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Assessment of dopamine receptor densities in the human brain with carbon-11-labeled N-methylspiperone.

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Assessment of Dopamine Receptor
Densities in the Human Brain with
Carbon-11-Labeled N-Methylspiperone
Henry N. Wagner, Jr, MD," H. Donald Burns, PhD," Robert F. Dannals, PhD," Dean F. Wong, MD,"
Bengt Langstrom, PhD,§ Timothy Duelfer, PhD,* J. James Frost, MD, PhD," Hayden T. Ravert, PhD,"
Jonathan M. Lmks, PhD," Shelley B. Rosenbloom, MD,? Scott E. Lukas, MD,"
Alfred V. Kramer, PhD,' and Michael J. Kuhar, PhD$
We describe the use of carbon-11-labeled 3-N-methylspiperone, a ligand that preferentially binds to dopamine receptors in vivo, to image the receptors by positron emission tomography scanning in baboons and, for the first time, in a
human. The method has now been used in 58 humans for noninvasive assessment of the state of brain dopamine
receptors under normal and pathological conditions.
Wagner H N Jr, Burns HD, Dannals RF,Wong DF, Langstrom B, Duelfer T, Frost JJ, Ravert HT, Links JM,
Rosenbloom SB, Lukas SE, Kramer AV, Kuhar MJ: Assessment of dopamine receptor densities in the
human brain with carbon- 11-labeled N-methylspiperone. Ann Neurol 15(suppl):S79-S84, 1984
In 1977 Sokoloff and colleagues I221 described the
carbon- 14-labeled deoxyglucose model for measuring
local cerebral glucose utilization and were able to quantify the relationship between the mental processes of
vision or hearing and regional glucose metabolism in
specific regions of the brain of rats and monkeys. In
1979 Reivich and associates 1191 extended the measurement of local cerebral glucose utilization to humans, based on the use of fluorine-18-labeled
fluorodeoxyglucose ("FDG) and positron emission tomography (PET). Soon thereafter, Reivich and colleagues [I81 at the University of Pennsylvania and at
Brookhaven National Laboratory were able to show
that sensory stimulation increased the rate of glucose
utilization in the involved regions of the human brain.
Visual, auditory, and somatosensory stimulation all resulted in increased regional glucose utilization. Subsequently, the "FDG method has been applied to the
study of patients with stroke, epilepsy, brain tumors,
and various types of dementia and mental illnesses
C9-11).
Another approach to the study of regional brain
function by PET is the use of oxygen-I5 to measure
regional cerebral blood flow and oxygen utilization {b,
23, 241. A major advantage of the oxygen-15 method
is that the short half-life of the tracer (2% minutes)
makes it possible to perform multiple determinations
in the same person over a period of several hours. For
example, Raichle and associates [I71 have made as
many as eight consecutive measurements of the
changes in regional cerebral blood volume resulting
from a subject's viewing of a light flashing at different
frequencies.
For the past five years our research has been directed
toward in vivo localization and quantification of
dopamine and opiate receptors in human beings. In our
original research proposal to the NINCDS in 1978, we
proposed to try to answer the following questions: (1)
Can we map the distribution of neuroreceptors,
specifically dopamine and opiate receptors, in the brain
of humans? (2) What are the factors that affect the
distribution and quantity of these receptors in various
regions of the brain? (3) Are there differences among
normal persons and among patients with selected
neurological and psychiatric diseases, such as parkinsonism, Huntington's chorea, tardive dyskinesia,
schizophrenia, chronic intractable pain states, and congenital insensitivity to pain? (4)Is it possible to monitor
From the *Division of Nuclear Medicine and the ?Division of
Neuroradiology, The Johns Hopkins Medical Institutions, Baltimore, MD 21205; the $Departments of Neuroscience, Pharmacology, and Experimental Therapeutics, and Psychiatry and Behavioral
Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; the $Institute of Chemistry, University of
Uppsala, S75121 Uppsala, Sweden 1; and the "National Institute of
Drug Abuse Addiction Research Center, Baltimore City Hospitals,
Baltimore, MD 21224.
