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In vivo quantitative imaging of dopamine receptors in human brain using positron emission tomography and [76Br]bromospiperone
European Journal of Pharmacology, 114 (1985) 267-272
267
Elsevier
IN VIVO QUANTITATIVE IMAGING OF D O P A M I N E R E C E P T O R S IN H U M A N BRAIN U S I N G
POSITRON EMISSION TOMOGRAPHY AND 176Br]BROMOSPIPERONE BERNARD MAZIERE *, CHRISTIAN LOC'H, JEAN-CLAUDE BARON, PANTS SGOUROPOULOS, NATHALIE DUQUESNOY, ROBERTA D'ANTONA and HENRI CAMBON Service Hospitalier FredericJoliot, Commissariat h l'EnergieAtomique, Dbpartement de Biologie, Hbpital d'Orsay, 91406 Orsay, France
Received 18 March 1985, revised MS received7 May 1985, accepted4 June 1985
B. MAZII~RE, C. LOC'H, J.-C. BARON, P. SGOUROPOULOS, N. DUQUESNOY, R. D'ANTONA and H. CAMBON, In riot quantitative imaging of dopamine receptors in human brain using positron emission tomography and [ZtBr]bromospiperone, European J. Pharmacol. 114 (1985) 267-272. The brain regional distribution and kinetics of [76Br]bromospiperone, a derivative of a neuroleptic (spiperone) labeled with the positron emitter bromine-76, were studied by time-of-flight tomography after i.v. injection in man. In a control subject the kinetic distribution study showed an accumulation of radioactivity which reached a maximum 3 h postinjection in the frontal cortex and cerebellum regions and 4-5 h postinjection in the basal ganglia. Thereafter the striatal activity remained essentially constant over a period of 25 h. In a group of 13 control subjects, the mean value for the striatum-to-cerebellum ratio, at 4.5 h postinjection, was 1.84 (S.D. 0.21). In two schizophrenics treated with high doses of haloperidol, this ratio was found to be only 1.22. These data indicate that radiolabeled bromospiperone is very suitable for human pharmacological or pathological investigations of the central dopaminergic system. Dopamine receptors
Positron tomography
[76Br]Bromospiperone
1. Introduction Post-mortem human studies have shown that changes in brain dopamine receptor densities and binding characteristics are correlated with various neurological diseases such as schizophrenia (Owen et al., 1978), Parkinson's disease (Reisine et al., 1977b; Lee et al., 1978) and Huntington's disease (Reisine et al., 1977a). Neuroleptic drugs such as spiperone, are known to bind extensively to dopamine and serotonin receptors in the rat brain (Laduron et al., 1978). In previous successful attempts to visualize these receptors in vivo by means of positron emission tomography (PET), spiperone and its methylated derivative were labeled with 11C (Fowler et al., 1982; Wagner et al., 1983; Burns et al., 1984) or 18F (Maeda et al., 1981; Kilbourn et al., 1984). Since it has been demon-
* To whom correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishers B.V.
strated that the substitution of bromine for hydrogen in the phenyl ring of the molecule introduces a minimal structural alteration without affecting pharmacological activity (Huang et al., 1980), spiperone has also been labeled with the bromine radioisotopes 76Br (Mazi&e et al., 1984) and 77Br (De Jesus et al., 1983). Using p-bromospiperone labeled with the positron emitter bromine-76 (T1/2 = 16.2 h), we have recently demonstrated in in vivo studies in rats and baboon that the striatal specific binding of this molecule [76Br]BSP mainly involved the dopamine receptors while the frontal cortical specific binding was due to a combination of dopamine and serotonin receptors (Mazitre et al., 1984). It was concluded from the baboon studies that [76Br]BSP was suitable for characterizing dopamine receptors in the basal ganglia by PET. Before using the method for pathophysiological investigations, the characteristics of its in vivo binding in normal subjects needed to be known.
268 We report here the first results of a study of the regional distribution and kinetics of [76Br]BSP in the human brain.
