; Pederson, J.; and Cohn, J.

Ventricular myocardial catecholamines in primates. J Lab Clin Med

106:205-210, 1985.

Rosenspire, K.; Kaka, M.; Jewett, D.; Van Dort, M.; Gildersleeve, D.;

Schwaiger, M.; and Wieland, D. Synthesis and preliminary

evaluation of [11C]meta-hydroxyephedrine: A false transmitter

agent for heart neuronal imaging. J Nucl Med 31:163-167, 1990.

Schafer, J.; Speilmann, R.; Schuchert, A.; Weber, K.; and Schlüter, M.

Iodine-123 meta-iodobenzylguanidine scintigraphy: A noninvasive

method to demonstrate myocardial adrenergic nervous system

disintegrity in patients with idiopathic dilated cardiomyopathy.

J Am Cull Cardiol 12:1252-1258, 1988.

Schwaiger, M.; Guibourg, H.; Rosenspire, K.; McClanahan, T.;

Gallagher, K.; Hutchins, G; and Wieland, D. Effect of regional

myocardial ischemia on sympathetic nervous system as assessed by

F-18 metaraminol. J Nucl Med 31(8):1352-1357, 1990a.

Schwaiger, M.; Hutchins, G.; Kalff, V.; Molina, E.; Rosenspire, K.;

Haka, M.; Deeb, M.; and Wieland, D. Evidence of regional

reinnervation of cardiac transplants by neuronal PET imaging. J Am

Coll Cardiol 15:224, 1990c.

Schwaiger, M.; Hutchins, G.; Rosenspire, K.; Haka, M.; and Wieland,

D. Quantitative evaluation of the sympathetic nervous system by

PET in patients with cardiomyopathy. J Nucl Med 31:792, 1990d.

Schwaiger, M.; Kalff, V.; Rosenspire, K.; Haka, M.; Molina, E.;

Hutchins, G.; Deeb, M.; Wolfe, E., Jr.; and Wieland, D. The

noninvasive evaluation of the sympathetic nervous system in the

human heart by PET. Circulation 82:457-464, 1990b .

Shapiro, B.; Copp, J.; Sisson, I.; Eyre, P.; Wallis, J.; and Beierwaltes,

W. Iodine-131 metaiodobenzylguanidine for the locating of

suspected pheochromocytoma: Experience in 400 cases. J Nucl Med

26:576-585, 1985.

Sisson, J.; Bolgos, G.; and Johnson, I. Measuring acute changes in

adrenergic nerve activity in the living animal. Am Heart J

121:1119-1123, 1990b.

106
Sisson, J.; Frager, M.; Valk, T.; Gross, M.; Swanson, D.; Wieland, D.;

Tobes, M.; Beierwaltes, W.; and Thompson, N. Scintigraphic

localization of pheochromocytoma. N Engl J Med 305:12-17, 1981.

Sisson, J.; Johnson, J.; Bolgos, G.; Lynch, J.; Uprichard, A.; Driscoll,

E.; Wieland, D.; and Lucchesi, B. Portrayal of adrenergic

denervation in the presence of myocardial infarction: A feasibility

study. Am J Physiol Imaging 5:151-166, 1990a

Sisson, J.; Lynch, J.; Johnson, J.; Jaques, S., Jr.; Wu, J.-L.; Bolgos, G.;

Lucchesi, B.; and Wieland, D. Scintigraphic detection of regional

disruption of adrenergic neurons in the heart. Am Heart J

116:67-76, 1988.

Sisson, J.; Shapiro, B.; Meyers, L.; Mallette, S.; Mangner, T.; Wieland,

D.; Glowniak, J.; Sherman, P.; and Beierwaltes, W. Metaiodobenzylguanidine to map scintigraphically the adrenergic nervous

system in man. J Nucl Med 28:1625-1636, 19876.

Sisson, J.; Wieland, D.; Sherman, P.; Mangner, T.; Tobes, M.; and

Jaques, S., Jr. Metaiodobenzylguanidine as an index of the

adrenergic nervous system integrity and function. J Nucl Med

28:1620-1624, 1987a.

Stiles, G.; Taylor, S.; and Lefkowitz, R. Human cardiac betaadrenergic receptors: Subtype heterogeneity delineated by direct

radioligand binding. Life Sci 33:467-473, 1983.

Syrota, A. Positron emission tomography: Evaluation of cardiac

receptors. In: Marcus, M.L.; Skorton, D.J.; Schelbert, H.R.; and

Wolf, G.L., eds. Braunwald, E., consulting ed. Cardiac Imaging: A

Companion of Braunwald’s Heart Disease. Philadelphia: W.B.

Sanders, 1990. pp. 1256-1270.

Waagstein, F.; Caidahl, K.; Wallentin, I.; Bergh, C-H.; and

Hjalmarson, A. Long-term B-blockade in dilated cardiomyopathy:

Effects of short- and long-term metoprolol treatment followed by

withdrawal and readministration of metoprolol. Circulation

80:551-563, 1989.

Wieland, D.; Brown, L.; Rogers, W.; Worthington, K.; Wu, J.-L.;

Clinthorne, N.; Otto, C.; Swanson, D; and Beierwaltes, W.

Myocardial imaging with a radioiodinated norepinephrine storage

analog. J Nucl Med 22:22-31, 1981a.

Wieland, D.; Brown, L.; Rogers, W.; Worthington, K.; Wu, J.-L.;

Clinthome, N.; Otto, C.; Swanson, D.; and Beierwaltes, W.

Myocardial imaging with a radioiodinated norepinephrine storage

analog. J Nucl Med 24:1127-1134, 1983.

107
Wieland, D.; Brown, L.; Tobes, M.; Rogers, W.; Marsh, D.; Mangner,

T.; Swanson, D.; and Beierwaltes, W. Imaging the primate adrenal

medulla with [123] and [131] metaiodobenzylguanidine: Concise

communication. J Nucl Med 22:358-364, 1981b.

Wieland, D.; Rosenspire, K.; Hutchins, G.; Van Dort, M.; Rothley, J.;

Mislankar, S.; Lee, H.; Massin, C.; Gildersleeve, D.; Sherman, P.;

and Schwaiger, M. Neuronal mapping of the heart with

6-[18F]fluorometaraminol. J Med Chem 33:956-964, 1990.

Wolpers, H.; Nguyen, N.; Rosenspire, K.; Haka, M.; Wieland, D.; and

Schwaiger, M. C-11 hydroxyephedrine as marker for neuronal

dysfunction in reperfused canine myocardium. Coronary Artery Dis

2:923-929, 1990.

ACKNOWLEDGMENTS

This work was done during the tenure of an established investigatorship from the American Heart Association and also was supported in

part by National Institutes of Health grants R01 HL41047-01 and R01

HL27555-06.

AUTHORS

Markus Schwaiger, M.D.

Professor of Internal Medicine

and

Director

Cardiovascular Nuclear Imaging

University of Michigan Medical Center

UH B1G505, Box 0028

1500 East Medical Center Drive

Ann Arbor, MI 48109-0028

James C. Sisson, M.D.

Professor of Internal Medicine

and

Associate Chief

Clinical Nuclear Medicine

108
Donald Wieland, M.D.

Professor of Internal Medicine and Research Scientist

and

Director

Radiopharmaceutical Chemistry

Division of Nuclear Medicine

Department of Internal Medicine

University of Michigan Medical Center

Ann Arbor, MI 48109

109

Development of PET/SPECT

Ligands for the Serotonin

Transporter

Ursula Scheffel, Robert F. Dannals, Makiko Suehiro,

George A. Ricaurte, F. Ivy Carroll, Michael J. Kuhar, and

Henry N. Wagner, Jr.

INTRODUCTION

There is clear evidence in rodents and nonhuman primates that the

widely abused “designer” drug, 3,4-methylenedioxymethamphetamine

(MDMA, “ecstasy”), causes loss of central serotonergic (5-hydroxytryptamine [5-HTJ]) neurons (Commins et al. 1986; Ricaurte et al.

1988a). Doses of MDMA that approximate those typically taken by

humans have been shown to be toxic to serotonergic nerve fibers in the

monkey brain (Ricaurte et al. 1988b; Slikker et al. 1988). Of similar

or greater concern are neurochemical studies showing that the anorectic

agent fenfluramine causes serotonergic damage in rodents as well as in

primates (Harvey et al. 1975; Schuster et al. 1986). Currently,

fenfluramine is a clinically prescribed drug in both the United States

and Europe. It is used to treat obesity with doses that closely

approach those that produce neurotoxic effects in animals (0.2 to 2.0

mg/kg). At this time, it is not known whether regeneration of 5-HT

neurons occurs in nonhuman primates and, if so, what the timeframe

for recovery is.

Long-term neurotoxic damage caused by MDMA and fenfluramine

appears to be restricted to the presynaptic element of the serotonergic

nerve fiber. Recent investigations on the effect of MDMA on

postsynaptic 5-HT2 receptors in the rat brain have shown that, although

there is an initial down-regulation of these postsynaptic receptors, it is

of short duration. Approximately 1 week after administration of

MDMA (eight subcutaneous injections [20 mg/kg], twice daily for 4

days), the number of 5-HT2-ligand binding sites returns to control

levels (Scheffel et al. 1992a). In contrast, 5-HT uptake sites, located

111
on presynaptic nerve endings in the rat brain, recover very slowly after

MDMA treatment: their number remains decreased for up to 12

months (Battaglia et al. 1988a).

Several methods have been employed to test for serotonin neurotoxicity in humans, including measurement of the serotonin metabolite

5-hydroxyindoleacetic acid in cerebrospinal fluid (Moir et al. 1970)

and endocrine challenge using intravenously administered

L-tryptophane (Heninger et al. 1984). All these tests are indirect and

cannot accurately reflect the state of the central serotonergic neuron.

Therefore, a presynaptic marker is needed to indicate serotonergic

neurotoxicity in the living human brain.

This chapter describes work performed in the authors’ laboratories in

the development and in vivo testing of radiolabeled ligands for the

serotonin uptake site suitable for single photon emission computed

tomography (SPECT) or positron emission tomography (PET) imaging.

Several compounds with known in vitro affinity for the 5-HT uptake

site were labeled with [

123I] for SPECT studies or with either [11C] or

[

18F] for investigations using PET. The radiotracers were characterized

in rodents for their in vivo kinetics, distribution, and specificity of

binding to the 5-HT uptake site.

PAROXETINE, N-[11C]METHYL PAROXETINE, AND

[

18F]FLUOROALKYLATED PAROXETINE ANALOGS

Paroxetine, first described in the late 1970s (Petersen et al. 1977; Buus

Lassen 1978), is an antidepressant drug with high affinity and selectivity for the 5-HT uptake site in platelets and presynaptic neurons in

the central nervous system (CNS) (Habert et al. 1985; Marcusson et al.

1988). In its tritiated form, paroxetine has been used to characterize

the binding to the 5-HT uptake site (Mellerup et al. 1983; Mellerup

and Plenge 1986) and to determine the density and distribution of

5-HT uptake sites in the rat and human brain (Battaglia et al. 19886;

Cortes et al. 1988).

Studies by Scheffel and Hartig (1989) indicated that [

3H]paroxetine is

a potent and selective agent for in vivo labeling of cerebral serotonin

uptake sites. Furthermore, Scheffel and Ricaurte (1990) demonstrated

112
that paroxetine can serve as an in vivo indicator of MDMA neurotoxicity. They concluded that labeling paroxetine with [

11C] (t1/2 = 20

min) or [18F] (t 1/2 = 110 min) would yield a useful PET tracer for

imaging and quantitating 5-HT uptake sites in the CNS of living mammals and humans, However, introduction of a positron emitter label

into the authentic paroxetine molecule has not been accomplished yet.

Several analogs of paroxetine that could be labeled more easily with

[

11C] or [18F] than authentic paroxetine were prepared (Villemagne et

al. 1989; Suehiro et al. 1991). However, in vitro binding assays

demonstrated a considerable loss of affinity of these analogs:

N-(methyl) paroxetine displaced [

3H]paroxetine binding to 5-HT uptake

sites 25 times less potently than authentic paroxetine; N-(3-fluoroethyl)

and N-(3-fluoropropyl) paroxetine had 424 and 1,220 times lower

potencies, respectively (table 1).

TABLE 1. Potencies of paroxetine and analogs in inhibiting

[

3H]paroxetine binding to 5-HT uptake sites in rat

cortical homogenates. Ki

 values are means of four

assays performed in triplicate.

Inhibitor Ki

(M)

Paroxetine* 5.9x10-11

N-(methyl) paroxetine† 1.5x10-9

N-(2-fluoroethyl) paroxetine* 2.5x10-8

N-(3-fluoropropyl) paroxetine* 7.2x10-8

SOURCE: *Suehiro et al. 1991; †Plenge et al. 1987

Similar results were obtained in in vivo studies using N-([11C]methyl)

paroxetine and N-(3-[18F]fluoropropyl) paroxetine (figure 1). After

intravenous (IV) injection of these high specific activity tracers into

mice, the radioactivity concentrations in different areas of the brain

were measured by dissecting the brain regions and weighing and

counting the tissue samples. Ratios of the radioactivities in the hypothalamus, an area known to contain high densities of 5-HT uptake

113
FIGURE 1.

KEY:

Comparison of the in vivo binding of [

3H]paroxetine with

that of its analogs, N-([11C]methyl) paroxetine and N-(3-

[

18F]fluoropropyl) paroxetine. Time course of

hypothalamus to cerebellar radioactivity ratios in the mouse

brain.

= [

3H]paroxetine; =N-([11C]methyl) paroxetine;

= N-(3-[18F]fluoropropyl) paroxetine

sites, and in the cerebellum, a region relatively devoid of binding sites,

were calculated to estimate total/nonspecific binding. With

N-([11C]methyl) paroxetine, the highest hypothalamus-to-cerebellar

ratio was 1.8:1 at 60 min after administration. For N-(3-

[

18F]fluoropropyl) paroxetine, the hypothalamus-to-cerebellar ratio

climbed to only 1.3:1 at 3 hours after injection. Ratios for

[

3H]paroxetine (Scheffel and Hartig 1989) were plotted for comparison.

Attempts to derivatize paroxetine for SPECT have been made. Mathis

and coworkers (1991) recently reported on several iodinated paroxetine

114
analogs, of which (±)-2’-[125I]iodo paroxetine appeared to be the most

promising. However, subsequent in vivo experiments showed that this

tracer penetrated the blood-brain barrier rather poorly, and nonspecific

binding was too high for the tracer to be useful for SPECT studies.

[

11C]CITALOPRAM, [11C]FLUOXETINE, AND CIS-[11C]DDPI

Citalopram, fluoxetine, and cis-N,N,-dimethyl-3-(2’,4’-dichlorophenyl)-

indanamine (cis-DDPI) are known to be potent and selective serotonin

uptake site blockers (Hyttel 1982; Fuller and Wong 1977; Bogeso et

al. 1985). These compounds were radiolabeled with [

11C] to assess

their potential use as PET ligands.

[

11C]Citalopram, [11C]fluoxetine, and cis-[11C]DDPI were synthesized

from their respective precursors and [

11C]iodomethane (Dannals et al.

1990; Kilboum 1988; Suehiro et al. 1992a). The biological activity of

these compounds was studied in mice (Scheffel et al. 1990; Suehiro et

al. 1992a). [

11C]Citalopram, [11C]fluoxetine, and cis-[11C]DDPI penetrated the blood-brain barrier well with 1.3, 3.1, and 3.6 percent of the

injected dose (%D), respectively, in the whole mouse brain at 15 min

after IV injection.

As shown in figure 2, [

11C]fluoxetine did not exhibit any specific

binding to 5-HT uptake sites during the time of observation (15 to 90

min after injection). At no time after administration did [

11C]fluoxetine

distribution agree with that of 5-HT uptake site densities, nor did

preinjection of the high-affinity uptake site blocker paroxetine (1

mg/kg) cause any inhibition of the accumulation of the tracer. The

reasons for the failure of [

11C]fluoxetine to bind to 5-HT uptake sites

in vivo are unknown. However, it is possible that metabolism of the

radioligand occurred before specific binding in the CNS could take

place. Conversely, cis-[11C]DDPI showed specific accumulation in

areas rich in 5HT uptake sites (e.g., the hypo-thalamus, olfactory

tubercles, and prefrontal cortex). This binding could be blocked 60 to

80 percent by preinjection of 1 mg paroxetine/ kg bodyweight. However, because nonspecific binding was high and the clearance of this

nonspecifically bound fraction was slow, the ratio between total to

nonspecific binding reached only 1.4:1 at 60 to 90 min after injection

(figure 2).

115
FIGURE 2. Hypothalamus-to-cerebellar ratios of [

11C] concentrations

at different times afer IV injection of 300 to 500 μCi of

either [

11C]fluoxetine, cis-[11C]DDPI, [11C]citalopram,

[

11C]RTI-SS, or trans(+)[11C]McN-5652-X into mice

weighing 25 to 35 g. The specific activity at time of

injection of each of the tracers was approximately 3,000

mCi/μmol. Data are means ±1 standard deviation, and

n = 3 to 4 mice for each time point.

[

11C]Citalopram was the most promising of the three candidate ligands.

Hypothalamus-to-cerebellar ratios rose to 2.0:1 at 60 min (figure 2).

Regional [

11C]citalopram distribution at this time was in agreement

with 5-HT uptake site densities, and preadministration of a blocking

dose of paroxetine did significantly inhibit tracer binding. Although

these results were encouraging, the ratios between specific and

nonspecific binding were not high enough to guarantee successful

imaging of 5-HT uptake sites by PET.