Address reprint requests to Dr Wagner, Division of Nuclear
Medicine, The Johns Hopkins Medical Institutions, 61 5 N Wolfe St,
Baltimore, M D 21205
s79
changes in the quantitative distribution of these
neuroreceptors, either in response to therapy or in the
natural progression of the disease?
Our proposal was based on the fact that the labeling
of biologically important compounds is greatly
facilitated by the use of positron-emitting radionuclides, such as carbon-1 1, the production of which required installation of an in-hospital cyclotron. Since the
award of the NINCDS grant in 1979, we have installed
a cyclotron and PET scanner in the Nuclear Medicine
Division of the Johns Hopkins Hospital. The cyclotron
(16 MeV protons and 8 MeV deuterons) is easily accessible to the patients to be studied.
Our approach is to synthesize radiolabeled drugs
that bind to specific neuroreceptors within the time
constraints associated with the 20 minute physical halflife of carbon- 11; we then quantitatively delineate the
distribution of dopamine and opiate receptors within
the brain. The first drug that we proposed to label with
carbon-11 was spiperone {2, 3, 13, 20, 211, a highly
specific binder to dopamine receptors. We also proposed to use carbon-1 1 diprenorphine, an antagonist of
opiate receptor activity { 161, to investigate the distribution and density of opiate receptors.
T o achieve the desired goal, a suitable radiotracer
with the following characteristics was needed:
1. The compound had to be labeled with a positronemitting radionuclide to permit the use of a PET
scanner for noninvasive measurements in humans.
2. The labeled drug had to cross the blood-brain barrier, permitting it to be administered by intravenous
injection.
3. The drug had to have a high chemical affinity for the
neurotransmitter under investigation.
4. The labeled drug had to dissociate slowly or not at
all from the receptors.
5. The drug had to be cleared rapidly from the blood
to increase the contrast between the regions containing receptors and the surrounding structures.
As in the case of the development of the deoxyglucose method, the approach to PET imaging was based
on successful prior research with autoradiography [7,
8). Prior to the in vivo labeling of receptors and the
autoradiograph, in vitro receptor binding techniques
had been developed. The in vitro assays involve the use
of trace quantities of radiolabeled drugs that have a
high affinity for specific types of neuroreceptors. The
characteristics of these receptors are: (1) The receptor
sites are limited in number, have a high affinity for
binding ligands, and are saturated at relatively low
amounts of the binding ligands. (2) The binding occurs
only where the specific type of receptor is located and
not elsewhere. For example, dopamine receptors are
S80 Annals of Neurology
Fig I . Chemical structure of carbon-1 1 -labeled 3-N-methylspiperone ( { I 'CINMSP).
found in highest concentrations in the caudate nucleus
and putamen. (3) The ligands are bound in amounts
known to produce certain specific pharmacological
effects.
Leyson and colleagues and Pert and co-workers [13,
161 and others El, 7, 81 found that the accumulation of
[3H]spiperone in the corpus striatum (caudate nucleus
and putamen) was stereospecific and was blocked by
other neuroleptic drugs in direct proportion to their
clinical effects. These researchers demonstrated a saturation phenomenon related to the dose of the administered ligand; that is, specific binding in the region
of the striatum plateaued as the dose increased, because of the limited number of binding sites. They also
found that {3H]spiperone was not metabolized after
binding to dopamine receptors, and that labeled drug
that was not specifically bound was rapidly cleared from
the brain and blood. In rats, 15 minutes after intravenous injection most of the labeled drug in the brain was
selectively bound to dopamine receptors, with only 20
to 25% remaining in the blood vessels and cerebrospinal fluid. Once the labeling had occurred and the
nonspecific binding and circulating drug had cleared
from the blood, localization of the bound drug was
achieved by autoradiography {7,81.
In order to image and quantify the location and concentration of dopamine receptors using PET, we synthesized the tracer carbon-1 1-labeled N-methylspiperone, or ["CINMSP (Fig l), by N-alkylation of
the neuroleptic drug spiperone with [ "Clmethyliodide, which was produced by an in-hospital cyclotron
(Model RNP-16, Scanditronix AB, Uppsala, Sweden).