2. Materials and methods
2.1. Preparation of the labeled radiopharmaceutical 76Br was prepared by irradiating an arsenic target with a beam of 30 MeV helium-3 ions. The target was dissolved in an acidic medium and after oxidation the radioactive bromine was distilled and trapped as bromide in diluted ammonia which was then taken to dryness. 76Br2, generated in situ by an HEOE-acetic acid mixture, was allowed to react for 10 rain with an excess of spiperone by means of an electrophilic substitution reaction. The radioactive bromo compound, separated from the cold precursor by high-performance liquid chromatography, was dissolved in dilute acetic acid. The solution was sterilized by filtration through a Millipore membrane and brought to isotonicity using diluted N a O H (final pH 4).
allows simultaneous acquisition of 7 slice images, 12 mm thick on average. The undetected space between slices is 3 mm. By means of bony landmarks and laser beams, the subject was carefully positioned so that the lowest slice, 1 cm above and parallel to the orbito-meatal (OM) plane, included the cerebellar hemispheres and the third slice (OM + 4 cm), the basal ganglia. After collection of transmission scan data via an external 68Ge source for subsequent correction of radiation attenuation effects, 1 mCi (37 MBq) of [76Br]BSP in isotonic acetate solution was injected in a brachial vein of the subject. For the initial kinetic distribution study, eight 30 to 60 min scans (accumulating a total of 300 000 counts typically for each brain level) were performed starting at the end of the injection and continuing for 26 h. For the subsequent brain distribution studies, two 30 min scans of each brain level were conducted 10 rain and 4.5 h after the injection of the radiopharmaceutical. Venous blood samples were collected 15 rain before the end of each scan and their radioactivity content assayed by means of a sodium iodide well counter.
2.2. Patients 2.4. Data analysis To determine the optimal time for quantitative uptake measurements a study of the variation with time of the regional distribution of [76Br]BSP in brain was performed on a 46-year-old healthy male volunteer. Brain biodistribution studies were subsequently performed on 12 control subjects and two schizophrenics under treatment with neuroleptics. The control subjects, 7 males and 5 females ranging from 39 to 87 years of age, were free from any known brain disease and were not taking medication suspected of interacting with dopamine receptors; the two schizophrenics, on the other hand, were stt~died 2 h after'intramuscular injection of a therapeutic dose (10 mg) of haloperidol. Informed consent was obtained from all patients.
2.3. P E T studies PET scans were performed by means of a timeof-flight tomograph (Soussaline et al., 1984) which
In the images with the highest total counts (that obtained 10 min postinjection for the slice at OM + 1 cm and that at 4.5 h post injection for the slice passing through the basal ganglia) the external border of the cerebellum and of the brain was taken empirically to be delineated by a contour representing, respectively, 60% and 30% of the maximum image intensity. With reference to a brain PET atlas (Mazziotta et al., 1981) a circular region of interest (ROI) was then placed visually over each cerebellar hemisphere (OM + 1 cm slice, ROI area = 28.6 cm 2), each striatal area (OM + 4 cm slice, ROI area = 3.7 cm 2) and over the frontal cortex (OM + 7 cm slice, ROI area = 7.1 cm2). Furthermore, a ROI was placed on the image displaying the basal ganglia structures (OM + 4 cm), corresponding to a contour equal to 80% of the maximum image intensity. When these structures were not visible (as with haloperidol-treated patients), the striatal ROIs placed on the images
269 from a control subject were copied on to those from the patient. The available software, which allowed automatic transfer of each ROI from one scan to another and correction for radioactive decay, produced values for a known time after injection of the fraction of the injected dose in each ROI. These data were used to calculate the frontal cortex-to-cerebellum radioactivity and the striatum-to-cerebellum radioactivity ratios for each study at each point in time.
3. Results
3.1. Radiopharmacetttical preparation Typically 5-6 mCi of 'no carrier added' [76Br]BSP were prepared under the above experimental conditions (radiochemical yield: 80%) with a specific activity varying from 0.5 to 2.0 Ci//~mol. Preliminary analytical and animal studies had shown that this radiopharmaceutical, prepared according to the protocol described, was sterile, pyrogen-free and free from any measurable chemical toxicity. The radionuclidic purity of [76Br]BSP was checked via 7-ray spectroscopy. At the time of injection the radioactive contaminants were 75Br and 77Br which amounted, respectively, to 0.5% and 1.6% of the total activity administered. Radiotoxicity was estimated by means of the results of a wholebody kinetic bio-distribution study performed on a baboon, showing that about 25% of the injected dose was concentrated in the liver and was eliminated with a biological half-life of 2.6 h. Using the classical model of the absorbed dose fractions (Smith et al., 1968) it was calculated that, for an i.v. injection of 1 mCi [76Br]BSP in man, the radiation dose absorbed by the liver and arising from positron and y-rays emissions and electron captures was 1.5 rad (1.5 cGy). The contribution of the radioactive contaminants [75Br] BSP and [77Br]BSP to this radiation dose was lower than 0.5%. Such a dose does not preclude PET studies in man.