116
TRANS(±)[11C]McN-5652-Z AND (+)[11C]McN-5652-X

Trans(±)McN-5652-Z was described by Shank and colleagues (1968)

as a highly potent serotonin uptake inhibitor. In addition, several

stereoisomers of this compound were isolated and tested for their

binding to presynaptic monoaminergic neurotransmitter uptake sites

(Shank et al. 1968). Trans(±)McN-5652-X was shown to exhibit the

highest affinity toward the 5-HT uptake site (Ki for inhibition of

[

3H]5-HT = 0.4 nM), followed by trans(±)McN-5652-X (Ki

 = 0.6 nM)

and cis McN-5655 (Ki = 16.6 nM); the trans(-)McN-5652-W isomer

was approximately 150 times less potent (Ki = 58.4 nM) than the

corresponding (+) isomer. All these compounds showed moderate in

vitro affinity for the norepinephrine (NE) uptake site (i.e., five times

lower than for the 5-HT uptake site) and little or no affinity for the

dopamine (DA) uptake site.

Trans(±)McN-5652-Z trans(+)McN-5652-X, trans(-)McN-5652-W, and

cis McN-5655-Z were labeled recently with [

11C] (Suehiro et al.

1992b, in press). The synthesis of these tracers was performed by

S-methylation of the respective precursors with [

11C]iodomethane. The

specific activity determined at the end of synthesis ranged from 3,300

to 4,250 mCi/μmol.

As shown in figure 2, [

11C]McN-5652-X, the trans(+) isomer, far

exceeded all previously tested radioligands in specific binding to the

5-HT transporter in the mouse brain. Ratios between hypothalamus

and cerebellar radioactivity concentrations reached 5.9:1 and 6.2:1,

respectively, at 60 and 90 min after IV injection of the tracer. In

contrast, [

11C]McN-5652-W, the trans(-) isomer, displayed little

specific binding. For this compound, the ratio of the radioactivity

concentration in the hypothalamus to the nonspecifically bound

radioactivity concentration in the cerebellum was 1.3:l at 60 min and

1.9:1 at 90 min after administration.

[

11C]McN-5652-Z, the racemic (±) mixture of the optical isomers (+)

and (-) [

11C]McN-5652, was prepared to study the selectivity of in vivo

binding to the 5-HT uptake site in the mouse model. Blocking doses

of drugs known to bind to specific monoamine neurotransmitter sites

were injected intravenously 5 min before tracer injection. Tissue-tocerebellar radioactivity ratios in different brain regions were deter117
FIGURE 3.

KEY:

SOURCE:

Effect of blocking doses (5 mg/kg) of paroxetine (specific for

the 5-HT uptake site), GBR 12909 (DA uptake site), and

desipramine (NE uptake site) on [

11C]McN-5652-Z binding.

The drugs were injected intravenously 5 minutes before

administration of the tracer. Tissue-to-cerebellar ratios

were determined 60 minutes after tracer injection. Data are

means ±1 standard deviation (n = 4).

OLF = olfactory tubercles; HYP = hypothalamus; HIPP =

hippocampus; STR = striatum; FTCTX = prefrontal cortex;

CTX = cortex; THAL = thalamus

Suehiro et al. 1993. Copyright 1993 by Society of Nuclear

Medicine.

mined 60 min after tracer injection and compared with those of salineinjected controls. The results of these studies are shown in figure 3.

Paroxetine (5 mg/kg) significantly decreased the specific binding of

[

11C]McN-5652-Z in all areas examined. In contrast, GBR 12909, a

DA uptake site-specific drug, and desipramine, which blocks the NE

118
uptake sites, had no effect. The results indicate that [

11C]McN-5652-Z

is a highly selective in vivo label for the serotonin transporter.

The ability of [

11C]McN-5652-Z to detect serotonergic damage in the

CNS was shown in rats lesioned with the known 5-HT neurotoxin

5,7-dihydroxytryptamine (5,7-DHT) (200 μg 5,7-DHT in 10 μL

0.01 percent ascorbic acid, injected intracerebroventricularly after NE

uptake sites were blocked by pretreatment of the animals with

desipramine [25 mg/kg, IV]) 13 days before the tracer study.

[

11C]McN-5652-Z was injected intravenously 60 min before sacrifice of

the animals. As shown in figure 4, specific [

11C]McN-5652-Z binding

(expressed as %D/g tissue minus %D/g in cerebellum) was reduced by

50 to 90 percent in different brain regions of the 5,7-DHT lesioned

rats. Moreover, the regional decreases in [

11C]McN-5652-Z binding

after 5,7-DHT treatment corresponded with those observed using

[

3H]paroxetine as the in vivo indicator. These results indicate that

[

11C]McN-5652-Z can be used to quantify the extent of serotonergic

damage.

[

125I]-RTI-55 AND [11C]-RTI-55

Recently, several cocaine analogs have been synthesized, radiolabeled,

and tested for their in vitro and in vivo affinities for monoaminergic

neurotransmitter uptake sites. (For a recent overview of this work, see

Boja et al. 1992a). Of these compounds, [

123/125I] 2ß-carbomethoxy-3ß-

(4-iodophenyl)tropane ([123/125I]RTI-55) (Carroll et al. 1991; Neumeyer

et al. 1991) was found to bind to both the DA and 5-HT transporters

in homogenate binding assays (Boja et al. 1992b) as well as after IV

injection in the brain of rodents and baboons (Cline et al. 1992; Innis

et al. 1991; Scheffel et al. 1992b). The affinity of RTI-55 was approximately 100 times greater than that of (-) cocaine for both the DA and

the 5-HT transporters (Boja et al. 1992b).

Studies in rats demonstrated that the in vivo distribution of

[

123I]RTI-55 in CNS areas rich in serotonin uptake sites correlated well

with regional densities of [

3H]serotonin uptake sites (figure 5). Maximal target-to-nontarget ratios (up to 7.4) in 5-HT uptake site-rich areas

in the rat brain were reached 5 hours after IV injection (figure 6).

However, as mentioned before, [

123I]RTI-55 labels not only 5-HT but

119
FIGURE 4. Effect of 5,7-DHT lesioning on the specific binding (%D/g

tissue-%D/g cerebellum) of [11C]McN-5652-Z at 60

minutes after tracer injection. Rat brains were lesioned by

intracerebroventricular injection of 5,7-DHT 13 days before

the trucer study. Data are means ±1 standard deviation,

n = 3.

KEY: OLF = olfactory tubercles; HYP = hypothalamus; HIPP =

hippocampus; STR = striatum; FTCTX = prefrontal cortex;

CTX = cortex; THAL = thalamus; S.COLL = superior

colliculi

SOURCE: Suehiro et al. 1993. Copyright 1993 by Society of Nuclear

Medicine.

DA transporters as well. At the time when [

123I]RTI-55 specific

binding in 5-HT uptake site-rich areas was highest (the hypothalamusto-cerebellar ratio was 7.4:1), the striatal-to-cerebellar ratio was 22.7:1.

120
FIGURE 5. Comparison between [

123I]RTI-55 concentrations (%D/g

tissue) in various regions of the rat brain at 2 hours after IV

injection and the proportional distribution of [

3H]serotonin

uptake in synaptosomal preparations of these regions as

published by Leysen (1985), where [

3H]serotonin uptake in

the frontal cortex is set at 100 percent.

KEY: CB = cerebellum, HIPP = hippocampus, THAL = thalamus,

FTCTX = prefrontal cortex, HYP = hypothalamus

Despite the fact that [

123I]RTI-55 is not selective for the serotonin

uptake site, it is anticipated that the tracer will be useful for quantitating 5-HT uptake sites in vivo by SPECT. In rats, neurotoxic doses

of fenfluramine caused decreases of 66 percent (in the hypothalamus)

to 83 percent (in the superior colliculi) of specific [

123I]RTI-55 binding

in all areas except in the striatum and the olfactory tubercles (regions

rich in DA transporters) (Scheffel et al. 1992b). In baboons, Innis and

colleagues (1991) and Laruelle and coworkers (1992) observed specific

121
FIGURE 6.

KEY:

SOURCE:

Time course of tissue-to-cerebellar radioactivity ratios in

selected areas of the rat brain after IV injection of

(

123I]RTI-55. Data are means ± SEM; n = 2 to 4 rats/time

point.

THAL = thalamus; CB = cerebellum; S.COLL = superior

colliculi; HYP = hypothalamus; FTCTX = prefrontal cortex;

CTX = cortex

Scheffel et al. 1992b. Copyright 1992 by Wiley-Liss.

binding of [

123I]RTI-55 in the midbrain that was associated primarily

with 5HT transporters. A more selective SPECT agent for the serotonin transporter has recently been described by Mathis and colleagues

(1992). Studies with [

125I]-iodo-6-nitroquipazine showed excellent

target-to-nontarget ratios in 5-HT transporter-rich regions of the rat

brain; a hypothalamus-to-cerebellar ratio of 6.3:1 at 6 hours after

injection was reported.

122
FIGURE 7. Time course for hypothalamus-to-cerebellar ratios in the

mouse brain after injection of either [

125I]RTI-5.5 or

[

11C]RTI-55

SOURCE: [

125]RTI-55 data are from Cline and colleagues (1992).

RTI-55 recently was labeled with [

11C] (Dannals, unpublished results),

and studies in mice were performed to compare the specificity and

selectivity of [

11C]RTI-55 binding to 5-HT uptake sites with that of

[

123I]RTI-55. Similar results were obtained with both tracers (figure 7)

[

11C]RTI-55 is being evaluated in nonhuman primates as a PET tracer

for 5-HT and DA uptake sites.

123
SUMMARY

There is a great need for PET and SPECT ligands with high affinity

and selectivity for the serotonin uptake site. These imaging agents

would be useful in screening human populations at risk (e.g., individuals exposed to neurotoxic amphetamines such as MDMA and

fenfluramine). Moreover, these radioligands would allow the study of

serotonergic function in the normal living human brain, and they also

would be useful in the examination of altered serotonergic neurotransmission in diseases such as depression and obsessive-compulsive

and other neuropsychiatric disorders.

Over the past several years, a number of radioligands have been

studied in several laboratories for their in vivo binding to 5-HT uptake

sites. Although [

3H]paroxetine showed promising binding characteristics, conversion of authentic paroxetine into a PET or SPECT

tracer turned out to be difficult and has not been achieved yet. Analogs of paroxetine displayed considerable loss of binding affinity and

were, therefore, not useful for imaging purposes. For [

11C]fluoxetine,

[

11C]citalopram, and cis-[11C]DDPI, target-to-nontarget (hypothalamusto-cerebellar) ratios remained less than 2.0:1 over a 90-min period after

injection. The most promising PET agents identified today are

[

11C]RTI-55 and [11C]McN-5652-X. [11C]RTI-55 labels both 5-HT and

DA uptake sites. [

11C]McN-5652-X is highly selective for 5-HT

uptake sites, and its distribution is consistent with the neuroanatomical

distribution of the 5-HT uptake site. Because [

11C]McN-5652-Z is a

racemic mixture of two stereoisomers, of which the (+) isomer (McN5652-X) binds to the 5-HT uptake site in vivo and the (-) isomer

(McN-5652-W) does not, the possibility exists that regional-specific

binding can be determined by subtracting nonspecific binding of the

(-) isomer from total radioactivity counts obtained with the (+) isomer.

[

11C]McN-5652-X is the best PET radioligand for the 5-HT uptake site

described thus far. This tracer warrants further testing in nonhuman

primates. Efforts are underway to obtain an investigational new drug

application for use of the tracer in humans.

Promising candidates as SPECT imaging agents for the 5-HT uptake

site are [

123I]RTI-55 and [123I]-iodo-6-nitroquipazine. Both agents are

under intense investigation in different laboratories in the United

States.

124
REFERENCES

Battaglia, G.; Yeh, S.Y.; and DeSouza, E.B. MDMA-induced

neurotoxicity: Parameters of degeneration and recovery of brain

serotonin neurons. Pharmacol Biochem Behav 29:269-274, 1988a.

Battaglia, G.; Yeh, S.Y.; O’Hearn, E.; Molliver, M.E.; Kuhar, M.J.;

and DeSouza, E.B. 3,4-Methylenedioxymethamphetamine and

3,4-methylenedioxyamphetamine destroy serotonin terminals in rat

brain: Quantification of neurodegeneration by measurement of

[

3H]paroxetine-labeled serotonin uptake sites. J Pharmacol Exp Ther

242:911-916, 1988b.

Bogeso, K.P.; Christensen, A.V.; Hyttel, J.; and Liljefors, T.

3-Phenyl-1-indanamines. Potential antidepressant activity and potent

inhibition of dopamine, norepinephrine and serotonin uptake. J Med

Chem 28:1817-1828, 1985.

Boja, J.W.; Cline, E.J.; Carroll, F.I.; Lewin, A.H.; Philip, A.; Dannals,

R.; Wong, D.; Scheffel, U.; and Kuhar, M.J. High potency cocaine

analogs: Neurochemical, imaging, and behavioral studies. In:

Kalivas, P.W., and Samson, H.H., eds. The Neurobiology of Drug

and Alcohol Addiction. Vol. 654, Annuals of the New York

Academy of Sciences. New York: The New York Academy of

Sciences, 1992a.

Boja, J.W.; Mitchell, W.M.; Patel, A.; Kopajtic, T.A.; Carroll, F.I.;

Lewin, A.H.; Abraham, P.; and Kuhar, M.J. High affinity binding

of [

125I]RTI-55 to dopamine and serotonin transporters in the rat

brain. Synapse 12:27-36, 1992b.

Buus Lassen, J. Potent and long-lasting potentiation of two

5-hydroxytryptophan-induced effects in mice by three selective

5-HT uptake inhibitors. Eur J Pharmacol 47:351-358, 1978.

Carroll, F.I.; Rahman, M.A.; Abraham, P.; Parham, K.; Lewin, A.H.;

Dannals, R.F.; Shaya, E.; Scheffel, U.; Wong, D.F.; Boja, J.W.; and

Kuhar, M.J. [

123I]313-(4-Iodophenyl)tropan-2ß-carboxylic acid methyl

ester (RTI-55), a unique cocaine receptor ligand for imaging the

dopamine and serotonin transporters in vivo. Med Chem Res

1:289-294, 1991.

Cline, E.J.; Scheffel, U.; Boja, J.W.; Mitchell, W.M.; Carroll, F.I.;

Rahman, A.; Lewin, A.H.; and Kuhar, M.J. In vivo binding of

[

125I]RTI-55 to dopamine transporters: Pharmacology and regional

distribution with autoradiography. Synapse 12:37-46, 1992.

125
Commins, D.L.; Vosmer, G.; Virus, R.M.; Woolverton, W.L.; Schuster,

C.R.; and Seiden, L.S. Biochemical and histological evidence that

methylenedioxymethamphetamine (MDMA) is toxic to neurons in

the rat brain. J Pharmocol Exp Ther 241:338-345, 1986.

Cortes, R.; Soriano, E.; Pazos, A.; Probst, A.; and Palacios, J.M.

Autoradiography of antidepressant binding sites in the human brain:

Localization using [

3H]imipramine and [3H]paroxetine. Neuroscience

27:473-496, 1988.

Dannals, R.F.; Ravert, H.T.; Wilson, A.A.; and Wagner, H.N., Jr.

Synthesis of a selective serotonin uptake inhibitor: [

11C]Citalopram.

Appl Radiat Isot 41:541-543, 1990.

Fuller, R.W., and Wong, D.T. Inhibition of serotonin reuptake. Fed

Proc 36:2154-2158, 1977.

Habert, E.; Graham, G.; Tahraoui, L.; Claustre, U.; and Langer, S.Z.

Characterization of [

3H]paroxetine binding in rat cortical

membranes. Eur J Pharmacol 118:107-114, 1985.

Harvey, J.; McMaster, S.; and Yunger, L. p-Chloroamphetamine:

Selective neurotoxic action in brain. Science 187:841-843, 1975.

Heninger, G.; Chamey, D.S.; and Stemberg, D.E. Serotonergic

function in depression: Prolactin response to intravenous tryptophan

in depressed patients and healthy subjects. Arch Gen Psychiatry

41:398-402, 1984.

Hyttel, J. Citalopram; basic and clinical studies. Pros Neuropsychopharmacology Biol Psychiatry 6:275-295, 1982.

Innis, R.; Baldwin, R.; Sybirska, E.; Zea, Y.; Laruelle, M.; Al-Tikriti,

M.; Chamey, D.; Zoghbi, S.; Smith, E.; Wisniewski, G.; Hoffer, P.;

Wang, S.; Milius, R.; and Neumeyer, J. Single photon emission

computed tomography imaging of monoamine reuptake sites in

primate brain with [

123I]CIT. Eur J Pharmacol 200:369-370, 1991.

Kilboum, M.R. Synthesis of carbon-11 labelled nisoxetine and

fluoxetine, monoamine reuptake inhibitors. (Abstract.) J Nucl Med

29:932, 1988.

Laruelle, M.; Baldwin, R.M.; Zea-Ponce, Y.; Zoghbi, S.S.; Al-Tikriti,

M.S.; Sybirska, E.H.; Malison, R.T.; Neumeyer, J.L.; Milius, R.;

Wong, S.; Smith, E.O.; Wisniewski, G.; Chamey, D.S.; Hoffer,

P.B.; and Innis, R.B. SPECT imaging of monoamine transporter

sites with 123I-CIT in nonhuman primates brain. Abstract. J Nucl

Med 33:945-946, 1992.

126
Leysen, J.L. Characterization of serotonin receptor binding sites. In:

Green, A.R., ed. Neuropharmacology of Serotonin. New York:

Oxford University Press, 1985. pp. 79-116.

Marcusson, J.O.; Bergstrom, M.; Eriksson, K.; and Ross, S.B. Characterization of [

3H]paroxetine binding in rat brain. J Neurochem 50:

1783-1790, 1988.

Mathis, C.; Biegon, A.; Taylor, S.; Enas, J.; Hanrahan, S.; and Jagust,

W. I-125-5-iodo-6-nitro-2-piperazinylquinoline: A potent and

selective ligand for the serotonin uptake complex. Eur J Pharmacol

210:103-104, 1992.

Mathis, C.A.; Gerdes, J.M.; Enas, J.D.; Havlik, S.; and Peroutka, S.J.

(±)-211-[I-125]iodoparoxetine: Synthesis and preliminary evaluation

of a presynaptic serotonin ligand. Abstract. J Nucl Med 32:965,

1991.