The entire synthesis was accomplished with sterile,
nonpyrogenic material that was ready for injection
within 40 minutes after the end of the cyclotron bombardment. Prior to injection, the product was purified and the specific activity was determined using a
reverse-phase liquid chromatographic column. The
specific activity was also determined by an in vitro competitive binding assay. The compound NMSP was previously described in a patent by Janssen [5].
A procedure for determining if the ligand is binding
to receptors in vivo is to test whether the regional
distribution of radioactivity after drug injection parallels the distribution of receptors and whether administration of excess, unlabeled, related drugs blocks this
Supplement to Volume 15, 1984
specific regional distribution. Rat striatum has very
high concentrations of dopamine receptors, while the
cerebellum has very low levels. Thus, high striaturd
cerebellum ratios of injected drug indicate the preferential labeling of dopamine receptors in vivo. The concentration of drug in the cerebellum is a measure of
nonspecific or non-receptor-associated drug in the
brain, while the striatal content reflects both specific
and nonspecific binding. Administration of excess, unlabeled neuroleptic drugs with high affinity for
dopamine receptors obliterates the regional distribution, as indicated by equal concentrations of the ligand
in the striatum and cerebellum.
From our in vitro experiments with receptorcontaining membranes obtained from the striatum of
rats, we found that NMSP has a binding affinity for
dopamine receptors similar to that of spiperone. In
these experiments, we found that NMSP had an inhibitory constant (Ki) of about 250 pmol (against hydrogen3-labeled spiperone), while the dissociation constant of
spiperone under the same conditions (KD) was about
190 pmol.
In preliminary in vivo studies, {"C)NMSP (with a
specific activity of 55 mCi/p,mol, in a dose of 10 pg per
kilogram of body weight) was injected into the tail
veins of mice. After killing the animals and dissecting
the brain regions, it was found that ratios of striatal to
cerebellar radioactivity were 15 : 1 to 2 1: 1 by 60 minutes after the injection. Coinjection of unlabeled spiperone lowered the striaturdcerebellum ratios by 70%
at spiperone doses of 150 pglkg without altering
cerebellar binding. These biochemical studies indicate that NMSP binds to dopamine receptors and is a
suitable ligand for labeling these receptors in vivo.
The next experiments involved PET imaging in
three studies of anesthetized baboons. All of the PET
scans were performed with a NeuroECAT scanner
(Ortec, Inc, Oak Ridge, TN). In these studies, intravenous injections of 16 mCi of ["CINMSP (10.4 mCi/
pmol) were administered. A preferential accumulation
of radioactivity in the caudate nucleus and putamen was
observed as early as 15 minutes after injection on all
occasions.
In one of these studies, two injections separated by
3 hours were given. The first injection employed
["C)NMSP with a specific activity of 10.4 mCi/pmol
in a dose of 21 pg per kilogram of body weight (Fig
2A). The injected tracer concentrated selectively in the
region of the caudate nucleus and putamen relative to
the rest of the brain. After the carbon-11 had been
allowed to decay for 3 hours (9 half-lives) to
insignificant levels, and while the baboon was still anesthetized, a second dose of {"C}NMSP was injected
intravenously. This time there was an added excess
(220 &kg) of unlabeled spiperone, which would com-
pete for binding to the receptors. The activity in this
instance did not accumulate preferentially in the region
of the caudate nucleus and putamen, although it again
accumulated in the eyes as it had previously (Fig 2B).
The reduction in binding in the caudate nucleus and
putamen elicited by unlabeled spiperone administration indicates pharmacological specificity of the binding
by dopamine receptors in this area (Table 1). The lack
of substantial reduction in activity in the eye region
indicates nonspecific binding in this area.
After the baboon experiments, the following experiment was performed with one of us (H. N. W.)
as the experimental subject. Twenty millicuries of
[l'C]NMSP was injected intravenously in the conscious subject with the eyes closed listening to music
through headphones while lying supine with the head
in the PET scanner. The specific activity of the
['lC)NMSP was 263 mCilpmo1, with an injected dose
of 70 pg of ["C)NMSP (approximately 0.9 pglkg).