3.2. Brain uptake kinetics Fig. 1 displays two tomographic images of the distribution of the radioactivity in brain slices at OM + 4 cm, taken at 10 min and 4.5 h after i.v. injection of 1 mCi [76Br]BSP in a healthy volunteer. As the preliminary baboon study led us to expect, the early image showed a perfusion-like distribution pattern with a higher activity in gray than in white matter. On the other hand, a clear delineation of the striatal structures was easily observed in the late image. At 4.5 h after the injection of 1 mCi [76Br]BSP about 350 000 counts were accumulated in 30 min in the section including the striatum and 220 000 and 260000 counts in 30 min in the sections displaying, respectively, the cerebellum and the frontal cortex. The root-mean-square percent uncertainty in the number of events in an average resolution cell (Budinger et al., 1978) was 6% for the striatum activity, and 9% and 10% respectively for the frontal cortex and cerebellum activities. W h a t e v e r form of ROI was used to determine the striatum activity, the radioactivity uptake data obtained did not differ significantly. The data (fig. 2) on radioactivity uptake per unit volume measured as a function of time in various brain structures during the kinetic distribution study show an accumulation which reached a maximum 3 h postinjection in the frontal cortex
Fig. 1. PET images of a human brain (OM + 4 cm) after i.v. injection of [76Br]BSP.Images obtained 10 to 25 min (A) and 4.5 to 5 h (B) after injection.
270
O
TABLE 1 Striatum-to-cerebellum (Str/Crb) and frontal cortex-to-cerebellum (Ctx/Crb) radioactivity concentration ratios in 13 control subjects and 2 haloperidol-treated shizophrenics, 4.5 h after the injection of 0.8 to 1.2 mCi [76Br]BSP (spec. act.: 0.5-2.0 Ci/t~ mol).
STRIATUM
3 T O~
_a
2
Subject Sex Age Str/Crb
a. ¢o
i
=..
Controls
x
1
-
o
;
WHOLE
A
BLOOD
.i.
lb HOURS
Fig. 2. Radioactivity uptake, expressed as a fraction of the dose injected (I.D.), measured in 1 cm3 of striatum (11), frontal cortex (0), cerebellum (e) and whole blood (A) as a function of time. Statistical uncertainties were calculated according to Budinger et al. (1978).
a n d c e r e b e l l u m regions a n d 4-5 h p o s t i n j e c t i o n in the b a s a l ganglia. T h e r e a f t e r the d e c a y - c o r r e c t e d activity in the s t r i a t u m r e m a i n e d essentially constant with time d u r i n g a p e r i o d of 25 h while the activity in the cortical region a n d in the c e r e b e l l u m d e c r e a s e d at a similar rate. T h e whole b l o o d radioactivity (corrected for decay) decreased d u r i n g the whole e x p e r i m e n t , b u t this decrease was slower t h a n in the cerebellum.
1 2 3 4 5 6 7 8 9 10 11 12 13 Treated 1 schizophrenics 2
M M M F M F M M M M F F F M M
39 46 58 59 59 63 67 72 72 76 79 81 87 49 33
2.10 2.23 1.78 1.85 1.96 1.63 1.75 1.85 2.03 1.73 1.45 1.72 1.80 1.24 1.21
Ctx/Crb 1.40 1.26 0.96 1.29 1.06 0.89 0.99 1.17 1.17 0.80 1.14 1.04 1.18 1.12 0.93
T h e m a x i m u m [76Br]BSP b r a i n uptake, which occurred 4-5 h after the a d m i n i s t r a t i o n of the r a d i o l i g a n d , was a b o u t 1.5% of the dose injected a n d the whole b l o o d c o n t r i b u t i o n to this q u a n t i t y was less than 1 × 1 0 - 2 % of the dose injected.
3.3. Brain activity distribution o <£
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,~I
FRONTAL
CORTEX
/ CEREBELLUM
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T h e s t r i a t u m - t o - c e r e b e l l u m a n d cortex-to-cereb e l l u m r a d i o a c t i v i t y ratios m e a s u r e d in the g r o u p o f c o n t r o l subjects a n d in the two h a l o p e r i d o l t r e a t e d p a t i e n t s are listed on table 1. In the c o n t r o l g r o u p the m e a n value for the striatum-to-cerebell u m ratio at 4.5 h p o s t injection was 1.84 (S.D. + 0.21). In the two t r e a t e d schizophrenics, these ratios were only 1.21 a n d 1.24 (values statistically different from the control, P < 0.05).
<(
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0
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10'
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4. Discussion
2b HOURS
Fig. 3. Stfiatum-to-ccrcbellum (O) and frontal cortex-to-cerebellum radioactivity concentration ratios (ll) as a function of
time. Root-mean-square uncertainties o n these ratios were calculated according to Budinger et al. (1978).