Mellerup, E.T., and Plenge, P. High affinity binding of [

3H]paroxetine

and [

3H]imipramine to rat neuronal membranes, Psychopharmacology 89:436-439, 1986.

Mellerup, E.T.; Plenge, P.; and Engelstoft, M. High affinity binding of

[

3H]paroxetine and [3H]imipramine to human platelet membranes.

Eur J Pharmacol 96:303-309, 1983.

Moir, A.T.; Ashcroft, G.W.; Crawford, T.B.; Eccleston, D.; and

Guldbert, H.C. Cerebral metabolites in cerebrospinal fluid as a

biochemical approach to the brain. Brain 93:357-368, 1970.

Neumeyer, J.L.; Wang, S.; Milius, R.A.; Baldwin, R.M.; Zea-Ponce,

Y.; Hoffer, P.B.; Chamey, D.S.; Malison, R.T.; Laruelle, M.; and

Innis, R.B. [

123I] 2ß-Carbomethoxy-3ß-(4-iodophenyl)tropane: High

affinity SPECT radiotracer of monoamine reuptake sites in brain.

J Med Chem 34:3144-3146, 1991.

Petersen, E.N.; Olsson, S.O.; and Squires, R.F. Effects of 5-HT uptake

inhibitors on the pressor response to 5-HT in the pithed rat. The

significance of the 5-HT blocking property. Eur J Pharmacol

43:209-215, 1977.

Plenge, P.; Mellerup, E.T.; Honore, T.; and LeFevre-Honore, P. The

activity of 25 paroxetine/femoxetine structure variants in various

reactions, assumed to be important for the effect of antidepressants.

J Pharm Pharmacol 38:877-882, 1987.

Ricaurte, G.A.; DeLanney, D.E.; Irwin, I.; and Langston, J.W. Toxic

effects of MDMA on central serotonergic neurons in the primate:

Importance of route and frequency of drug administration. Brain

Res 446:165-168, 1988a.

127
Ricaurte, G.A.; Forno, L.S.; Wilson, M.A.; and DeLanney, L.E. (±)3,4-

Methylenedioxymethamphetamine (MDMA) selectively damages

central serotonergic neurons in non-human primates. JAMA

260:51-55, 1988b.

Scheffel, U.; Dannals, R.F.; Cline, E.J.; Ricaurte, G.A.; Carroll, F.I.;

Abraham, P.; Lewis, A.H.; and Kuhar, M.J. [

123/125I]RTI-55, an in

vivo label for the serotonin transporter. Synapse 11:134-139, 19926.

Scheffel, U.; Dannals, R.F.; Suehiro, M.; Ravert, H.T.; Stathis, M.; and

Wagner, H.N., Jr. Evaluation of 11C-citalopram and 11C-fluoxetine as

in vivo ligands for the serotonin uptake site. Abstract. J Nucl Med

31:883, 1990.

Scheffel, U., and Hartig, P.R. In vivo labeling of serotonin uptake sites

with [

3H]paroxetine. J Neurochem 52:1605-1612, 1989.

Scheffel, U.; Lever, J.R.; Stathis, M.; and Ricaurte, G.A. Repeated

administration of MDMA causes transient down-regulation of 5-HT2

receptors: In vitro and in vivo studies using the ligand Nl-methyl2-[125I]-LSD. Neuropharmacology 31:881-893, 1992a.

Scheffel, U., and Ricaurte, G.A. Paroxetine as an in vivo indicator of

3,4-methylenedioxymethamphetamine neurotoxicity: A presynaptic

serotonergic positron emission tomography ligand? Brain Res

527:89-95, 1990.

Schuster, C.R.; Lewis, M.; and Seiden, L.S. Fenfluramine

neurotoxicity . Psychopharmacol Bull 22:148-151, 1986.

Shank, R.P.; Vaught, J.L.; Pelley, K.A.; Settler, P.E.; McComsey, D.F.;

and Maryanoff, B.E. McN-5652: A highly potent inhibitor of

serotonin uptake. J Phannacol Exp Ther 247:1032-1038, 1968.

Slikker, W.; Ali, SF.; Scallet, A.C.; Frith, C.H.; Newport, G.D.; and

Bailey, J.R. Neurochemical and neurohistological alterations in the

rat and monkey produced by orally administered methylenedioxymethamphetamine (MDMA). Toxicol Appl Pharmacol

94:448-457, 1988.

Suehiro, M.; Ravert, H.T.; Dannals, R.F.; Scheffel, U.; and Wagner,

H.N., Jr. Synthesis of a radiotracer for studying serotonin uptake

sites with positron emission tomography: [

11C]McN-5652.

J Labelled Comp Radiopharm 31:841-848, 1992b.

Suehiro, M.; Scheffel, U.; Dannals, R.F.; Ravert, H.T.; and Wagner,

H.N., Jr. [

11C](+)McN5652 as a radiotracer for imaging serotonin

uptake sites with PET. Life Sci, in press.

128
Suehiro, M.; Scheffel, U.; Dannals, R.F.; Ricaurte, G.A.; Ravert, H.T.;

and Wagner, H.N., Jr. A PET radiotracer for studying serotonin

uptake sites: [

11C]McN-5652-Z. J Nucl Med 34:120-127, 1993.

Suehiro, M.; Scheffel, U.; Dannals, R.F.; Wilson, A.A.; Ravert, H.T.;

and Wagner, H.N., Jr. A new radiotracer for in vivo labeling of

serotonin uptake sites by PET, cis-N,N[11C]dimethyl-3-(2’,4’-

dichlorophenyl)-indanamine, cis-[11C]DDPI. Nucl Med Biol 19:549-

553, 1992a.

Suehiro, M.; Wilson, A.A.; Scheffel, U.; Dannals, R.F.; Ravert, H.T.;

and Wagner, H.N., Jr. Radiosynthesis and evaluation of N-(3-

[

18F]fluoropropyl) paroxetine as a radiotracer for in vivo labeling of

serotonin uptake sites by PET. Nucl Med Biol 18:791-796, 1991.

Villemagne, V.L.; Dannals, R.F.; Sanchez-Roa, P.M.; Scheffel, U.;

Ravert, H.T.; Wilson, A.A.; Natarajan, T.K.; Frost, J.J.; and

Wagner, H.N., Jr. PET imaging of central serotonin re-uptake sites

in the baboon brain with [

11C]-N-Me-Paroxetine. Abstract. Paper

presented at the Society of Nuclear Medicine, St. Louis, MO, June

13-16, 1989.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grants DA05477, NS-15080, and CA-32845. Marigo Stathis and Christine

Steinert provided work in biodistribution studies in rodents and

assistance with baboon studies. Robert Smoot assisted with the

operation of the cyclotron. Dr. Hayden T. Ravert helped to prepare

the [

11C] and [18F] tracers.

AUTHORS

Ursula Scheffel, Sc.D.

Assistant Professor

Robert F. Dannals, Ph.D.

Associate Professor

Makiko Suehiro, Ph.D.

Research Associate

129
Henry N. Wagner, Jr., M.D.

Professor

Division of Nuclear Medicine

Department of Radiology

The Johns Hopkins Medical Institutions

615 North Wolfe Street

Baltimore, MD 21205

George A. Ricaurte, M.D., Ph.D.

Assistant Professor

Department of Neurology

The Johns Hopkins Medical Institutions

Baltimore, MD 21224

F. Ivy Carroll, Ph.D.

Director for Organic and Medicinal Chemistry

Research Triangle Institute

Research Triangle Park, NC 27709

Michael J. Kuhar, Ph.D.

Professor

Neuroscience Branch

National Institute on Drug Abuse

Addiction Research Center

Baltimore, MD 21224

130
SPECT Imaging of Dopamine and

Serotonin Transporters in

Nonhuman Primate Brain

Marc Laruelle, Ronald M. Baldwin, and Robert B. Innis

INTRODUCTION

Since the recent introduction of high affinity iodinated ligands and

high-resolution, brain-dedicated, single photon emission computerized

tomography (SPECT) devices, SPECT has been used to visualize

various neuroreceptors, including the acetylcholine muscarinic receptor

(Eckelman et al. 1985), the dopamine D2 receptor (Kung et al. 1989,

1990), and the benzodiazepine receptor (Innis et al. 1991a).

The ability to visualize and quantify dopamine (DA) and serotonin

(5-HT) transporters presumably would lead to several clinical

applications. Alteration of these transporters has been described in

Parkinson’s disease (Cash et al. 1985; Raisman et al. 1986; Janowsky

et al. 1987; Hirai et al. 1988; Maloteaux et al. 1988; Kaufman and

Madras 1991; Niznik et al. 1991), Alzheimer’s disease (Allard et al.

1990), suicide victims (Stanley et al. 1982), patients with major

affective disorders (Perry et al. 1983; Crow et al. 1984; Paul et al.

1984), and schizophrenia (Joyce et al. 1993; Laruelle et al., in press-a).

In addition to their association with pathophysiological processes, DA

and 5-HT transporters are implicated in the mechanism of some

antidepressant drugs (Glowinsky and Axelrod 1964) and addictive

substances like cocaine (Kuhar et al. 1991). Therefore, radiolabeled

tracers suitable for visualization and quantification of these sites may

provide valuable tools for the diagnosis and treatment of several

neuropsychiatric disorders.

Multiple saturable binding sites for [

3H]cocaine have been identified in

the central nervous system, the majority of which being associated

with DA, 5-HT, and norepinephrine (NE) transporters (Reith et al.

1980, 1983). Some of the cocaine phenyltropane congeners are more

potent than cocaine itself in behavioral stimulation, monoamine uptake

131
inhibition, and affinity for the monoamine transporters (Clarke et al.

1973; Madras et al. 1989a; Ritz et al. 1990; Balster et al. 1991).

For example, the radiolabeled phenyltropane fluoro derivative

[

3H]2P-carbomethoxy-3ß-(4-fluorophenyl)tropane (WIN 35, 428, also

designated CFT, figure 1) is used as a potent in vitro ligand for the

DA transporter (Madras et al. 1989a).

The search for an iodinated analog of CFT led to the development of

methyl 3ß-(4-iodophenyl)tropane-2ß-carboxylate (figure 1), designated

as ß-CIT in analogy to CFT (Neumeyer et al. 1991) and by the code

number RTI-55 (Carroll et al. 1991). This chapter presents the major

steps in the development of radiolabeled ß-CIT as a SPECT tracer for

visualization of the DA and 5-HT transporters in nonhuman primates:

in vitro binding studies, radiolabeling, pharmacokinetics, metabolites

analysis, brain uptake kinetics, and in vivo pharmacology. This work

has been published previously in a series of articles by the authors and

others (Boja et al. 1991, 1992; Carroll et al. 1991; Innis et al. 1991b ;

Neumeyer et al. 1991; Cline et al. 1992; Scheffel et al. 1992; Shaya et

al. 1992; Baldwin et al. 1993; Laruelle et al. 1993a).

METHODS

Binding Studies

Binding studies were performed with unlabeled ß-CIT. The affinity of

ß-CIT for DA and 5-HT transporters was evaluated by measuring the

IC50 of ß-CIT for displacing [

3H]CFT from DA transporters and

[

3H]paroxetine from 5-HT transporters in baboon striatum and rat

cortex, respectively. Assays were performed according to previously

published methods (Laruelle et al. 1988; Madras et al. 1989b ). ß-CIT

was compared with cocaine, CFT, and CIT (the C-2 epimer of

ß-CIT).

Radiolabeling

[

123I]ß-CIT was prepared by oxidative iododestannylation of the

corresponding tributylstannyl precursor (Neumeyer et al. 1991;

Baldwin et al. 1993), and, in turn, was synthesized from ß-CIT by

palladium catalyzed reaction with the bis(trialkyl)distannane

132
FIGURE 1. Chemical formula of cocaine and its phenyltropane

congeners, CFT and ß-CIT

(Azizian et al. 1981). The radiolabeling of the precursor with 123I was

performed using the following method: To a vial containing 50 μg

(0.094 μmol) methyl 3ß-(4-tributylstannylphenyl)tropane-2ßcarboxylate was added, in the following order, 50 μL ethanol, 150 μL

0.5 M H3PO4, 125-500 μL (20-30 mCi) Na123I solution, and 100 μL

(4.2 μmol) 0.32 percent peracetic acid (freshly prepared from 32

percent CH3CO3H). After 20-30 min at room temperature, 50 μL

(5 mg) 100 mg/mL aqueous NaHSO3 solution was added, and the vial

headspace was purged into charcoal with a stream of N2 or Ar for

10 min. Saturated NaHCO3

 solution (1 mL) was added, and the mixture was extracted with 3x1 mL ethyl acetate; the combined extracts

were passed through a small column of anhydrous Na2SO4

 and

133
evaporated on a rotary evaporator with a stream of N2

 at a bath temperature of 37 °C. The residue was dissolved in 50 μL CH3OH, mixed

with 100 μL mobile phase, and injected into the High Performance

Liquid Chromatography (HPLC). The fraction eluting at the retention

time of ß-CIT was collected in a 50 mL pear-shaped flask containing

100 μL (100 μg; 5.7 μmol) 1 mg/mL L-ascorbic acid. The solvent was

evaporated as above, and the residue was dissolved in 400 μL ethanol

and diluted with 6-8 ml 0.9 percent NaCl. The final solution (approximately 5 percent ethanol and 0.1 mM ascorbic acid at a pH of 5-5.5)

was filtered through a 0.2 μ membrane filter into a sterile 10 mL

serum vial or 10 mL syringe. The radiochemical purity was measured

by HPLC immediately after preparation and again after storage at room

temperature for 15-28 h. Specific activity was measured by comparing

the UV response of the sample with that of known concentrations of

ß-CIT. Radiolabeling yield was calculated as the fraction extracted by

ethyl acetate times the fraction of product in the extract measured by

HPLC. Radiochemical yield was calculated by dividing the radioactivity in the final purified product, corrected for decay (T1/2 13.2 h)

by the amount of starting [

123I]NaI. Radioactivities were measured in a

gamma ionization chamber calibrated for each geometry and vessel

configuration.

Anesthesia Procedure

Pharmacokinetic and SPECT brain imaging experiments were performed with 11-14 kg baboons (Papio anubis) and 6-7 kg vervet

monkeys (Cercopiftzecus aethiops). Fasted animals were immobilized

with ketamine (10 mg/kg intramuscular [i.m.]) and anesthetized with

2.5 percent isoflurane through an endotracheal tube. Glycopyrrolate

(10 μg/kg i.m.), a long-acting peripheral anticholinergic drug, was

administered at the beginning of the study to decrease respiratory and

digestive secretions. Vital signs were monitored every 30 min, and the

temperature was kept constant at 36.5-37.5 °C with heated water

blankets. An IV perfusion line with 0.9 percent NaCl was maintained

during the experiment and used for the injection of radiotracer and

nonradioactive drugs. The animal’s head was immobilized within the

camera gantry with a “bean bag” that hardens on evacuation.

134
Pharmacokinetics and Plasma Metabolites Analysis

Plasma pharmacokinetics were studied in one baboon and two vervet

monkeys, with each animal studied in two separate experiments 28-33

days apart. [

123I]ß-CIT (9.8-13.5 mCi for the baboon and 3.2-6.5 mCi

for the vervets) was injected intravenously. Arterial blood samples

were collected every 10 s up to 2 min and at longer intervals thereafter, up to 300-360 min after administration of the tracer. Selected

venous samples were taken from the contralateral vein.

Plasma was extracted with ethyl acetate, and selected samples were

analyzed by HPLC, estimating intermediate values by linear interpolation. Control samples incubated with whole blood and treated in

the same way were used to calculate the recovery coefficient for

[

123I]ß-CIT and to verify stability of the compound in blood under the

storage conditions (18-22 h at 4 °C). Extraction values for plasma

samples were corrected by dividing by the recovery coefficient. The

precision of the extraction method, measured by extracting five

repeated aliquots of a single large plasma sample from one baboon

experiment, was 0.5 percent expressed as the coefficient of variation.

Control experiments in which [

123I]ß-CIT was added to whole blood

and subjected to the metabolite extraction procedure produced a

recovery of 92.0±0.6 percent (n = 6). The stability of the compound

in blood was demonstrated in each experiment by adding one aliquot

to a sample of blood at the time of administration to the animal and

comparing the result after overnight storage with that of a fresh sample

added at the time of metabolite analysis. The content of parent

[

123I]ß-CIT was similar in both samples, averaging 92.6±0.5 percent

versus 91.3±0.6 percent (n = 6) at an average time interval of 20.3±

0.5 h between the first and second incubation. Plasma protein binding

was measured by ultrafiltration with membranes having a 30,000 mol

wt cutoff. A control sample consisting of tracer diluted with 0.9 percent NaCl was run concurrently.

Plasma total parent concentrations were fitted to a three exponential

function with a weighted least squares program. The half-life T1/2

derived from the relation T1/2 = (ln 2)/ with as the rate constant of

each exponential.

135
SPECT Data Acquisition and Analysis

Data were acquired with two brain-dedicated SPECT cameras: the

multislice CERASPECT device (Digital Scintigraphics, Cambridge;

Holman et al. 1990) and the single slice 810X Brain Imager

(Strichman Medical Equipment, Medfield, MA; Stoddart and Stoddart

1979). The injected dose of [

123I]ß-CIT was 6.52±.5 mCi, with these

and subsequent data expressed as mean ± SEM.

Scans were acquired in continuous mode for 3 min throughout the

experiments. Scanning experiments (n = 39) lasted 329±8 min

yielding 94±3 acquisitions per experiment. Tomographic backprojection and image reconstruction were performed as previously

described (Laruelle et al. 1993a). Attenuation correction was performed assuming uniform attenuation equal to that of water (attenuation coefficient μ = 0.150 cm2

/g) within an ellipse drawn around the

brain (Chang 1987). Four regions of interest (ROIs) were examined

on both Strichman and CERASPECT scans (striatum, occipital, hypothalamic/midbrain area, and cerebellum). In addition, frontal pole was

examined on the CERASPECT scans. The ROIs were positioned by

reference to the activity distribution, using a baboon brain atlas (Riche

et al. 1988) and magnetic resonance imaging (MRI) scans, performed

in three of the animals and coregistered to the SPECT images (Malison

et al., in press). ROI activities were decay corrected to the time of

injection.