There was a progressive increase with time in the caudate/cerebellum ratio of activity, as visualized by serial
PET scans (Table 2). This increase has been observed
repeatedly in animal studies and is due mainly to the
reduction of nonspecific binding in the cerebellum and
the remainder of the brain [I, 4 , 8 , 12). Figure 3 shows
two of the PET images. The basal ganglia were again
seen, with slightly less activity in the rest of the brain.
Activity in other regions, particularly in the cerebral
cortex, .could reflect serotonin-2 receptors, which are
also labeled by neuroleptic drugs.
Since spiperone is known to have a substantial
affinity for 5-hydroxytryptamine-2 (5HT-2) receptors
[l4), we tested NMSP to determine its affinity for
these receptors. From the in vitro binding experiments
with rat frontal cortex, we found that NMSP had a K,
for 5HT-2 receptors (against [3H)spiperone) of about
1.3 nmol while spiperone has a K D for 5HT-2 receptors
of about 0.8 nmol under the same conditions. Thus,
NMSP has a high affinity for 5HT-2 receptors, although the affinity is somewhat less than that for
spiperone. While some of the radioactivity in the PET
scans is presumably localized to 5HT-2 receptors, the
bulk of the activity is bound to dopamine receptors.
This pattern is indicated by the fact that the activity
concentrates highly in the caudate nucleus and putamen, as compared to the cortex; the pattern parallels
the distribution of dopamine receptors rather than
5HT-2 receptors, which are found in about equal concentrations in the caudate nucleus and the remainder of
the cortex in several species {IS).
In both the human and baboon studies the planes
imaged by PET were selected on the basis of computed
tomographic (CT) images. The location of the high
concentrations of activity within the brain slice corresponded to the location of the basal ganglia as deter-
Wagner et al: Imaging Dopamine Receptors S81
Fig 2. PET images of baboon brain following intravenous injection of carbon-l 1-labeled 3-N-methylspiperone({"C)NMSP). A
9-year-old 30 kg male Papio anubis, obtained from Primate Imports, was initially immobilized with ketamine hydrochloride
(250 mg, intramuscularlyi,anesthetized with sodium pentobarbital(15 mgikg, intravenously),and received atropine (0.2 mg,
intramuscularly).The animal was positioned using anatomical
and surface markings such that the middle slice of the
NeuroECAT passed through the caudate heads and cerebellum.
High-resolution-mode cfull width at half maximum, 9 mm) acquisitions were obtained using septa and shadow shields and a
Shepp and Logan filter. Calculated attenuation corrections were
made using an elliptical region of interest. (A) The PET scan obtained 40 to 60 minutes after injection shows relatively increased
activity in the basal ganglia following the injection of
{I'C)NMSP (16 mCi, with a spec& activity d 10.4 mCilpmolj
in the baboon. (B) The same PET section at a time 40 to 60
minutes following the injection of an excess of unlabeled
spiperone (6.6 mg, or 222 kglkgi, along with {"C)NMSP.
This image shows unifrm uptake throughout the brain, as
compared to (A).This uniformity indicates blockade of the
dopamine receptors by the unlabeled drug, resulting in little or
no specific receptor binding in the area ofthe caudate nucleus.
Both displayed images are scaled by the computer t o a fixed
maximum brightness, giving an artificial appearance of higher
counts in the whole brain in (B). The actual counts, however,
are considerably lower than those in (A) (see Table I ) .
Table 1 . Carbon-1 1-Lubeled N-Metbylspiperone Levels in Baboon Caudate Nucleus and Cerebellum"
Time of acquisition
Condition
Mean cpm per pixel
Caudate nucleus
Cerebellum
Caudatekerebellum ratio
Scan 1
Scan 2
40 to 60 minutes after first injection
40 to 60 minutes after second injection
[WINMSP
alone
["CINMSP with excess, unlabeled
spiperone
303
140
2.03
73
88
1.05
'Regions for quantitation were selected from the video monitor with a cursor and were verified by comparison to computed tomographic scans.
All counts are corrected back to time of injection.