T o resolve in vivo specific a n d non-specific b i n d i n g of a d o p a m i n e r g i c radioligand, it is generally a s s u m e d that r a d i o a c t i v i t y in the c e r e b e l l u m m e a s u r e s non-specific b i n d i n g while the activity in
271 other brain structures reflects regional total binding (i.e. specific plus non-specific). Thus the specific binding for each brain structure can be expressed as the ratio of the concentration of the radioactivity for that structure to that of the cerebellum. Due to the steady washout of the non-specifically bound radioactivity (e.g. in the cerebellum), this ratio increases continuously for the striatum (fig. 3) and the highest value (4.8), corresponding to a specific binding of 75% of the total binding was observed 25 h post injection. However, due to the decay of 76Br, the statistical uncertainty of the measurement of this ratio was at a minimum 4-5 h after the administration of the radioligand, and this imaging time was thus chosen for subsequent brain distribution studies. A comparison of the' striatal (dopamine receptors) and frontal cortical (dopamine and serotonin receptors) radioactivity curves (fig. 3) suggest a significantly lower affinity of [76Br]BSP a n d / o r density of receptors for serotonin than for dopamine receptors. Partial volume effects are known to lower considerably the fraction of the true isotope concentration recovered in small structures (e.g. striatum) (Mazziotta et al., 1981). As these errors were not corrected for in our calculations, the real values of the striatum or frontal cortex-to-cerebellum activity ratios were notably underestimated. However, assuming that size and shape of striatum were roughly similar from subject to subject, the extent of this underestimation was essentially constant. Among the control group, a significant difference was observed (t-test, P < 0.05) between the mean values of the striatum-to-cerebellum activity ratios in the male (1.93) and female (1.69) subgroups. Due to differences in mean age and to the small number of subjects in each of these subgroups, it was not possible to establish whether this difference is, or is not due to different age-related specific binding decreases in the male and female subgroups (Wong et al., 1984). The low values for the ratios found in the two haloperidol-treated schizophrenics had been anticipated. There was a 70% decrease in specific binding in striatum (compared to that in controls) which is presumably due to partial occupation of
the dopamine receptors by the previously administrated haloperidol. From our PET radioactivity measurements, it can be calculated that i.v. injection of 1 mCi [76Br]BSP (spec. act. 1 C i / # m o l ) leads to a neuroleptic concentration of about 3 × 10-2 pmol per g striatum 4.5 h post administration. Human postmortem studies (Bokobza et al., 1984) have shown that the striatal dopamine receptor concentration, measured using [3H]spiperone, is about 6.0 pmol per g tissue. Since [3H]spiperone and [76Br]BSP have very similar in vitro specific binding characteristics for striatal membranes (Mazi6re et al., 1985) this indicates that, under our experimental conditions, only a small fraction (about 0.5%) of the striatal receptors was occupied by the radioligand and that non-specific binding of BSP was kept minimal. Dosimetric calculations have shown that [76Br]BSP can be used safely in man. However for clinical applications, 75Br, another positron emitter bromine isotope should be preferred over 76Br as label. Indeed as 75Br has more suitable emission characteristics and a shorter half-life (T1/2 = 1.6 h), the irradiation of the patient would be divided by a factor of 2. This study, which confirms the results of our previous animal experiments clearly indicates that radiolabeled bromospiperone is suitable for human pharmacological and pathological investigations of the central dopaminergic system.
Acknowledgements We wish to thank the SHFJ staff for its collaboration, N. De Blecker for typing the manuscript and Dr. B. Pate for language corrections.
References Bokobza, B., M. Ruberg, B. Scanon, F. Javoy-Agid and Y. Agid (1984) [3H]Spiperone binding, dopamine and HVA concentrations in Parkinson's disease and supranuclear palsy, European J. Pharmacol. 99, 167. Budinger, T.F., S.E. Derenzo, W.L. Greenberg, G.T. Gullberg and R.H. Huesman (1978) Quantitative potentials of dynamic emission computed tomography, J. Nucl. Med. 19, 309.