Pharmacological Selectivity

The pharmacological nature of in vivo [

123I]ß-CIT binding was

investigated by displacement studies, achieved by IV injections of

pharmacological doses of nonradioactive compounds with known

pharmacological properties.

[

123I]ß-CIT binding to DA, 5-HT, and NE transporters was evaluated

with four non-cocaine-related monoamine uptake inhibitors: LU

19-005, a nonspecific monoamine uptake inhibitor (Hyttel and Larsen

1985); GBR 12909, a specific DA uptake inhibitor (Andersen 1989);

citalopram, a specific 5-HT uptake inhibitor (Hyttel 1982); and maprotiline, a specific NE uptake inhibitor (Hyttel 1982). Displacement

experiments were analyzed by comparing the ROI activity after

136
injection of the displacing agent to the projected ROI activity in the

absence of its injection, estimated using the kinetic parameters derived

from the control scans.

In Vivo Potencies of Cocaine Analogs

The authors investigated the ability of [

123I]ß-CIT SPECT imaging to

provide quantitative information about the level of in vivo site occupancy reached by IV injection of cocaine (n = 5), CFT (n = 4), and

ß-CIT (n = 8). The level of site occupancy induced by the injection

of a given dose of the drug was estimated as the difference between

the level of [

123I]ß-CIT specific uptake before injection and the level at

which the uptake curve stabilized after injection. The percentage displacements (P) were logit transformed as ln[P/(100-P)] and plotted

versus 1n dose or 1n cumulative dose in case of multiple injections.

The dose inducing 50 percent site occupancy (ED50) was calculated as

the x-axis intercept and expressed in μmol/kg. The Hill coefficient

was calculated as the slope of the regression line divided by 2.303.

The relationship between the in vivo ED50 and the in vitro IC50 was

investigated by linear regression analysis (IC50-3.8 nM), where IC50 is

the concentration of drug required to displace 50 percent of the specifically bound radiotracer.

RESULTS

Binding Studies

ß-CIT showed high potency for displacing both [

3H]CFT (IC50 = 1.6

nM) from DA transporter sites and [

3H]paroxetine from 5-HT transporter sites (IC50 = 3.8 nM; table 1). The binding was stereoselective:

the affinity of the isomer a-CIT was much lower (IC50 of 87.6 nM and

210 nM for DA and 5-HT transporters, respectively). ß-CIT was 10

times and 100 times more potent than CFT for DA transporters (IC50 =

15.3 nM) and 5-HT transporters (IC50 = 479 nM), respectively. ß-CIT

also displayed greater potency than cocaine at each of these sites (IC50

of 221 nM and 207 nM for DA and 5-HT transporters, respectively).

137
TABLE 1. ß-CIT, CFT, and Cocaine: Comparison between in vitro

ID50 for DA and 5-HT transporters and in vivo ED50 for

displacing striatal [123I]ß-CIT specific binding

ß-CIT

CFT

In vitro In vitro

[

3H]CFT [3H]paroxetine

IC50

a

IC50

b

nM nM

1.6 ± 0.15 3.8 ± 0.53

15.3 ± 1.2 479 ± 59

In vivo

[

123I]ß-CIT

ED50

c

μmol/kg

0.11

0.40

Cocaine 221 ± 14 207 ± 66 9.6

aMeasured in baboon caudate homogenates membranes, according to

Madras et al. (1989b); n = 4.

bMeasured in rat cortical homogenates membranes, according to

Laruelle et al. (1988); n = 4.

cMeasured in baboon striatum with SPECT and Logit-log transform of

displacement data (see Methods).

Radiolabeling

The radiolabeling yield averaged 65.2±2.3 percent (n = 18), and overall radiochemical yield averaged 54.4±2.2 percent. The radiolabeled

product was stable for at least 28 h at room temperature in normal

saline containing about 5 percent ethanol and 0.1 mM ascorbic acid;

radiochemical purity averaged 97.5±0.5 percent immediately after

preparation and 96.0±0.6 percent measured 21.1±0.8 h later. The

specific activity of the final product was estimated to be 500-1,200

Ci/mmol, due to the presence in the tributylstannyl precursor of about

7 mol percent ß-CIT that was carried through the radiosynthesis and

detected by HPLC of the tributylstannyl precursor.

138
Pharmacokinetics and Plasma Metabolites Analysis

Protein Binding. Plasma protein binding of [

123I]ß-CIT measured in

vitro by ultrafiltration was 74.8±l.4 percent (n = 6), whether incubated

in plasma or in whole blood for up to 22 h. Control binding of tracer

to the ultrafiltration membrane in the absence of plasma was 6.2±0.2

percent (n = 6).

Metabolites. Plasma metabolites were characterized by solvent

extraction followed by HPLC of the organic extract. The major metabolite was a polar, nonextractable fraction that increased to > 50

percent of the plasma activity by 30-45 min postinjection (p.i.). An

extractable metabolite, less lipophilic than ß-CIT on HPLC, also was

observed, increasing to about 4 percent of the plasma activity after

2-3 h.

Plasma Kinetics. After IV administration of [

123I]ß-CIT, peak arterial

plasma levels of total 123I and of unmetabolized parent were reached

within 40-80 sec. In two experiments in which both arterial and

venous samples were taken, venous plasma levels initially were lower

than arterial levels but converged within 2 min in one experiment and

within 30 min in the other. Arterial plasma concentration of parent

compound was observed to match a triexponential function, with a

terminal half-life of 77-166 min in baboons and 108-153 min in

vervets.

Brain Uptake

Brain activity was concentrated in two areas. The most superior

activity concentrations overlaid the right and left striatum. A second

area of activity was detected consistently on the midline about 15 mm

below the level of the striatal slice. This activity extended inferiorly

and posteriorly over about 12 mm. This area was identified by reference to brain atlas diagrams and MRI coregistered scans (Malison et

al., in press), as located at the level of the lower diencephalon

(including the hypothalamus) and the mesencephalon (including the

substantia nigra, the raphe nuclei, and the colliculi). Since the

anatomical resolution of the camera did not allow differentiating the

individual contributions of each of these structures to the activity, the

ROI placed on the anterior/superior part of this area was labeled as

139
hypothalamic/midbrain ROI. Typical time activity curves in the

striatum, hypothalamic/midbrain, occipital, frontal, and cerebellar

regions are shown in figure 2.

Striatum. Striatum time activity curves were characterized by a

prolonged phase of increasing activity, peaking at about 142±10 min

and followed by an almost negligible washout (0.6±0.2 percent/h).

The target-to-background (i.e., striatal-to-cerebellar) ratio increased

consistently over the course of the experiments reaching 7.33±0.88 at

300 min. Despite the stability of the striatal plateau phase, [

123I]ß-CIT

uptake was found to be reversible as the injection of an excess dose of

nonradioactive ß-CIT at 180 min (figure 3) induced a displacement of

striatal activity to a level similar to the cerebellar activity. This experiment showed that, at 180 min, more than 75 percent of striatal activity

was associated with saturable sites.

Hypothalamic/Midbrain Area. Activity in the hypothalamic/midbrain

area reached a peak at 47±2 min at a level equivalent to 63±3 percent

of the striatal peak activity. Activity decreased thereafter at a rate of

14.4±0.9 percent/h. Despite the fact that the absolute number of

counts reached a peak value at 47±2 min, the target-to-background

ratio continued to increase for a longer period to 1.69±0.10 at 60 min,

2.57±0.18 at 180 min, and 2.32±0.38 at 300 min. Injection of nonradioactive ß-CIT at 180 min displaced at least 50 percent of the

activity in the hypothalamic/midbrain area (figure 3).

Occipital, Frontal, and Cerebellar Areas. The time course of

activities in these three regions was similar. Activities peaked within

the first 30 min. Peak activities in these regions also were within a

similar range; the occipital had values (46±3 percent) slightly higher

than the cerebellar and frontal regions (36+3 percent and 38±3 percent,

respectively). The washout rate was high in all three regions (20 to 30

percent/h). The injection of nonradioactive ß-CIT at 180 min did

notaffect the activity in these cortical areas, suggesting that, at that

time, most of the activity was not associated with saturable sites.

140
FIGURE 2. Kinetic of brain uptake of [

123I]ß-CIT in various regions

of the baboon’s brain, following the injection of 3.2 mCi

[

123I]ß-UT: striatum, average of left and right (closed

circles), hypothalamic/midbrain area (open circles),

occipital pole (closed squares), frontal pole (open

squares), and cerebellum (open triangles). The striaral

activity increased during the first three hours and

remained stable thereafter. Hypothalamic/midbrain

activity exhibited a peak between 45 and 60 min and then

washed out at a rate of 14 percent of the peak value per

h. Occipital, frontal, and cerebellar activities displayed

similar kinetics, characterized by an early peak (20-30

min), followed by a rapid washout (20-30 percent of peak

value per h). Data were acquired on the CERASPECT

camera and are expressed in CPM/pixel.

141
FIGURE 3. Effects of injection of receptor saturating dose of ß-CIT

on [

123I]ß-CIT uptake in baboon. Nonradioactive ß-CIT

(0.94 μmol/kg IV) was injected IV 210 min (arrow)

following the injection of 6.0 mCi [

123I]ß-CIT and caused

displacement of 70-80 percent striatal activity and 50-60

percent of hypothalamic/midbrain activity. No effects

were observed in the cortical and cerebellar regions.

Data were acquired on the CERASPECT camera and are

expressed in CPM/pixel.

Pharmacological Selectivity

The pharmacological identity of the saturable sites associated with

[

123I]ß-CIT uptake in the striatum was studied by injection of nonradioactive drugs during the plateau phase of the striatal uptake

(180-200 min, “late injections”). Uptake in the hypothalamic/midbrain

142
area was investigated more specifically by injections performed shortly

after peak time (60 min, “early injections”).

LU 19-005. Injection of LU 19-005 (3 μmol/kg IV), a nonspecific

uptake inhibitor, at 180 min displaced both the striatal and hypothalamic/midbrain activities.

GBR 12909. Injections of GBR 12909 (3.4 and 6.8 μmol/kg IV)

were performed at 200 min in two experiments and induced 47-50

percent displacement of striatal [

123I]ß-CIT (figure 4). The hypothalamic/midbrain [

123I]ß-CIT binding was unaffected, as demonstrated

by an unchanged washout rate after the injection (13.3 percent/h).

Citalopram. Injection of citalopram (7.4 μmol/kg IV) at 180 min had

no noticeable affect on striatal activity. However, it induced a marked

increase in washout rate of the hypothalamic/midbrain area from 13.6

percent/h before the injection to 35.5 percent/h during the 60 min

following the injection (figure 5). In three other experiments, citalopram was injected after 60 min at 7.4, 11.1, and 14.8 μmol/kg IV,

respectively. The hypothalamic/midbrain washout rates, calculated

during 60 min following the injection, increased to 45, 34, and 41

percent/h, respectively.

Maprotiline. Injection of maprotiline (13.9 μmol/kg IV) at 60 min

failed to induce apparent changes in the uptake kinetics in either

striatal or hypothalamic/midbrain areas.

In Vivo Potencies of Cocaine Analogs

The prolonged phase of stable striatal uptake allowed the authors to

perform up to three displacements in a single experiment. Since

thespontaneous washout was negligible, no correction was needed in

computing the percentage of displacement. Striatal specific binding

was defined operationally as the difference between striatal and

cerebellar activity. The validity of this subtraction technique in the

late phase of the scan was supported by displacement experiments with

high doses of ß-CIT, as the level of nondisplaceable striatal activity

was similar to the cerebellar activity (figure 3).

143
FIGURE 4. Effects of the selective DA uptake inhibitor GBR 12909 on

[

123I]ß-CIT uptake in baboon. GBR 12909 (6.8 μmol/kg)

was injected IV 180 min after in injection of 4.1 mCi

[

123I]b-CIT and induced a displacement of 49 percent of

striatal activity. Hypothalamic/midbrain area was

unaffected by the injection of GBR 12909, as the washout

rate was unchanged by the injection. Cerebellum activity

was unaffected. Data were acquired on the 810X Brain

Imager camera and are expressed in SMU/pixel, with

SMU corresponding to the count rate recorded from a

untform flood source containing 100 Bq/ml of 99mTc point

source.

Results of five experiments with a total of eight injections of various

doses of nonradioactive cocaine were analyzed with the logit-log plot

method (figure 6). Linear regression (r2 = 0.92) allowed calculation of

the ED50 as the inverse logarithm of the x-axis intercept (cocaine ED50

= 9.6 μmol/kg). The ED50 of ß-CIT and CFT were calculated with a

144
FIGURE 5. Effects of the selective 5-HT uptake inhibitor citalopram

on [

123I]ß-CIT uptake in baboon striatum (closed circles),

hypothalamic/midbrain area (open circles), and

cerebellum (open triangles). Citalopram (7.4 μmol/kg)

was injected IV 180 min after the injection of 13.5 mCi

[

123I]ß-CIT and induced an increase in the

hypothalamic/midbrain activity washout from 13.6

percent/h before the injection to 35.5 percent/h during the

60 min period after the injection. Striatal and cerebellar

activities were unaffected by the injection of citalopram.

Data were acquired on the CERASPECT camera and are

expressed in CPM/pixel.

similar method and found to be 0.11 and 0.40 μmol/kg, respectively.

The relationship between the in vivo ED50 of the three investigated

cocaine-like drugs and their in vitro IC50 for displacing [

3H]CFT from

DA transporters in monkey striatum appeared linear (r2 = 0.96; figure

7). There was no apparent relationship with their in vitro IC50 for

displacing [

3H]paroxetine from 5-HT transporters in rat cortical

membranes (r2 = 0.27).

145
FIGURE 6. Logit-log plot of four displacements performed with various

doses of CFT. Data were acquired during four scanning

experiments. To linearize the relationship between the

injected dose and site occupancy, the percent displacement

(P) was logit transformted as ln[P/(100-P)] and plotted

versus ln dose. In this graph, 50 percent site occupancy

corresponded to the 0 value on the y axis. Data were

analyzed by linear regression (r2 = 0.96). The inverse

logarithm of the x intercept of the regression line was used

as the estimation of the ED50 (0.40 μmol/kg).

DISCUSSION

These studies suggest that [

123I]ß-CIT is a potentially useful ligand to

label DA and 5-HT transporters in the living human brain.

146
FIGURE 7. Relarionship between the in vivo ED50 (μmol/kg) of

cocaine, CFT, and ß-UT for displacing [123I]ß-CIT

binding in monkey striatum and their in vitro IC50(nM) for

displacing [3H]CFT from DA transporters in monkey

striatum. There was a significant relationship between in

vivo ED50 values and in vitro [

3H]CFT IC50 values (r2

 =

0.96). Such a relationship was not found between the in

vivo ED50 values and [

3H]paroxetine IC50 values (r2

 =

0.27).

Binding Studies

ß-CIT displayed a similar high affinity for both DA and 5-HT transporters. Further pharmacological characterization with [

125I]ß-CIT

demonstrated a good correlation between the potency of various drugs

to inhibit DA uptake and to displace [

125I]ß-CIT binding in rat striatum

(Boja et al. 1992). In the cortex, a similar correlation was found with

the potency of various drugs to inhibit 5-HT uptake (Boja et al. 1992).

The authors recently observed that displacement of [

125I]ß-CIT binding

in rat and monkey striatum by citalopram was best fit with a two-site

model, the first site corresponding to 5-10 percent of the binding

147
(Laruelle et al., in press- b). Since the IC50 of this first site was close

to the affinity of citalopram for 5-HT transporter, the authors

believethat 5-10 percent of [

125I]ß-CIT in vitro striatal binding is

related to the 5-HT transporter. This value is in accordance with the

density ratio of [

3H]GBR 12909 and [3H]paroxetine binding sites in

human striatum (Laruelle et al. 1988; Marcusson and Erikson 1988;

Backstrom et al. 1989; De Keyser et al. 1989).

Pharmacokinetics

Studies of clearance from arterial plasma of the parent compound

showed slow terminal half-lives (77-166 min in baboons and 108-153

min in vervets). Misra and colleagues (1977) reported a plasma

terminal half-life of [

3H]cocaine in rhesus monkey of 79 min. Thus,

these data suggested that ß-CIT might be metabolized more slowly

than cocaine. The major metabolite was a polar, nonextractable

fraction, and, thus, is expected not to cross the blood-brain barrier.

This was confirmed by experiments in which monkeys (n = 3

[2 vervets and 1 baboon]) were euthanized 90-120 min after

[

125I]ß-CIT injection. The parent compound represented 75 to 90

percent of the activity in the caudate but only 10 to 30 percent of the

activity in the plasma.

Brain Uptake

The total brain uptake of the tracer was high, as attested by a high

count rate within the field of view. The high brain uptake was

measured more accurately with whole body dosimetry (Baldwin et al.

1993) that showed that 6 to 7 percent of the activity was present in the

brain during the first h postinjection.

The activity was concentrated in the striatum and the midbrain/ hypothalamic area. This localization was confirmed with ex vivo autoradiographic studies in three animals performed at 120 min after injection of

[

123I]ß-CIT. In these experiments, the authors found high levels of

activities in the striatum, the hypothalamus, the colliculi, the substantia

nigra, and the raphe nuclei.

The striatal activity increased for a prolonged period (2 to 3 h) and

showed a remarkably stable plateau phase thereafter, with a negligible

148
washout phase (< 1 percent/h). As a result, the striatum-to-cerebellum

ratio was very high at the end of the study (about 7 to 1). In contrast,

the hypothalamic/midbrain activity peaked and washed out more

quickly.