582
Annals of Neurology
Supplement to VoIume 15, 1984
Table 2. Carbon-11-Labeled N-Metbylspiperone Levels
in Human Caudate Nucleus and Cerebellum"
Scan 1
~
Time of acquisition after
injection (min)
Mean cpm per pixel
Caudate nucleus
Cerebellum
Caudatekerebellum ratio
Scan 3
Scan 2
~~~
20-30
40-60
70- 130
110
63
1.8
117
42
2.8
86
20
4.4
"All counts are corrected back ro time of injection.
mined by the CT scans. The caudatelcerebellum ratio
of radioactivity was 4 : 1 between 70 and 130 minutes
after injection in the human and 2: 1 between 40 and
60 minutes after injection in baboons. Three simultaneous planes (slices each 32 mm apart) were used for
the PET scans. For the human studies the cerebellum
was on a plane 32 mm caudal to the plane with the
caudate nucleus (see Fig 3) and was used for comparison with the region of the caudate nucleus.
The recovery coefficient for our NeuroECAT is
about 0.5 for the caudate nucleus, which means that
the concentration in a region this size is underestimated by a factor of 2 because of the limited spatial
resolution of the system. We assume that the recovery
coefficient for a region the size of the cerebellum is
1.0 (i.e., no underestimation of activity) because of
its size compared to the resolution capability of the
NeuroECAT. Thus, the actual caudate/cerebellum
ratios are believed to be on the order of at least 4 : 1 in
the baboon at 40 to 60 minutes and 8: 1 in the human
at 70 to 130 minutes after injection.
Thus, ["CINMSP, a compound with a high affinity
for dopamine receptors in vitro, localizes to the basal
ganglia, a region with a high density of dopamine receptors, after in vivo administration in primates. The
accumulation in the basal ganglia is blocked by administering other nonradiolabeled dopamine receptorblocking drugs in excess. Taken together, these data
indicate that the imaged radioactivity reflects densities
of dopamine receptors. Given this demonstration that
it is possible to image the distribution of dopamine
receptors in the human brain, it may now be feasible to
assess in human beings the role of dopamine receptors
in the actions of numerous psychotropic drugs and in
disorders such as schizophrenia, tardive dyslunesia,
parkinsonism, and Huntington's chorea.
Addendum
Since submission of this manuscript, some of the results reported here have been published elsewhere (Wagner HN,
Burns HD, Dannals RF, et al: Imaging dopamine receptors in
the human brain. Science 221:1264-1266, 1983).
Fig 3. PET scan of a human subject follwing intravenous injection of carbon-1 1-labeled 3-N-methylspiperone.A 56-year-old
conscious male was positioned in the PET scanner using a headbolder. The middle slice ofthe PET scan contains a section of the
brain where the basal ganglia are located (A and B) , The scan
was carried out in the same way as with the baboons. Images obtained at 40 t o 60 minutes postinjection (A)and at 70 to 130
minutes postinjection (B) show high accumulation of activity in
the basal ganglia relative to the rest of the brain.
Wagner et al: Imaging Dopamine Receptors
S83
and atrophy in Huntington's disease determined by "FDG and
computed tomographic scan. Ann Neurol 12:425-434, 1982
12. Laduron P, Leysen J: Specific in vivo binding of neurolepric
drugs in rat brain. Biochem Pharmacol 26:1003-1007, I977
13. Leysen JE, Gommeren W, Laduron PM: Spiperone: a ligand of
choice for neuroleptic receptors. 1. l n e t i c s and characteristics
of in vitro binding. Biochem Pharmacol 27:307, 1978
14. Peroutka SJ, Snyder SH: Multiple serotonin receptors: differential binding of IiH] 5-hydroxytryptamine, ['Hllysergic acid
diethylamide and [ 'Hlspiroperidol. Mol Pharmacol 16:687,
The authors thank D. Koller, J. Rhine, and M. Bryan for help with
the PET srudies; T. K. Natarajan and F. Gilbart for cyclotron assistance; L. Widerman for CT assistance; G . Hopkins and H. Drew for
in vitro assistance; J. Anderson for animal assistance; and J. Reyes
and D. Weimer for manuscript preparation. The authors are especially grateful to D r Solomon H . Snyder for his advice and help with
experiments and with the preparation of this manuscript.
Supported by Grants CA32845, NS15080, CA09199, and
MH0053 from the US Public Health Service.
1979
15. Peroutka SJ, Snyder SH: Two distinct serotonin receptors: re-
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