Assessment of Dopamine Receptor Densities in the Human Brain with

Carbon- 1 1-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-1 1-labeled 3-N-methylspiperone, a ligand that preferentially binds to dopamine recep- tors 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- 1 1-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 quan- tify 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 mea- surement of local cerebral glucose utilization to hu- mans, based on the use of fluorine-18-labeled fluorodeoxyglucose ("FDG) and positron emission to- mography (PET). Soon thereafter, Reivich and col- leagues [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 re- sulted in increased regional glucose utilization. Subse- quently, 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 parkin- sonism, Huntington's chorea, tardive dyskinesia, schizophrenia, chronic intractable pain states, and con- genital insensitivity to pain? (4) Is it possible to monitor

From the *Division of Nuclear Medicine and the ?Division of Address reprint requests to Dr Wagner, Division of Nuclear Neuroradiology, The Johns Hopkins Medical Institutions, Balti- Medicine, The Johns Hopkins Medical Institutions, 61 5 N Wolfe St, more, MD 21205; the $Departments of Neuroscience, Pharmacol- Baltimore, MD 21205 ogy, and Experimental Therapeutics, and Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Balti- more, 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.

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 radionu- clides, such as carbon-1 1, the production of which re- quired 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 acces- sible 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 half- life 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 pro- posed to use carbon-1 1 diprenorphine, an antagonist of opiate receptor activity { 161, to investigate the distribu- tion and density of opiate receptors.

To achieve the desired goal, a suitable radiotracer with the following characteristics was needed:

1. The compound had to be labeled with a positron- emitting radionuclide to permit the use of a PET scanner for noninvasive measurements in humans.

2. The labeled drug had to cross the blood-brain bar- rier, 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 con- taining receptors and the surrounding structures.

As in the case of the development of the deoxyglu- cose 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

Fig I . Chemical structure of carbon-1 1 -labeled 3-N-methyl- spiperone ( { 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 sat- uration phenomenon related to the dose of the ad- ministered ligand; that is, specific binding in the region of the striatum plateaued as the dose increased, be- cause 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 intrave- nous 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 cerebrospi- nal 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 con- centration of dopamine receptors using PET, we syn- thesized the tracer carbon-1 1-labeled N-methylspi- perone, or ["CINMSP (Fig l) , by N-alkylation of the neuroleptic drug spiperone with [ "Clmethylio- dide, 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 bom- bardment. Prior to injection, the product was puri- fied and the specific activity was determined using a reverse-phase liquid chromatographic column. The specific activity was also determined by an in vitro com- petitive binding assay. The compound NMSP was pre- viously 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 paral- lels the distribution of receptors and whether adminis- tration of excess, unlabeled, related drugs blocks this

S80 Annals of Neurology 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 prefer- ential labeling of dopamine receptors in vivo. The con- centration 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, un- labeled neuroleptic drugs with high affinity for dopamine receptors obliterates the regional distribu- tion, as indicated by equal concentrations of the ligand in the striatum and cerebellum.

From our in vitro experiments with receptor- containing 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 inhibi- tory constant (Ki) of about 250 pmol (against hydrogen- 3-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 min- utes after the injection. Coinjection of unlabeled spipe- rone lowered the striaturdcerebellum ratios by 70% at spiperone doses of 150 pglkg without altering cerebellar binding. These biochemical studies indi- cate 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, intrave- nous 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 anes- thetized, 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 administra- tion 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 ex- periment was performed with one of us (H. N. W.) as the experimental subject. Twenty millicuries of [l'C]NMSP was injected intravenously in the con- scious 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 cau- date/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, al- though 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 puta- men, as compared to the cortex; the pattern parallels the distribution of dopamine receptors rather than 5HT-2 receptors, which are found in about equal con- centrations 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 corre- sponded 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 injec- tion of carbon-l 1 -labeled 3-N-methylspiperone ({"C)NMSP). A 9-year-old 30 kg male Papio anubis, obtained from Primate Im- ports, was initially immobilized with ketamine hydrochloride (250 mg, intramuscularlyi, anesthetized with sodium pentobar- bital(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) ac- quisitions 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 ob- tained 40 t o 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"

Scan 1 Scan 2

Time of acquisition Condition

Mean cpm per pixel Caudate nucleus Cerebellum

Caudatekerebellum ratio

40 to 60 minutes after first injection [WINMSP alone

40 to 60 minutes after second injection ["CINMSP with excess, unlabeled

spiperone

303 140 2.03

7 3 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 Scan 2 Scan 3 ~ ~~~

Time of acquisition after 20-30 40-60 70- 130 injection (min)

Mean cpm per pixel Caudate nucleus 110 117 86 Cerebellum 63 42 20

Caudatekerebellum ratio 1.8 2.8 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 simul- taneous 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 compari- son 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 underes- timated 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 re- ceptors, after in vivo administration in primates. The accumulation in the basal ganglia is blocked by admin- istering other nonradiolabeled dopamine receptor- blocking 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 re- ported 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 injec- tion of carbon-1 1 -labeled 3-N-methylspiperone. A 56-year-old conscious male was positioned in the PET scanner using a head- bolder. 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 ob- tained 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

The authors thank D. Koller, J. Rhine, and M. Bryan for help with the PET srudies; T. K. Natarajan and F. Gilbart for cyclotron assis- tance; 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 espe- cially 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.

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