Pharmacological Specificity

Displacement experiments performed with unlabeled ß-CIT and the

nonselective monoamine inhibitor LU 19-005 demonstrated that

activity in the striatum and midbrain/hypothalamic area was associated

with binding to saturable sites. Selective monoamine uptake inhibitors

were then used to define the pharmacological identity of these sites.

At 300 min, the striatal activity was displaceable by GBR 12909, a DA

selective drug, but not by citalopram, a 5-HT selective drug. This

indicates that, at 300 min, the striatal uptake was primarily associated

with DA transporters. The inverse was observed in the hypothalamic/

midbrain area, showing that the activity in this region was associated

predominantly with 5-HT transporters. These results confirmed similar

in vivo observations in rodents (Cline et al. 1992; Scheffel et al. 1992).

The absence of a citalopram effect in the striatum contrasted with in

vitro experiments in which citalopram displaced about 10 percent of

[

125I]ß-CIT equilibrium binding in the monkey caudate. The in vivo

conditions are, however, very different from the in vitro equilibrium

conditions. The rapid washout of the midbrain/hypothalamic area

suggested that the uptake associated with 5-HT transporters washed out

more quickly than the uptake associated with DA transporter. Thus,

the component of striatal uptake associated with 5-HT transporter

might have washed away at 300 min when the displacement

experiments were performed.

Citalopram was the only drug found to be effective in increasing the

washout rate in the hypothalamic/midbrain area. This is consistent

with the fact that 5-HT transporters are more abundant than DA

transporters in this region (Cortes et al. 1988; Laruelle et al. 1988;

De Keyser et al. 1989). DA transporters are, however, present in the

substantia nigra, but [

123I]ß-CIT binding to these sites was not demonstrated by the GBR 12909 injections. The interpretation of displacement experiments was more difficult in the hypothalamic/midbrain area

149
than in the striatum because of the absence of a prolonged plateau

phase. This difficulty could be overcome by inducing a state of prolonged equilibrium by a constant infusion of the radiotracer. This

paradigm, applied successfully for [

123I]iomazenil (Laruelle et al.

1993b), does allow a stable baseline of activity that increases the

sensitivity to displacement experiments.

In Vivo Potencies of Cocaine Analogs

The stable baseline of [

123I]ß-CIT activity in the striatum allowed

calculating the receptor occupancy ED50 of three related compounds:

ß-CIT, cocaine, and CFT. There was a good relationship between the

in vivo ED50 of these compounds and their in vitro IC50 for displacing

[

3H]CFT (r2

 = 0.96) but not with their IC50 for displacing

[

3H]paroxetine (r2

 = 0.27). Several conclusions can be made from this

series of experiments. First, in vivo [

123I]ß-CIT binding in striatum is

associated predominantly with DA transporters. Second, the affinity

for the DA transporters is the main factor that explains the differences

in ED50 between these drugs. Differences in peripheral clearance and

blood-brain barrier permeability, if any, do not seem to have a significant contribution to the observed ED50 differences. Third, this paradigm has potential useful applications in humans to the study of the

relationship between behavioral effects of cocaine-related drugs and

kinetics of transporter occupancy.

[

123I]ß-ClT as a SPECT Radiotracer

[

123I]ß-CIT appears to be a promising SPECT radiotracer for labeling

DA and 5-HT transporters. Some of the characteristics of the ligand

are clearly advantageous: high brain uptake, slow peripheral clearance,

low levels of metabolites in the brain, and low nonspecific binding and

corresponding high levels of target-to-background ratio. Other characteristics (e.g., selectivity and high affinity) can be interpreted as positive or negative, depending on the intended application.

The lack of specificity for either the DA or the 5-HT transporter is not

an ideal situation for the quantification of these sites in each brain

region. However, it appears that binding to these transporters has a

different kinetic profile, such that the striatal signal becomes predominantly associated with DA transporters after 180 min, and the peak of

150
uptake in the hypothalamic/midbrain activity is predominantly associated with 5-HT transporters. Thus, binding to each of these sites can

be distinguished both anatomically and kinetically. If this observation

is replicated in humans, it opens the opportunity to obtain information

on both systems in one scanning session. This might be cost effective

in clinical situations when alterations of both sites have been described, as in Parkinson’s disease or in chronic cocaine abuse. More

specific ligands, such as [

123I]isopropyl-CIT ([123I]RTI-121; Scheffel et

al. 1993) or [

123I]5-iodo-6-nitroquipazine (Mathis et al. 1992), are

presently under development and might broaden the range of options

available to the clinician.

The high affinity of [

123I]ß-CIT for the DA transporter, inducing a

prolonged plateau phase in the striatum, is an ideal situation for

pharmacological challenge tests, as performed here with cocaine

analogs. This kinetic profile, however, creates a difficulty for the

quantification of the binding sites. A ligand with a high tissue

equilibrium volume of distribution (i.e., a high maximal number of

binding sites (Bmax)-to-equilibrium binding constant [KD] ratio) is not

likely to reach equilibrium during the timeframe of the imaging session

(Gjedde and Wong 1990), and equilibrium is required for measurement

of Bmax and KD. In the absence of equilibrium, only the rate of association to the receptor can be measured (the product Bmax kon, with kon

being the association rate constant).

Furthermore, if equilibrium is not reached and if the association to the

receptor is rapid as compared to the blood-brain barrier permeability,

the activity in the region of interest reflects the blood flow more than

the density of receptors, as observed with high affinity ligands like

[

123I]qunuclidinyl benzilate (QNB) (Gibson et al. 1984) and

[N-methyl-11C]scopolamine (Vora et al. 1983; Frey et al. 1987). Under

these conditions, little quantitative information can be gained by

empirical methods, such as ratio methods, as opposed to model-based

methods (Carson 1991) that are more difficult to implement in the

clinical setting. Additional experiments comparing empirical methods

to model-based methods with [

123I]ß-CIT are needed to explore this

issue.

151
CONCLUSION

In conclusion, [

123I]ß-CIT appears to be a promising ligand for

visualization and pharmacological studies of DA and 5-HT transporters. More experiments are needed to assess the protocol and

methods necessary for measurement of the density and affinity of these

sites. Development of these methods and evaluation of the tracer in

humans currently are being performed in the authors’ laboratory.

REFERENCES

Allard, P.; Alafuzoff, I.; Carlsson, A.; Erikson, K.; Ericson, E.;

Gottfries, C.G.; and Marcusson, J.O. Loss of dopamine uptake sites

labeled with [

3H]GBR-12935 in Alzheimer’s disease. Eur Neural

30:181-185, 1990.

Andersen, P.H. The dopamine uptake inhibitor GBR 12909: Selectivity

and molecular mechanism of action. Eur J Pharmacol 166:493-504,

1989.

Azizian, H.; Eabom, C.; and Pidcock, A. Synthesis of organotrialkylstannanes. The reaction between organic halides and

hexaalkyldistannanes in the presence of palladium complexes.

J Organomet Chem 215:49-58, 1981.

Bäckström, I.; Bergstrom, M.; and Marcusson, J. High affinity

[

3H]paroxetine binding to serotonin uptake sites in human brain.

Brain Res 486:261-265, 1989.

Baldwin, R.M.; Zea-Ponce, Y.; Zoghbi, S.S.; Laruelle, M.; Al-Tikriti,

M.S.; Sybriska, E.H.; Malison, R.T.; Neumeyer, J.L.; Milius, R.A.;

Wang, S.; Stabin, M.; Smith, E.O.; Chamey, D.S.; Hoffer, P.B.; and

Innis, R.B. Evaluation of the monoamine uptake site ligand

[

123I]methyl 3ß-(4-iodophenyl)tropane-2ß-carboxylate ([123I]ß-CIT)

in nonhuman primates: Pharmacokinetics, biodistribution, and

SPECT imaging coregistered with MRI. Nucl Med Biol 20:597-606,

1993.

Balster, R.L.; Caroll, F.I.; Graham, J.H.; Mansbach, R.S.; Rahman,

M.A.; Philip, A.; Lewin, A.H.; and Showalter, V.M. Potent

substituted-3-ß-phenyltropane analogs of cocaine have cocaine-like

discriminative stimulus effects. Drug Alcohol Depend 29:145-151,

1991.

152
Boja, J.W.; Mitchell, W.M.; Patel, A.; Kopajtic, T.A.; Carroll, F.I.;

Lewin, A.H.; Abraham, P.; and Kuhar, M.J. High-affinity binding

of [

125I]RTI-55 to dopamine and serotonin transporters in rat brain.

Synapse 12:27-36, 1992.

Boja, J.W.; Patel, A.; Carroll, F.I.; Rahman, M.A.; Philip, A.; Lewin,

A.H.; Kopajtic, T.A.; and Kuhar, M.J. [

125I]RTI-55: A potent ligand

for dopamine transporters. Eur J Pharmacol 194: 133-134, 1991.

Carroll, F.I.; Gao, Y.; Rahman, M.A.; Abraham, P.; Parham, K.;

Lewin, A.H.; Boja, J.W. and Kuhar, M.J. Synthesis, ligand binding,

QSAR, and CoMFA study of 3ß-(p-substituted phenyl)tropane2ß-carboxylic acid methyl esters. J Med Chem 34:27 19-2725, 1991.

Carson, R.E. Precision and accuracy considerations of physiological

quantitation in PET. J Cereb Blood Flow Metab 11:A45-A50, 1991.

Cash, R.; Raisman, R.; Ploska, A.; and Agid, A. High and low affinity

3H-imipramine binding sites in control and Parkinsonian brains. Eur

J Pharmacol 117:71-80, 1985.

Chang, L.T. A method for attenuation correction in computed tomography. IEEE Trans Natl Sci NS-25:638-643, 1987.

Clarke, R.L.; Daum, S.J.; Gambino, A.J.; Aceto, M.D.; Pearl, J.;

Levitt, M.; Cumiskey, W.R.; and Bogado, E.F. Compounds

affecting the central nervous system: IV. 3ß-Phenyltropane-2-

carboxylic esters and analogs. J Med Chem 16:1260-1267, 1973.

Cline, E.J.; Scheffel, U.; Boja, J.W.; Mitchell, W.M.; Caroll, F.I.;

Abraham, P.; Lewin, A.H.; and Kuhar, M.J. In vivo binding of

[

125I]RTI-55 to dopamine transporters: Pharmacology and regional

distribution with autoradiography. Synapse 12:37-46, 1992.

Cortes, R.; Soranoi, E.; Pazos, A.; Probst, A.; and Palacios, J.M.

Autoradiography of antidepressant binding sites in the human brain:

Localization using [

3H]imipramine and [3H]paroxetine. Neuroscience

27:473-496, 1988.

Crow, T.J.; Cross, A.J.; Cooper, S.J.; Deakin, J.F.W.; Ferrier, I.N.;

Johnson, J.A.; Joseph, M.H.; Owen F.; Poulter, M.; Lofthouse, R.;

Corsellis, J.A.N.; Chambers, D.R.; Blessed, G.; Perry, E.K.; and

Perry, E.H. Neurotransmitter receptors and monoamine metabolites

in the patients with Alzheimer-type dementia and depression and

suicides. Neuropharmacology 23:1561-1569, 1984.

De Keyser, J.; De Baecker, J.P.; Ebinger, G.; and Vauquelin, G.

[

3H]GBR 12935 binding to dopamine uptake sites in the human

brain. J Neurochem 53:1400-1404, 1989.

153
Eckelman, W.C.; Rzeszotarski, W.J.; Gibson, R.E.; Francis, B.; and

Reba, R.C. Use of 3-quinulidinyl 4-iodobenzilate as a receptor

binding radiotracer. J Nucl Med 26:637-642, 1985.

Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.E.; and Agranoff, B.W.

Quantitative in vivo receptor binding: III. Tracer kinetic modeling

of muscarinic cholinergic receptor binding. Proc Natl Acad Sci

U S A 82:6711-6715, 1987.

Gibson, R.E.; Weckstein, D.J.; Jagoda, E.M.; Rzeszotarski, W.J.; Reba,

R.C.; and Eckelman, W.C. The characteristics of I-125 4-IQNB and

H-3 QNB in vivo and in vitro. J Nucl Med 25:214-222, 1984.

Gjedde, A., and Wong, D.F. Modeling neuroreceptor binding of

radioligands in vivo. In: Frost, J.J., and Wagner, H.N., Jr., eds.

Quantitative Imaging: Neuroreceptors, Neurotransmitters, and

Enzymes. New York: Raven Press, 1990. pp. 51-79.

Glowinsky, J., and Axelrod, J. Inhibition of uptake of tritiated

noradrenaline in intact rat brain by imipramine and structurally

related compounds. Nature 204:107-114, 1964.

Hirai, M.; Kitamura, N.; Hashumoto, T.; Nakai, T.; Mita, T.;

Shirakawa, O.; Yamadori, T.; Amano, T.; Noguchi-Kuno, S.A.; and

Tanaka, C. [

3H]GBR-12935 binding sites in human striatal membranes: Binding characteristics and changes in Parkinsonians and

schizophrenics. Jpn J Pharmacol 47:237-243, 1988.

Holman, B.L.; Carvalho, P.A.; Zimmerman, R.E.; Johnson, K.A.;

Tumeh, S.S.; Smith, A.P.; and Genna, S. Brain perfusion SPECT

using an annular single crystal camera: Initial clinical experience.

J Nucl Med 31:1456-1461, 1990.

Hyttel, J. Citalopram: Pharmacological profile of a specific serotonin

uptake inhibitor with antidepressant activity. Prog Neuropsychopharmacol Biol Psychiatry 6:277-295, 1982.

Hyttel, J., and Larsen, J.-J. Neurochemical profile of Lu 19-005 a

potent inhibitor of uptake of dopamine, noradrenaline, and serotonin. J Neurochem 44:1615-1621, 1985.

Innis, R.; Baldwin, R.; Sybirska, E.; Zea, Y.; Laruelle, M.; Al-Tikriti,

M.; Chamey, D.; Zoghbi, S.; Smith, E.; Wisniewski, G.; Hoffer, P.;

Wang, S.; Milius, R.; and Neumeyer, J. Single photon emission

computed tomography imaging of monoamine reuptake sites in

primate brain with [

123I]CIT. Eur J Pharmacol 200:369-370, 1991b .

154
Innis, R.; Zoghbi, S.; Johnson, E.; Woods, SW.; Al-Tikriti, M.;

Baldwin, R.; Seibyl, J.; Malison, R.; Zubal, G.; Chamey, D.;

Heninger, G.; and Hoffer, P. SPECT imaging of the benzodiazepine

receptor in non-human primate brain with [

123I]Ro 16-0154. Eur J

Pharmacol 193:249, 1991a.

Janowsky, A.; Vocci, F.; Berger, P.; Angel, I.; Zelnick, N.; Kleinman,

J.; Skolnick, P.; and Paul, S.M. [

3H]GBR-12935 binding to the

dopamine transporter is decreased in the caudate nucleus in

Parkinson’s disease. J Neurochem 49:617-621, 1987.

Joyce, J.N.; Shane, A.; Lexow, N.; Winokur, A.; Casanova, M.F.; and

Kleinman, J. E. Serotonin uptake sites and serotonin receptors are

altered in the limbic system of schizophrenics. Neuropsychopharmacology 8:315-336, 1993.

Kaufman, M.J., and Madras, B.K. Severe depletion of cocaine

recognition sites associated with the dopamine transporter in

Parkinson’s diseased striatum. Synapse 49:43-49, 1991.

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

the reinforcing properties of cocaine. Trends Neurosci 14:299-302,

1991.

Kung, M.-P.; Kung, H.F.; Billings, J.; Yang, Y.; Murphy, R.A.; and

Alavi, A. Characterization of IBF as a new selective dopamine D-2

receptor imaging agent. J Nucl Med 31:648-654, 1990.

Kung, H.F.; Pan, S.; Kung, M.-P.; Billings, J.; Kasliwal, R.; Reilley,

J.; and Alavi, A. In vitro and in vivo evaluation of [

123I]IBZM: A

potential CNS D-2 dopamine receptor imaging agent. J Nucl Med

30:88-92, 1989.

Laruelle, M.; Abi-Dargham, A.; Casanova, M.F.; Toti, R.; Weinberger,

D.R.; and Kleinman, J.E. Selective abnormalities of prefrontal

serotonergic receptors in schizophrenia: A postmortem study. Arch

Gen Psychiatry, in press-a.

Laruelle, M.; Abi-Dargham, A.; Rattner, Z.; Al-Tikriti, M.; Zea-Ponce,

Y.; Zoghbi, S.S.; Chamey, D.S.; Price, J.; Frost, J.J.; Hoffer, P.B.;

Baldwin, R.M.; and Innis, R.B. Single photon emission computed

tomography measurement of benzodiazepine receptor number and

affinity in primates brain: a constant infusion paradigm with

[

123I]iomazenil. Eur J Pharmacol 230:119-123, 1993b.

155
Laruelle, M.; Baldwin, R.M.; Malison, R.T.; Zea-Ponce, Y.; Zoghbi,

S.S.; Al-Tikriti, M.; Sybriska, E.H.; Zimmerman, R.C.; Wisnieski,

G.; Neumeyer, J.L.; Milius, R.A.; Wang, R.A.; Smith, E.O.; Roth,

R.H.; Chamey, D.; Hoffer, P.B.; and Innis, R.B. SPECT imaging of

dopamine and serotonin transporters with [

123I]ß-CIT: Pharmacological characterization of brain uptake in nonhuman primates.

Synapse 13:295-309, 1993a.

Laruelle, M.; Giddings, S.S.; Zea-Ponce, Y.; Chamey, D.S.; Neumeyer,

J.L.; Baldwin, R.M.; and Innis, R.B. [

125I]ß-CIT in vitro binding to

dopamine and serotonin transporters under “physiological” conditions. J Neurochem, in press-b.

Laruelle, M.; Vanisberg, M.-A.; and Maloteaux, J.-M. Regional and

subcellular localization in human brain of [

3H] paroxetine binding a

marker of serotonin uptake sites. Biol Psychiatry 24:299-309, 1988.

Madras, B.K.; Fahey, M.A.; Bergman, J.; Canfield, D.R.; and

Spealman, R.D. Effects of cocaine and related drugs in non human

primates: I. [

3H]Cocaine binding sites in caudate-putamen.

J Phannacol Exp Ther 251:131-141, 1989a.

Madras, B.K.; Spealman, R.D.; Fahey, M.A.; Neumeyer, J.L.; Saha,

J.K.; and Milius, R.A. Cocaine receptors labeled by [

3H]-2ßcarbomethoxy-3ß-(4-fluorophenyl)tropane. Mel Pharmacol

36:5 18-524, 1989b.

Malison, R.T.; Miller, E.G.; Greene, R.; Hoffer, P.B.; McCarthy, G.;

and Innis, R.B. Computer-assisted coregistration of multislice

SPECT and MRI images by fixed external fiducials. J Comput

Assist Tomogr, in press.

Maloteaux, J.M.; Vanisberg, M.A.; Laterre, C.; Javoy-Agid, F.; Agid,

Y.; and Laduron, P.M. [

3H]GBR-12935 binding to dopamine uptake

sites: Subcellular localization and reduction in Parkinson’s disease

and progressive supranuclear palsy. Eur J Pharmacol 156:331-340,

1988.

Marcusson, J., and Erikson, K. [

3H]GBR-12935 binding to dopamine

uptake sites in the human brain. Brain Res 457:122-129, 1988.

Mathis, C.A.; Biegon, A.; Taylor, S.E.; Enas, J.E.; and Hanrahan, SM.

[

125I]5-Iodo-6-nitro-2-piperazinylquino1ine: A potent and selective

ligand for the serotonin uptake complex. Eur J Pharmacol

210:103-104, 1992.

156
Misra, A.L.; Giri, V.V.; Patel, M.N.; Alluri, V.R.; and Mule, S.J.

Disposition and metabolism of [

3H]cocaine in acutely and

chronically treated monkeys. Drug Alcohol Depend 2:261-272,

1977.

Neumeyer, J.L.; Wang, S.; Milius, R.A.; Baldwin, R.M.; Zea-Ponce,

Y.; Hoffer, P.B.; Sybirska, E.; Al-Tikriti, M.; Laruelle, M.; and

Innis, R.B. [

123I]2ß-carbomethoxy-3ß-(4-iodophenyl)tropane

(ß-CIT): High affinity SPECT radiotracer of monoamine reuptake

sites in brain. J Med Chem 34:3144-3146, 1991.

Niznik, H.B.; Fogel, E.F.; Fassos, F.F.; and Seeman, P. The dopamine

transporter is absent in Parkinsonian putamen and reduced in the

caudate nucleus. J Neurochem 56:192-198, 1991.

Paul, S.M.; Rehavi, M.; Skolnick, P.; and Goodwin, F.K. High affinity

binding of antidepressants to a biogenic amine transport site in

human brain and platelet: Studies in depression. In: Post, R.M., and

Ballinger, J.C., eds. Neurobiology of Mood Disorders. Baltimore:

Williams & Wilkins, 1984. pp. 846-853.

Perry, E.K.; Marshall, E.F.; Blessed, G.; Tomlinson, B.E.; and Perry,

R.H. Decreased imipramine binding in the brains of patients with

depressive illness. Br J Psychiatry 142:188-192, 1983.

Raisman, R.; Cash, R.; and Agid, Y. Parkinson’s disease: Decreased

density of 3H-imipramine and 3H-paroxetine binding sites in

putamen. Neurology 36:556-560, 1986.

Reith, M.E.A.; Sershen, H.; Allen, D.L.; and Lajtha, A. A portion of

[

3H]cocaine binding in brain is associated with serotonergic neurons.

Mol Pharmacol 23:600-606, 1983.

Reith, M.E.A.; Sershen, H.; and Lajtha, A. Saturable [

3H]cocaine

binding in central nervous system of mouse. Life Sci 27:1055-1062,

1980.

Riche, D.; Hantraye, P.; Guibert, B.; Naquet, R.; Loch’h, C.; and

Mazière, M. Anatomical atlas of the baboon’s brain in the

orbito-meatal plane used in experimental positron emission

tomography. Brain Res Bull 20:283-301, 1988.

Ritz, M.C.; Cone, E.J.; and Kuhar, M.J. Cocaine inhibition of ligand

binding at dopamine, norepinephrine, and serotonin transporters: A

structure activity study. Life Sci 46:635-645, 1990.

Scheffel, U.; Dannals, R.F.; Cline, E.J.; Ricaurte, G.A.; Carrel, F.I.;

Abraham, P.; Lewin, A.H.; and Kuhar, M.J. [

123/125I]RTI-55, an in

vivo label for the serotonin transporter. Synapse 11:134-139, 1992.

157
Scheffel, U.; Dannals, R.F.; Wong, D.F.; Yokoi, F.; Carroll, F.I.; and

Kuhar, M.J. Dopamine transporter imaging with novel, selective

cocaine analogs. Neuroreport 3:969-972, 1993.

Shaya, E.K.; Scheffel, U.; Dannals, R.F.; Ricaurte, G.A.; Carrol, F.I.;

Wagner, H.N.; Kuhar, M.J.; and Wong, D.F. In vivo imaging of

dopamine reuptake sites in the primate brain using single photon

emission computed tomography (SPECT) and Iodine- 123 labeled

RTI-55. Synapse 10:169-172, 1992.

Stanley, M.; Virgilio, J.; and Gershon, S. Tritiated imipramine binding

sites are decreased in the frontal cortex of suicides. Science

216:1337-1339, 1982.

Stoddart, H.F., and Stoddart, H.A. A new development in single

gamma transaxial tomography Union Carbide focused collimator

scanner. IEEE Trans Nucl Sci 26:2710-2712, 1979.

Vora, M.M.; Finn, R.D.; Boothe, D.R.; Liskowsky, D.R.; and Potter,

L.T. [N-methyl-11C]-scopolamine: synthesis and biodistribution in

rat brain. J Labelled Comp Radiopharm 20:1229-1236, 1983.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the help of members of the

Neurochemical Brain Imaging Program, Department of Psychiatry and

Section of Nuclear Medicine, Yale School of Medicine, and Research

Biochemicals Int. (Natick, MA) for providing the stannyl precursor of

[

123I]ß-CIT.

AUTHORS

Marc Laruelle, M.D.

Assistant Professor

Ronald M. Baldwin, Ph.D.

Research Scientist

158
Robert B. Innis, M.D., Ph.D.

Associate Professor

Department of Psychiatry

Yale School of Medicine

West Haven VA Medical Center/116A2

West Haven, CT 06516

159

Cerebral Blood Flow Changes

With Acute Cocaine Intoxication:

Clinical Correlations With

SPECT, CT, and MRI

I. Mena, R.J. Giombetti, B.L. Miller, K. Garrett,

J. Villanueva-Meyer, C. Mody, and M.A. Goldberg

INTRODUCTION

Fourteen consecutive patients with acute cocaine intoxication and

neurological symptoms were studied using computerized axial tomography (CT) and single photon emission computerized tomography

(SPECT). Symptoms included transient ischemic attacks, stroke,

seizures, and confusional states. Cerebral blood flow (CBF) was

imaged with 99mTechnetium [99mTc] d,l, hexamethylpropyleneamineoxime (HMPAO) and was measured using Xenon-133 (133Xe). Focal,

multifocal, or diffuse areas of hypoperfusion were found in all 14

patients with either [99mTc]HMPAO SPECT or 133Xe. Multiple

small, superficial, or deep areas of hypoperfusion causing a “scalloped”

appearance on SPECT were seen in 12 patients, and this pattern is

observed frequently in cocaine abuse. The mean CBF measured with

133Xe was diminished at 43.6+3.4 ml/100 g/min compared to 55.1+1.4

ml/100 g/mm in normals. Brain SPECT was useful in the evaluation

of patients who were symptomatic from cocaine abuse, and cortical

and subcortical vascular abnormalities not seen on CT or magnetic

resonance imaging (MRI) were often found with SPECT.

Cocaine continues to be a major public health problem in the United

States, with as many as 5 million daily users (U.S. Department of

Health and Human Services 1990). While the general medical complications of cocaine have been reported extensively, neurological disease

has received less attention and only recently have cocaine-induced

seizures, transient ischemic attacks (TIA), and strokes been described

(Brust and Richter 1977; Caplan et al. 1981; Mody et al. 1988; Levine

and Welch 1988; Levine et al. 1990; Root and Rowbotham 1988).

161
Although vasospasm has been proposed as a possible mechanism for

cocaine-induced TIA and ischemic stroke, angiography is often normal

in these patients (Mody et al. 1988). The combined use of the 133Xe

washout to quantify CBF and [99mTc]HMPAO SPECT to obtain a

high-resolution image of CBF may help delineate the pathophysiological mechanisms associated with cocaine-induced neurological

disease.

The authors evaluated CBF changes in 14 patients with neurological

symptoms due to acute cocaine intoxication. CBF was assessed

quantitatively with 133Xe, while high-resolution tomographic images

of CBF were obtained with [99mTc]HMPAO. Clinical diagnoses were

correlated with CT, MRI, and SPECT.

PATIENTS

Patients entering the emergency room at the Harbor-University of

California, Los Angeles (UCLA) Medical Center with cocaine intoxication and neurological symptomatology were referred for study. In

all, cocaine abuse (mostly “crack” cocaine) had occurred within the

past 48 hours, and subjects with a history of polysubstance abuse were

excluded. The history of cocaine abuse was obtained from the patient

and relatives or friends, and urine toxicology was performed. All

patients were evaluated with neurological examinations, CT or MRI,

and SPECT. Cerebral angiography and lumbar puncture were performed when clinically indicated.

Regional CBF (rCBF)

133Xe was used for absolute measurements of rCBF. 1110 Megabecquerel (MBq) (30 millicuries [mCi]) of 133Xe was injected

intravenously, and CBF was measured using a lo-probe system in a

self-contained bedside structure. After intravenous injection of 133Xe,

measurements were performed for 10 minutes. The slow and fast

component of the cerebral washout was calculated, beginning at the

time when the exhaled air dropped to 20 percent of maximum. The

slow exponential was subtracted from the observed brain curves, and a

fast component was defined. CBF was derived using the inert gas

equation as defined initially by Kety and Schmidt (1945), and the

162
results were expressed in ml/100 g/min. CBF was calculated for all

patients, and a mean and standard error of the 10-probe measurements

was reported.

The authors used [99mTc]HMPAO as a cerebral perfusion imaging

molecule for the study (Neirinckx et al. 1987; Sharp et al. 1986).

Each patient received 740 MBq (20 mCi) of [99mTc]HMPAO, injected

intravenously within 20 minutes after reconstitution. SPECT scanning

started 2 hours after injection to clear soft tissue background activity.

[99mTc]HMPAO brain SPECT was performed using a large field-ofview rotating gamma camera with a truncated detector. The camera

was equipped with a high-resolution parallel-hole collimator and

operated on a step-and-shoot modality. The orbit was circular-360

degrees. Sixty images (one image for every 6-degree step) were

gathered. Each image took 30 seconds, and total acquisition time was

30 minutes. Reconstruction was performed using backprojection

reconstruction with Hanning filtering. Transaxial, coronal, and sagittal

sets of images were obtained. Each set had 14 to 16 images,

depending on the volume of the brain. The images were displayed

using a color scale that denoted abnormally diminished uptakedefined by a drop below 60 percent of maximal brain activity.

The [99mTc]HMPAO brain SPECT lesions were categorized as

demon-strating: no abnormalities, multiple superficial cortical lesions,

multi-focal deep periventricular lesions (periventricular scalloping), or

focal wedge-shaped hypoperfusion. Lesions also were defined as to

their location. Abnormalities were described qualitatively by a radiologist blinded to the patients’ clinical state. A data base collection of 10

age-matched normal volunteers (age 32±5 years) was used as a

reference.

RESULTS

Clinical Description

Fourteen patients were enrolled in the study. The patients’ problems

included ischemic stroke (4), TIA (3), brain hemorrhage (2), generalized seizures (2), and confusional states (3). The mean patient age

was 32.9±2.7 standard error of the mean (SEM) years, and there were

163
eight females and six males. Overall, the patients were free of preexisting medical conditions, although one had chronic hypertension

(patient #1) and another had a history of migraine headaches (patient #5).

lschemic Stroke. Three patients with ischemic strokes had cerebral

localization verified by CT or MRI (patients #2, #3, and #4) while a

fourth (patient #1) had normal CT and MRI but an abnormal SPECT.

Patient #3 had a right frontal (anterior cerebral artery distribution)

infarct, patient #2 had bilateral occipital (posterior cerebral artery)

infarcts, and patient #4 had a pontine infarct. Patient #1 had a

suspected right hemispheric stroke because he developed left hemiparesis with hemisensory loss to pinprick. However, head CT, MRI,

and cerebral angiography were all normal, and focal abnormalities that

correlated with clinical symptoms were evident only on SPECT.

T/A. Two patients developed symptoms of middle cerebral artery

distribution TIA (patients #6 and #7). Symptoms and focal deficits

had resolved by the time of neurological consultation in both patients

(within 8 hours). Patient #6 had both a CT and cerebral angiography

that were normal. Patient #7 also had a normal CT, but angiography

was not performed. In both, SPECT was abnormal.

A third patient (#5) had TIA-like symptoms minutes after intranasal

cocaine abuse. The episode started with scintillating scotoma described as “clear, moving needles” and was followed by right visual

field loss, word-finding difficulty, and right upper extremity numbness

that lasted 20 minutes. No headache was associated with this episode,

but the patient had a history of two previous episodes of visual disturbances followed by hemicranial headaches not associated with

cocaine abuse. The patient’s CT was normal, and a diagnosis of

cocaine-induced migraine versus TIA was made.

Cerebral Hemorrhage. Two patients (#8 and #9) had large bleeds in

the basal ganglia (#8) and thalamus (#9) demonstrated with CT.

Although the hemorrhages occurred in places typical for hypertensive

hemorrhage, neither patient had a history of hypertension.

Confusional States. Three patients (#10, #11, and #12) presented

with confusional states characterized by drowsiness, inattention, and

164
fluctuating level of consciousness. All had normal head CT, and all

improved to baseline mental status within 48 hours.

Seizures. Two patients (#13 and #14) came to the hospital due to

generalized tonic-clonic seizures. Neither patient had a history of

seizures, and both the neurological examination, electroencephalogram, and head CT were normal.

SPECT

Absolute CBF was measured in all patients with 133Xe. Mean CBF

was 43.6+3.4 ml/100 g/min compared to a CBF of 55.1+1.4 ml/100

g/min in a group of age-matched normals. Nine patients had markedly

diminished CBF (< 45 ml/100 g/min) while in five it was > 45 ml/100

g/min. All 14 patients had abnormal cerebral perfusion with

[99mTc]HMPAO SPECT. Multiple superficial or deep lesions

(periventricular scalloping) occurred in 12 patients (86 percent). The

most common focal cortical lesions were unilateral parietal-occipital

lesions (43 percent) and bilateral frontal lesions (29 percent). The

SPECT results are summarized in table 1.

Two patients did not demonstrate cortical scalloping, one had a pontine

stroke (patient #4) and the other had a confusional state (patient #10).

Patient #10 came to the hospital in a stupor, but she was alert and

oriented when her SPECT was performed 10 hours after the onset of

symptoms. At that time, she was irritable, hostile, and disinhibited.

These symptoms resolved within 48 hours. On SPECT, scalloping was

not found, although CBF measured with 133Xe was extremely low at

21 ml/100 g/min and brain SPECT revealed large areas of frontal

hypoperfusion bilaterally.

There was not a one-to-one correlation between clinical symptoms, CT,

MRI, and SPECT, although SPECT appeared extremely sensitive to

symptoms of focal ischemia. In only one stroke patient (#2) did

findings on CT, MRI, and SPECT strongly correlate, although SPECT

also revealed temporal hypoperfusion and diffuse scalloping. Patient

#1 had a grossly abnormal SPECT with marked right parietal

165
TABLE 1. Demographic data

166
a

rCBF in ml/100 g/min

hHMPAO SPECT location of hypoperfusion

KEY: L = left; R = right; NL = normal; B/L = bilateral; ND = not

done; PAR = parietal; TEMP = temporal; PCA = posterior

cerebral artery; OCC = occipital; ACA = anterior cerebral

artery; MCA = middle cerebral artery

hypoperfusion that corresponded to his clinical stroke, even though his

CT and MRI were normal. Also, severe periventricular scalloping and

small cortical perfusion deficits were seen. Patient #3 had multiple

deficits on SPECT, but the findings did not correlate with abnormalities on CT and MRI, which fit better with his clinical syndrome.

Patient #4 had posterior circulation hypoperfusion on SPECT that was

consistent with the pontine stroke seen on CT. All three patients (#5,

#6, and #7) with TIA had cerebral hypoperfusion that correlated with

clinical symptoms, although CT was normal in all three. In patients

with cerebral hemorrhage (#8 and #9), diffuse cerebral hypoperfusion

was seen in addition to the decreased flow at the site of hemorrhage.

In those patients with confusional states or seizures (#10 to #14)

diffuse and multifocal deficits were found in all, except in patient #10.

There were no clinical symptoms or neurological deficits to correspond

to brain SPECT abnormalities, but rather these findings appear to be

evidence of the diffuse cerebral effects of cocaine.

167
DISCUSSION

Acute cocaine intoxication caused both generalized and focally diminished CBF. Mean CBF was 43.6 ml/100 g/min, and in nine it was

diminished markedly at < 45 ml/100 g/min. In all 14 patients abnormalities were visualized with [99mTc]HMPAO. The most striking

abnormalities seen were multifocal small cortical and sometimes deep

areas of hypoperfusion. A characteristic and unusual scalloping pattern

of periventricular perfusion was noted. The authors previously had

seen this perfusion pattern in patients with both cerebral vasculitis and

with acquired immunodeficiency syndrome (AIDS) dementia syndrome. It was not present in any of the control patients.

Recent findings show vasoconstriction in coronary vessels during

catheterization after small doses of cocaine (Lange et al. 1989; Kaye

and Fainstat 1987; Krendel et al. 1990), and brain SPECT appears to

be a sensitive way to detect similar phenomena in cerebral vessels.

The authors suspect that this periventricular subcortical hypoperfusion

(scalloping) is the manifestation of cerebral vasoconstriction and is a

characteristic of vasoconstrictive agents such as cocaine. The scalloping may be due to the constriction of small- and medium-sized

arteries, although for reasons that are still undetermined. The deep

white matter appears to show selective involvement with cocaine. A

second hypothesis is that focal areas of hypoperfusion are secondary to

clusters of cells damaged during the acute, intense, prolonged vasoconstriction induced by cocaine. Primary hypofunction will induce a

focal secondary hypoperfusion.

Also, large wedge-shaped cortical perfusion defects were seen frequently. These deficits appeared to be caused by vasoconstriction of

larger arteries or confluence of multiple constricted smaller arteries,

This pattern was found in three of four stroke patients and two patients

with TIA. Unlike the scalloping and multifocal cortical deficits that

had uncertain clinical significance, these large SPECT deficits corresponded to focal clinical symptomatology. These wedge-shaped areas

of hypoperfusion are seen in patients with other causes of cerebral

ischemia.

168
Frontal lobe hypopetfusion was found in over one-quarter of the

patients, and the patients with this abnormality were irritable and

disinhibited-characteristics of patients with frontal lobe dysfunction

(Stuss and Benson 1986; Miller et al. 1989). A previous study of CBF

in cocaine abusers was performed by Volkow and colleagues (1988),

who used positron emission tomography (PET) to assess cerebral

perfusion with oxygen-labeled water. Patients for that study were

recruited from a drug rehabilitation center and were, by definition,

without major medical or neurological illness. The patients in this

study differed in this respect because all had neurological symptoms.

In Volkow’s study, diffuse patchy areas of hypoperfusion were

observed, and marked hypoperfusion was seen in the prefrontal cortex.

Repeat scans performed 10 days later revealed persistent deficits. The

authors’ findings of multifocal hypoperfusion and frontal hypoperfusion appear consistent with this earlier PET work. More recent

studies by Volkow and colleagues (1991) in subjects during the first

week of withdrawal from cocaine showed increased metabolism of

glucose in the orbital-frontal cortex and basal ganglia.

Like Volkow and colleagues (1991), the authors found a substantial

group of patients with frontal hypoperfusion. Although these changes

may have been secondary to alterations in metabolism induced by the

cocaine, it is possible that ischemia vasospasm involving frontal lobes

may have been the primary event responsible for these patients’

behavioral changes (Miller et al. 1989). Cocaine intoxication often

leads to behavioral disinhibition, and it is conceivable that these

behavioral disturbances are due to frontal lobe dysfunction induced by

focal ischemia. Alternately, the frontal hypoperfusion could have been

a premorbid characteristic of the subjects who will abuse cocaine,

although this seems unlikely. A third explanation would be that the

hypoperfusion was secondary to a metabolic disturbance induced by

changes in neurotransmitters associated with cocaine.

Both of the patients with seizures had brief generalized tonic-clonic

spells, but both were clinically normal by the time that SPECT was

performed. The SPECT findings in these patients were nonspecific

and revealed diffuse areas of hypoperfusion and scallopingabnormalities that were seen in patients with other diagnoses. Neither

169
of the patients had focal seizures where SPECT has been reported to

show focal hypoperfusion in the postictal state (Katz et al. 1990).

SUMMARY AND CONCLUSIONS

In summary, these data suggest that widespread primary or secondary

cerebral vasoconstriction is common in patients with neurological

complications from cocaine. In most patients, SPECT showed widespread hypoperfusion in regions that had no clear clinical significance

(e.g., the periventricular area). In many, the SPECT was performed

more than 24 hours after the onset of neurological symptomatology.

These findings raise several questions.

It has been assumed that these SPECT changes in patients with acute

neurological symptoms are temporary, although it will be important to

determine whether these areas of hypoperfusion persist after symptoms

have abated. Recently, Holman and colleagues (1991) found multifocal and deep areas of hypoperfusion with SPECT in 16 of 18

patients with a history of chronic cocaine abuse. Although most of the

subjects tested positive for cocaine, several had abstained from cocaine

use for weeks prior to the study. All 18 subjects had neuropsychological deficits, 13 mild and 5 moderate. Similarly, Pascual-Leone and

colleagues (1991) have shown that CT scan atrophy strongly correlates

with the duration of cocaine abuse, suggesting that brain injury may

occur with continued use of cocaine. It is the authors’ concern that

cocaine abuse might produce permanent changes in cerebral perfusion.

In conclusion, brain SPECT was found to be a useful procedure in the

evaluation of acute cocaine intoxication. Brain SPECT revealed focal

cortical lesions not seen on head CT or MRI, which corresponded to

clinical deficits. In addition, [99mTc]HMPAO brain SPECT had a

characteristic scalloped appearance, and this may be a marker for acute

intoxication with cocaine. This study further supports the contention

that cocaine causes neurological disease by its vasoconstrictive action.

170
REFERENCES

Brust, J., and Richter, R.W. A stroke associated with cocaine abuse.

N Y State J Med 77:1473-1475, 1977.

Caplan, L.R.; Hier, D.B.; and Banks, G. Stroke and drug abuse. Stroke

13:869-872, 1981.

Cregler, L.L., and Mark, H. Medical complications of cocaine abuse.

N Engl J Med 3 15:1495-1500, 1986.

Holman, B.L.; Carvalho, P.A.; Mendelson, J.; Teoh, S.K.; Nardin, R.;

Hallgring, E.; Hebben, N.; and Johnson, K.A. Brain perfusion is

abnormal in cocaine-dependent polydrug users: A study using

technetium-99m-HMPAO and ASPECT. J Nucl Med 32:1206-1210,

1991.

Katz, A.; Bose, A.; Lind, S.J.; and Spencer, S.S. SPECT in patients

with epilepsia partialis continua. Neurology 40:1848-1850, 1990.

Kaye, B.R., and Fainstat, M. Cerebral vasculitis associated with

cocaine abuse. JAMA 258:2104-2106, 1987.

Kety, S.S., and Schmidt, C.F. The determination of cerebral blood flow

in man by the use of nitrous oxide in low concentrations. Am J

Physiol 143:53-66, 1945.

Krendel, D.A.; Ditter, S.M.; Frankel, M.R.; and Ross, W.K. Biopsyproven cerebral vasculitis associated with cocaine abuse. Neurology

40:1092-1094, 1990.

Lange, R.A.; Cigarroa, R.G.; Yancy, C.W. Jr.; Willard, J.E.; Popma,

J.J.; Sills, M.N.; McBride, W.; Kim, A.S.; and Hillis, L.D. Cocaineinduced coronary-artery vasoconstriction. N Engl J Med 321:1557-

1562, 1989.

Levine, S.L., and Welch, K.M.A. Cocaine and stroke. Stroke 19:779-

783, 1988.

Levine, S.R.; Brust, J.C.M.; Futtrell, N.; Ho, K.L.; Blake, D.; Millikan,

C.H.; Brass, L.M.; Fayad, P.; Schultz, L.R.; and Selwa, J.F.

Cerebrovascular complications of the use of the “crack” form of

alkaloidal cocaine. N Engl J Med 323:699-704, 1990.

Miller, B.L.; Garrett, K.; Leedom, L.; and Djenderedjian, A. Neurospect in cocaine abuse: rCBF and HMPAO findings. Clin Nucl Med

14:12, 1989.

171
Mody, C.K.; Miller, B.L.; McIntyre, H.B.; Cobb, S.K.; and Goldberg,

M.A. Neurological complications of cocaine abuse. Neurology

38:1189-1193, 1988.

Neirinckx, R.D.; Canning, L.R.; Piper, I.M.; Nowotnik, D.P.; Pickett,

R.D.; Holmes, R.A.; Volkert, W.A.; Forster, A.M.; Weisner, P.S.;

Marriott, J.A.; and Chaplin, S.B. Technetium-99md,l HMPAO: A

new radiopharmaceutical for SPECT imaging of regional cerebral

blood perfusion. J Nucl Med 28:191-202, 1987.

Pascual-Leone, A.; Dhuna, A.; and Anderson, D.C. Cerebral atrophy in

habitual cocaine abusers: a planimetric CT study. Neurology 41:34-

38, 1991.

Pitts, D.K., and Marwah, J. Autonomic effects of cocaine. Can J

Physiol Pharmacol 67:1167-1176, 1989.

Root, R.K., and Rowbotham, M. Neurologic aspects of cocaine abuse.

West J Med 149:442-448, 1988.

Sharp, P.F.; Smith, F.W.; Gemmell, H.G.; Lyall, D.; Evans, N.T.;

Gvozdanovic, D.; Davidson, J.; Tyrrell, D.A.; Pickett, R.D.; and

Neirinckx, R.D. Technetium-99m HM-PAO steroisomers as

potential agents for imaging regional cerebral blood flow: Human

volunteer studies. J Nucl Med 27: 171-177, 1986.

Stuss, D.T., and Benson, D.F. The Frontal Lobes. New York: Raven

Press, 1986.

U.S. Department of Health and Human Services. Cocaine induces

vascular changes in brain: Demonstration by SPECT. News Release.

August 31, 1990.

Volkow, N.D.; Fowler, J.S.; and Wolf, A.P. Changes in brain glucose

metabolism in cocaine dependence and withdrawal. Am J Psychiatry

148:621-626, 1991.

Volkow, N.D.; Mullani, N.; Gould, K.; Adler, S.; and Krajewski, K.

Cerebral blood flow in chronic cocaine users: a study using positron

emission tomography. Br J Psychiatry 152:641-648, 1988.

172
AUTHORS

I. Mena, M.D.

Division of Nuclear Medicine

R.J. Giombetti, M.D.

Department of Neurology

B.L. Miller, M.D.

Department of Neurology

K. Garrett, CNMT

Division of Nuclear Medicine

J. Villanueva-Meyer, M.D.

Division of Nuclear Medicine

C. Mody, M.D.

Department of Neurology

M.A. Goldberg, Ph.D., M.D.

Department of Neurology

UCLA School of Medicine

Harbor-UCLA Medical Center

Torrance, CA 90509

173

An Introduction to Magnetic

Resonance Methods for Clinical

Research

Gerald G. Blackwell

INTRODUCTION

Nuclear magnetic resonance (NMR) analytical methods have been an

integral part of basic biochemistry laboratories since NMR’s introduction approximately 40 years ago. In recent years, however, these

powerful methods have found a niche in diagnostic medical imaging

and basic biomedical research. The purpose of this brief chapter is to

introduce the reader to basic NMR methods so as to foster thought for

applications in the arena of drug development.

BASIC PRINCIPLES

NMR methods exploit the magnetic properties of certain atomic nuclei

(Saini et al. 1988). Nuclei possessing an odd number of protons,

neutrons, or both have a small electromagnetic field associated with

their nuclear spin. Different nuclear species spin at different frequencies, but, importantly, all nuclei of a given element will spin at

precisely the same frequency. This frequency is described by the

Larmor equation: frequency = gyromagnetic ratio x magnetic field

strength. The Larmor frequency for atomic nuclei occurs in the

radiofrequency range of the electromagnetic spectrum, and specialized

instruments enable researchers to interact with atomic nuclei using this

nonionizing radiation (Doyle and Blackwell 1992).

The instruments designed to acquire magnetic resonance images and

spectra are composed of a magnet set, a coil capable of both transmitting and receiving radiofrequency signals, and a computer that

coordinates and integrates instructions to the instrument. The magnet

set determines the external field strength and also can be manipulated

to produce transient gradients that allow spatial localization. The

175
radiofrequency coil is “tuned” to interrogate the desired nuclear

species, and the computer faithfully records the resultant signal (Doyle

and Blackwell 1992).

The nucleus most easily interrogated by NMR methods is hydrogen.

The hydrogen nucleus is widely distributed in all organic compounds,

and its electromagnetic signal can be detected easily. Other species

that are NMR “visible” and of potential medical relevance are given in

table 1. Specialized techniques can produce three-dimensional displays

of nuclear magnetic resonance distribution (magnetic resonance

imaging-MRI) or, alternatively, displays of nuclear composition and

concentration (magnetic resonance spectroscopy). Most medical

research to date has focused on the imaging of hydrogen nuclei and

spectroscopy of phosphorus nuclei.

TABLE 1. NMR “visible” nuclei of potential medical relevance

*MHz/T = Megahertz/Tesla

176
It must be recognized that the appearance of magnetic resonance

images and spectra is dependent upon a complex interaction between

equipment acquisition parameters and the local chemical environment

of the nucleus in question. Detailed descriptions of these principles

are beyond the scope of this article, and the interested reader can

consult several comprehensive reviews and texts (Higgins et al. 1992;

Bottomley et al. 1984; Pykett 1982; Horowitz 1989; Nelson et al.

1984).

MAGNETIC RESONANCE IMAGING

The first human magnetic resonance images were obtained approximately 20 years ago, but, through the collaboration of basic scientists

and clinicians, MRI applications have expanded rapidly (Lauterbur

1973). The clarity and resolution of magnetic resonance images are

unparalleled, and MRI is established firmly as the imaging method of

choice for diseases involving many organ systems. The advantages of

MRI, compared with other imaging techniques, include the ability to

manipulate image contrast by changing acquisition parameters and the

ability to assess function and flow. Tissue contrast also can be

improved in certain situations by the use of paramagnetic contrast

agents (Canby et al. 1987). Most clinical imaging to date has focused

on central nervous system (CNS) and musculoskeletal applications.

Magnetic resonance images are degraded by motion, and applications

to organ systems such as the cardiovascular system have been slower

to develop. Gating techniques and ultrafast imaging sequences reduce

motion artifact, and applications for this technology are expanding

(Lotan et al. 1988; Blackwell et al. 1992).

The CNS is well suited for study by MRI. Unprecedented insight into

both normal and abnormal brain structure has been provided using

current clinical instruments. Preliminary work in the author’s laboratory using a very high field strength 4.1 tesla instrument offers hope

that new frontiers in human CNS imaging will be opened. The tissue

contrast and spatial resolution achieved at this high field strength may

exceed what is obtainable on standard clinical instruments (figure 1).

Continued research may add to existing knowledge of structure and

function relationships within the CNS.

177
FIGURE 1.

SOURCE:

Inversion recovery gradient echo image of the human brain

obtained at a field strength of 4.1 tesla. Slice thickness is 3

mm with an in-plane resolution of 450 microns. Tissue

contrast and spatial resolution may be improved using this

highfield strength.

Image courtesy of Dr. Hoby Hetherington, Center for

Nuclear Imaging Research, University of Alabama at

Birmingham.

178
MAGNETIC RESONANCE SPECTROSCOPY

In addition to providing high-resolution images, biochemical

information also can be obtained noninvasively using magnetic

resonance spectroscopy. Many chemical moieties can be interrogated

using the nuclei shown in table 1. Because of the central role of

phosphorus-containing compounds in cellular energetics, much of the

work to date has focused on phosphorus-31 spectroscopy (Bottomley et

al. 1990; Robitaille et al. 1989; Schaefer et al. 1989). Using magnetic

resonance techniques, measurements can be made of compounds such

as phosphocreatine (PCr), inorganic phosphate (Pi), and adenosine

triphosphate (ATP). Figure 2 is an example of a normal in vivo

phosphorus spectrum from human myocardium.

In vivo and in vitro spectroscopy research also is being performed

utilizing compounds that contain hydrogen, 13C, and 23Na (Barany et al.

1988; Evanochko et al. 1987; Reeves et al. 1989). Many organic

compounds (e.g., lipids and lactate) can be studied through hydrogen

and carbon spectroscopy, and sodium spectroscopy may facilitate

insight into intracellular and extracellular fluid distribution and

membrane integrity.

Realizing that acquisitions may be manipulated to obtain either images

or spectra, researchers recently have focused on providing images of

different compounds, so-called spectroscopic imaging. Figure 3 is an

example of such an image. With spectroscopic imaging, anatomic

maps can be derived of a specific chemical moiety. Much work has

been carried out at lower field strengths, but research using high field

strength instruments with their inherently improved signal-to-noise

ratio is ongoing in the author’s laboratory and other laboratories. It is

anticipated that the combination of high field strength instruments and

better radiofrequency coil design will greatly advance these techniques.

SUMMARY

It is very early in the era of exploiting the power of magnetic resonance methods. Great strides have been made in both diagnostic

medical imaging and spectroscopy. Using high field strength

instruments, normal and abnormal biochemistry within specific CNS

179
FIGURE 2. Normal phosphorous spectrum from the human myocardium

obtained at 1.5 tesla. Note separate peaks for the three

phosphorus atoms in ATP.

KEY: Pi = inorganic phosphate; PCr = phosphocreatine; ATP =

adenosine triphosphate

SOURCE: Reproduced from Blackwell, G.; Cranney, G.; and Pohost,

G., eds. MRI: Cardiovascular System. New York: Gower

Medical Publishing, 1992.

regions and their response to interventions ultimately may be discemible. With technological improvements, it is to be expected that image

quality will improve, and in vivo spectroscopy may move rapidly from

the research environment into the clinical arena.

180
FIGURE 3.

SOURCE:

Spectroscopic image of the human brain obtained at 4.1

tesla. The right panel is a standard transverse scout image.

The left panel represents distribution of the compound Nacetylaspartate within this slice. Voxel resolution 0.5 cm3

.

Using this technology, anatomic distribution of many

different compounds can be discerned.

Image courtesy of Dr. Hoby Hetherington, Center for

Nuclear Imaging Research, University of Alabama at

Birmingham.

181
REFERENCES

Barany, M.; Langer, B.G.; Glick, R.P.; Venkatasubramonion, P.N.;

Wilbur, A.C.; and Spigos, D.G. In vivo H-l spectroscopy in humans

at 1.5 T. Radiology 167:839-844, 1988.

Blackwell, G.; Doyle, M.; and Cranney, G. Cardiovascular MRI

techniques. In: Blackwell, G.; Cranney, G.; and Pohost, G., eds.

MRI: Cardiovascular System. New York: Gower Medical

Publishing, 1992.

Bottomley, P.A.; Foster, T.H.; Argersinger, R.E.; and Pfeifer, L.M. A

review of normal tissue hydrogen NMR relaxation times and

mechanisms from 1-100 MHz: Dependence on tissue type, NMR

frequency, temperature, species, excision, and age. Am Assoc Phys

Med 11:425-448, 1984.

Bottomley, P.A.; Hardy, C.J.; and Roemer, P.B. Phosphate metabolite

imaging and concentration measurements in human heart by nuclear

magnetic resonance. Magn Reson Med 14:425-434, 1990.

Canby, R.C.; Elgavish, G.A.; and Pohost, G.M. Paramagnetic NMR

contrast agents for cardiovascular imaging. New Concepts Card

Imaging 3:315-342, 1987.

Doyle, M., and Blackwell, G. Basic principles of MRI. In: Blackwell,

G.; Cranney, G.; and Pohost, G. eds. MRI: Cardiovascular System.

New York: Gower Medical Publishing, 1992.

Evanochko, W.T.; Reeves, R.C.; Sakai, T.T.; Canby, R.C.; and Pohost,

G.M. Proton NMR spectroscopy in myocardial ischemic insult.

Magn Reson Med 5:23-31, 1987.

Higgins, C.B.; Hricak, H.; and Helms, C.A., eds. Magnetic Resonance

Imaging of the Body. New York: Raven Press, 1992.

Horowitz, A.L. MRI Physics for Physicians. New York: SpringerVerlag, 1989.

Lauterbur, P.C. Image formation by induced local interactions:

Examples employing nuclear magnetic resonance. Nature 242:

190, 1973.

Lotan, C.S.; Cranney, G.B.; and Pohost, G.M. Cine NMR: An

emerging technology. Echocardiography 5:373-382, 1988.

Nelson, T.R.; Hendrick, R.E.; and Hendee, W.R. Selection of pulse

sequences producing maximum tissue contrast in magnetic

resonance imaging. Magn Reson Imaging 2:285-294, 1984.

Pykett, I.L. NMR imaging in medicine. Sci Am 246:78-89, 1982.

182
Reeves, R.C.; Evanochko, W.T.; Canby, R.C.; McMillin, J.B.; and

Pohost, G.M. Demonstration of increased myocardial lipid with

postischemic dysfunction (“myocardial stunning”) by proton nuclear

magnetic resonance spectroscopy. J Am Coll Cardiol 13:739, 1989.

Robitaille, P.M.; Lew, B.; Merkle, H.; Sublett E.; Lindstrom, P.;

From, A.H.; Garwood, M.; Bache, R.J.; and Ugurbil, K. Transmural

metabolite distribution in regional myocardial ischemia as studied

with 31P NMR. Magn Reson Med 10:108-110, 1989.

Saini, S.; Frankel, R.B.; Stark, D.D.; and Ferrucci, J.T. Magnetism: A

primer and review. AJR 150:735-744, 1988.

Schaefer, S.; Camacho, A.; Gober, J.M.; Obregon, R.G.; DeGroot,

M.A.; Botvinick, E.H.; Massie, B.; and Weiner, M.W. Response of

myocardial metabolites to graded regional ischemia: 31P NMR

spectroscopy of porcine myocardium in vivo. Circ Res 64:968-976,

1989.

AUTHOR

Gerald G. Blackwell, M.D.

1808 Seventh Avenue South

Cardiovascular NMR Laboratory

D105 Diabetes Hospital

University of Alabama at Birmingham

Birmingham, AL 35294-0012

183

monograph series

While limited supplies last, single copies of the monographs may be obtained

free of charge from the National Clearinghouse for Alcohol and Drug

Information (NCADI). Please contact NCADI also for information about

availability of coming issues and other publications of the National Institute

on Drug Abuse relevant to drug abuse research.

Additional copies may be purchased from the U.S. Government Printing

Office (GPO) and/or the National Technical Information Service (NTIS) as

indicated. NTIS prices are for paper copy; add $3.00 handling charge for

each order. Microfiche copies are also available from NTIS. Prices from

either source are subject to change.

Addresses are:

NCADI

National Clearinghouse for Alcohol and Drug Information

P.O. Box 2345

Rockville, MD 20852

(301) 468-2600

(800) 729-6686

GPO

Superintendent of Documents

U.S. Government Printing Office

P.O. Box 371954

Pittsburgh, PA 15220-7954

(202) 738-3238

FAX (202) 512-2233

NTIS

National Technical Information Service

U.S. Department of Commerce

Springfield, VA 22161

(703) 487-4650

For information on availability of NIDA Research Monographs from 1975-

1993 and those not listed, write to NIDA, Community and Professional

Education Branch, Room 10A-39, 5600 Fishers Lane, Rockville, MD 20857.

185
26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor,

Ph.D., ed. (Reprint from 1979 Surgeon General’s Report on Smoking and

Health.)

NCADI #M26 NTIS PB #80-118755/AS $23

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS.

Richard L. Hawks, Ph.D., ed.

NCADI #M42 NTIS PB #83-136044/AS $23

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF

ABUSE. John Grabowski, Ph.D., ed.

NCADI #M50 NTIS PB #85-150381/AS $23

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL

AND ABUSE LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas,

Ph.D., eds.

NCADI #M52 NTIS PB #85-150373/AS $23

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION.

John Grabowski, Ph.D., and Sharon M. Hall, Ph.D., eds.

NCADI #M53 NTIS PB #89-123186/AS $23

54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles W.

Sharp, Ph.D., ed.

NCADI #M54 NTIS PB #89-103279/AS $39

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR

PREVENTION. Coryl LaRue Jones, Ph.D., and Robert J. Battjes,

D.S.W., eds.

NCADI #M56 NTIS PB #89-123160/AS $31

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECTIVES. Nicholas J. Kozel, MS., and Edgar H. Adams, M.S.,

eds.

NCADI #M61 NTIS PB #89-131866/AS $31

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger

M. Brown, Ph.D., and David P. Friedman, Ph.D., eds.

NCADI #M62 NTIS PB #89-130660/AS $23

186
63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG

CHILDREN AND ADOLESCENTS. Catherine S. Bell, MS., and

Robert J. Battjes, D.S.W., eds.

NCADI #M63 NTIS PB #89-103287/AS $31

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

NCADI #M64 NTIS PB #89-131858/AS $31

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A.

Ray, Ph.D., and Monique C. Braude, Ph.D., eds.

NCADI #M65 NTIS PB #89-130637/AS $23

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka,

Ph.D.; Gene Bamett, Ph.D.; and Richard L. Hawks, Ph.D., eds.

NCADI #M69 NTIS PB #89-158422/AS $39

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY,

BIOSYNTHESIS, AND ANALYSIS. Rao S. Rapaka, Ph.D., and Richard

L. Hawks, Ph.D., eds.

NCADI #M70 NTIS PB #89-158430/AS $45

72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims,

Ph.D., and Carl G. Leukefeld, D.S.W., eds.

NCADI #M72 NTIS PB #89-151963/AS $3 1

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

Stephen I. Szara, M.D., D.Sc., ed.

NCADI #M74 NTIS PB #89-151989/AS $23

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO

DRUGS. David P. Friedman, Ph.D., and Doris H. Clouet, Ph.D., eds.

NCADI #M78 NTIS PB #88-245683/AS $31

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE

CANNABINOIDS. Rao S. Rapaka, Ph.D., and Alexandros Makriyannis,

Ph.D., eds.

NCADI #M79 NTIS PB #89-109201/AS $31

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

NATIONAL AND INTERNATIONAL PERSPECTIVES. Robert J.

Battjes, D.S.W., and Roy W. Pickens, Ph.D., eds.

NCADI #M80 NTIS PB #88-236138/AS $31

187
82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

David P. Friedman, Ph.D., eds.

NCADI #M82 NTIS PB #88-245691/AS $23

83 HEALTH HAZARDS OF NITRITE INHALANTS. Harry W. Haverkos,

M.D., and John A. Dougherty, Ph.D., eds.

NCADI #M83 NTIS PB #89-125496/AS $23

84 LEARNING FACTORS IN SUBSTANCE ABUSE. Barbara A. Ray,

Ph.D., ed.

NCADI #M84 NTIS PB #89-125504/AS $31

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE. Raquel A.

Crider, Ph.D., and Beatrice A. Rouse, Ph.D., eds.

NCADI #M85 NTIS PB #89-123178/AS $31

87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and Bhola

N. Dhawan, M.D., eds.

NCADI #M87 NTIS PB #89-158430/AS $45

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY. Doris H.

Clouet, Ph.D.; Khursheed Asghar, Ph.D.; and Roger M. Brown, Ph.D.,

eds.

NCADI #M88 NTIS PB #89-125512/AS $39

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE. Roy W.

Pickens, Ph.D., and Dace S. Svikis, B.A., eds.

NCADI #M89 NTIS PB #89-125520/AS $23

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

NCADI #M92 NTIS PB #90-148933/AS $45

94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND

RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D., and Errol

De Souza, Ph.D., eds.

NCADI #M94 NTIS PB #90-148958/AS $39

95 PROBLEMS OF DRUG DEPENDENCE, 1989. PROCEEDINGS OF

THE 5 1 ST ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON

PROBLEMS OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D.,

ed.

NCADI #M95 NTIS PB #0-237660/AS $67

188
96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY,

IMMUNOLOGY, AND AIDS. Phuong Thi Kim Pham, Ph.D., and

Kenner Rice, Ph.D., eds.

NCADI #M96 NTIS PB #90-237678/AS $31

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND

MEMORY. Lynda Erinoff, Ph.D., ed.

NCADI #M97 NTIS PB #90-237686/AS $31

98 THE COLLECTION AND INTERPRETATION OF DATA FROM

HIDDEN POPULATIONS. Elizabeth Y. Lambert, MS., ed.

NCADI #M98 NTIS PB #90-237694/AS $23

99 RESEARCH FINDINGS ON SMOKING OF ABUSED

SUBSTANCES. C. Nora Chiang, Ph.D., and Richard L. Hawks, Ph.D.,

eds.

NCADI #M99 NTIS PB #91-141119/AS $23

100 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION

DATA. VOLUME II. Steven W. Gust, Ph.D.; J. Michael Walsh,

Ph.D.; Linda B. Thomas, B.S.; and Dennis J. Crouch, M.B.A., eds.

GPO Stock #017-024-01458-3 $8 NCADI #M100

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR. John

W. Spencer, Ph.D., and John J. Boren, Ph.D., eds.

GPO Stock #017-024-01426-7 $6 NTIS PB #91-172858/AS $31

NCADI #M101

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and Lynda

Erinoff, Ph.D., eds.

GPO Stock #017-024-01425-7 $8 NTIS PB #91-172866/AS $31

NCADI #M 102

106 IMPROVING DRUG ABUSE TREATMENT. Roy W. Pickens, Ph.D.;

Carl G. Leukefeld, D.S.W.; and Charles R. Schuster, Ph.D., eds.

GPO Stock #017-024-01439-7 $12 NTIS PB #92-105873

NCADI #M106 Paperback $50 Microfiche $19

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and

William J. Bukoski, Ph.D., eds.

GPO Stock #017-024-01441-9 $9 NTIS PB #92-160985

NCADI #M107 Paperback $35 Microfiche $17

189
108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING

MECHANISMS. Pushpa Thadani, Ph.D., ed.

GPO Stock #017-024-01446-0 $7 NTIS PB #92-106608

NCADI #M108 Paperback $35 Microfiche $17

109 LONGITUDINAL STUDIES OF HIV INFECTION IN INTRAVENOUS DRUG USERS: METHODOLOGICAL ISSUES IN

NATURAL HISTORY RESEARCH. Peter Hartsock, Dr.P.H., and

Sander G. Genser, M.D., M.P.H., eds.

GPO Stock #017-024-01445-l $4.50 NTIS PB #92-106616

NCADI #M109 Paperback $26 Microfiche $12.50

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH:

VOLUME I. RECEPTOR CLONING, NEUROTRANSMITTER

EXPRESSION, AND MOLECULAR GENETICS. Theresa N.H. Lee,

Ph.D., ed.

NCADI #M111 NTIS PB #92-135743

Paperback $35 Microfiche $17

112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG

ABUSE RESEARCH. Rao S. Rapaka, Ph.D.; Alexandros Makriyannis,

Ph.D.; and Michael J. Kuhar, Ph.D., eds.

GPO Stock #017-024-01455-9 $11 NTIS PB #92-155449

NCADI #M112 Paperback $44 Microfiche $17.50

113 ECONOMIC COSTS, COST EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT. William S.

Cartwright, Ph.D., and James M. Kaple, Ph.D., eds.

NCADI #M113 NTIS PB #92-155795

Paperback $35 Microfiche $17

114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON

EFFECTS OF PRENATAL EXPOSURE TO DRUG ABUSE.

M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

GPO Stock #017-024-01459-1 $12 NTIS PB #92-146216

NCADI #M114 Paperback $43 Microfiche $17

115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND

IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H., and Nicholas J.

Kozel, M.S., eds.

GPO Stock #017-024-01460-5 $4 NCADI #M115

190
116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE

RESEARCH. Richard A. Glennon, Ph.D.; Torbjörn U.C. Järbe, Ph.D.;

and Jerry Frankenheim, Ph.D., eds.

GPO Stock #017-024-01470-2 $13 NCADI #M116

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY, PREVENTION,

AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN

AND THEIR CHILDREN. M. Marlyne Kilbey, Ph.D., and Khursheed

Asghar, Ph.D., eds.

GPO Stock #017-024-01472-9 $12 NCADI #M117

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. Carl G.

Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #017-024-01473-7 $16 NCADI #M118

120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE

BLOOD-BRAIN BARRIER. Jerry Frankenheim, Ph.D., and Roger M.

Brown, Ph.D., eds.

GPO Stock #017-024-01481-8 $10 NCADI #M120

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR

OPIOID DEPENDENCE. Jack D. Blaine, Ph.D., ed.

GPO Stock #017-024-01482-6 $5 NCADI #M121

123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

TREATMENT. Heinz Sorer, Ph.D., ed.

GPO #017-024-01501-6 $6.50 NCADI #M123

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Roger M. Brown, Ph.D., and Joseph Fracella, Ph.D.

eds.

GPO Stock #017-024-01492-3 $9 NCADI #M124

125 ACTIVATION OF IMMEDIATE EARLY GENES BY DRUGS OF

ABUSE. Reinhard Grzanna, Ph.D., and Roger M. Brown, Ph.D., eds.

GPO #017-024-01503-2 $7.50 NCADI #M125

126 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH.

VOLUME II: STRUCTURE, FUNCTION, AND EXPRESSION.

Theresa N.H. Lee, Ph.D., ed. NCADI #M126

191
127 PROGRESS AND ISSUES IN CASE MANAGEMENT. Rebecca

Sager Ashery, D.S.W., ed.

GPO Stock #017-024-01490-7 $14 NCADI #M127

128 STATISTICAL ISSUES IN CLINICAL TRIALS FOR TREATMENT

OF OPIATE DEPENDENCE. Ram B. Jain, Ph.D., ed.

GPO Stock #017-024-01491-5 $6.50 NCADI #M128

129 INHALANT ABUSE: A VOLATILE RESEARCH AGENDA.

Charles W. Sharp, Ph.D.; Fred Beauvais, Ph.D.; and Richard Spence,

Ph.D., eds.

GPO Stock #017-024-01496-6 $12 NCADI #M129

130 DRUG ABUSE AMONG MINORITY YOUTH: ADVANCES IN

RESEARCH AND METHODOLOGY. Mario R. De La Rosa, Ph.D.,

and Juan-Luis Recio Adrados, Ph.D., eds.

GPO #017-024-01506-7 $14 NCADI #M130

131 IMPACT OF PRESCRIPTION DRUG DIVERSION CONTROL

SYSTEMS ON MEDICAL PRACTICE AND PATIENT CARE.

James R. Cooper, Ph.D.; Dorynne J. Czechowicz, M.D.; Stephen P.

Molinari, J.D., R.Ph.; and Robert C. Peterson, Ph.D., eds.

GPO #017-024-01505-9 $14 NCADI #M131

132 PROBLEMS OF DRUG DEPENDENCE, 1992: PROCEEDINGS OF

THE 54TH ANNUAL SCIENTIFIC MEETING OF THE COLLEGE

ON PROBLEMS OF DRUG DEPENDENCE. Louis Harris, Ph.D., ed.

GPO Stock #017-024-01502-4 $23 NCADI #M132

133 SIGMA, PCP, AND NMDA RECEPTORS. Errol B. De Souza, Ph.D.;

Doris H. Clouet, Ph.D.; and Edythe D. London, Ph.D., eds.

NCADI #M133

134 MEDICATIONS DEVELOPMENT: DRUG DISCOVERY,

DATABASES, AND COMPUTER-AIDED DRUG DESIGN. Rao S.

Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO #017-024-01511-3 $11 NCADI #M134

135 COCAINE TREATMENT: RESEARCH AND CLINICAL

PERSPECTIVES. Frank M. Tims, Ph.D., and Carl G. Leukefeld,

D.S.W., eds.

GPO #017-024-01520-2 $11 NCADI #M135

192
136 ASSESSING NEUROTOXICITY OF DRUGS OF ABUSE. Lynda

Erinoff, Ph.D., ed.

GPO #017-024-01518-1 $11 NCADI #M136

137 BEHAVIORAL TREATMENTS FOR DRUG ABUSE AND

DEPENDENCE. Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.; and

John J. Boren, Ph.D., eds.

GPO #017-024-01519-9 NCADI #M137

193

U.S. GOVERNMENT PRINTING OFFICE: 1994 300-973/80006
NIH Publication No. 94-3728

Printed 1994