272 Burns, H.D., R.F. Dannals, B. Langstrom, H.T. Ravert, S.E. Zeyman, T. Duelfert, D.F. Wong, J.J. Frost, M.J. Kuhar and H.N. Wagner (1984) (3-N-[llC]methyl) spiperone, a ligand binding to dopamine receptors. Radiochemical synthesis and biodistribution studies in mice, J. Nucl. Med. 25, 1222. De Jesus, O.T., A.M. Friedman, A. Prasad and J.R. Revenaugh (1983) Preparation and purification of 77Br-labelled pbromospiroperidol suitable for in vivo dopamine receptor studies, J. Label. Comp. Radiopharm. 20, 745. Fowler, J.S., C.D. Arnett, A.P. Wolf, R.R. MacGregor, E.F. Norton and A.M. Findley (1982) [11C]Spiroperidol: synthesis, specific activity determination and biodistribution in mice, J. Nucl. Med. 23, 437. Huang, C.C., A.M. Friedman, R. So, M. Simonovic and H.Y. Meltzer (1980) Synthesis and biological evaluation of pbromospiperone as potential neuroleptic drug, J. Pharm Sci. 69, 984. Kilbourn, M.R., M.J. Welch, C.S. Dence, T.J. Tewson, H. Saji and M. Maeda (1984) Carrier-added and no carrier-added synthesis of [lSF]spiroperidol and [18F]haloperidol, Int. J. Radiat. Isot. 35, 591. Laduron, P.M., P.F.M. Janssen and J.E. Leysen (1978) Characterization of specific in vivo binding of neuroleptic drugs in rat brain, Life Sci. 33, 581. Lee, T., P. Seemann, A. Rajput, I.J. Farley and O. Hornykiewicz (1978) Receptor basis for dopaminergic supersensitivity in Parkinson's disease, Nature 273, 59. Maeda, M., T.J. Tewson and M.J. Welch (1981) Synthesis of high specific activity [18F]spiroperidol for dopamine receptor studies, J. Label. Comp. Radiopharm. 18, 102. Mazi~re, B., C. Loc'h, P. Hantraye, R. Guillon, N. Duquesnoy, F. Soussaline, R. Naquet, D. Comar and M. Mazi~re (1984) 76Bromospiroperidol: A new tool for quantitative in vivo imaging of neuroleptic receptors, Life Sci. 35, 1349.
Mazi&e, B., C. Loc'h and O. Stulzaft (1985) 76Bromospiroperidol: synthesis and in vitro binding characteristics. (submitted). Mazziotta, J.C., M.E. Phelps, J. Miller and C.E. Kuhl (1981) Quantitation in positron emission computed tomography: 5. Physical anatomical effects, J. Comput. Ass. Tomog. 5: 734. Owen, F., T.J. Cross, T.J. Crow, A. Longden, M. Poulter and C.J. Riley (1978) Increased dopamine-receptor sensitivity in schizophrenia, Lancet ii, 223. Reisine, T.D., J.Z. Fields, L.Z. Stern, P.C. Johnson, E.D. Bird and H.I. Yamamura (1977a) Alterations in dopaminergie receptors in Huntington's disease, Life Sci. 21, 1223. Reisine, T.D., J.Z. Fields, H.I. Yamamura, E.D. Bird, E. Spokes, P.S. Schreiner and S.J. Enna (1977b) Neurotransmitter receptor alterations in Parkinson's disease, Life Sci., 21, 335. Smith, E.M., G.L. Brown and W.H. Ellet (1968) Radiation dosimetry. Principles of Nuclear Medicine, Saunders Company, Philadelphia, ed. Wagner, Jr. Soussaline, F., R. Campagnolo, B. Verrey, B. Bendriem, A. Bouvier, J.L. Lecomte and D. Comar (1984) Physical characterization of a time-of-flight positron emission tomography system for whole-body quantitative studies, J. Nucl. Med. 25, P46. Wagner Jr., H.N., D.H. Burns, R.F. Dannals, D.F. Wong, B. Langstrom, T. Duelfer, J.J. Frost, H.T. Ravert, J.M. Links, B.B. Rosenbloom, S.E. Lukas, A.V. Kramer and M.T. Kuhar (1983) Imaging dopamine receptors in the human brain by positron tomography, Science 221, 1224. Wong, D.F., H.N. Wagner, Jr. R.F. Dannals, J.M. Links, J. Frost, H.T. Ravert, A.A. Wilson, A.E. Rosenbaum, A. Gjedde, K.H. Douglass, J.O. Petronis, M.F. Folstein, J.K.T. Toung, H.O. Burns and M.J. Kuhar (1984) Effects of age on dopamine and serotonin receptors measured by Positron Tomography in the living human brain, Science, 1393.
267
Elsevier
IN VIVO QUANTITATIVE IMAGING OF D O P A M I N E R E C E P T O R S IN H U M A N BRAIN U S I N G
POSITRON EMISSION TOMOGRAPHY AND 176Br]BROMOSPIPERONE BERNARD MAZIERE *, CHRISTIAN LOC'H, JEAN-CLAUDE BARON, PANTS SGOUROPOULOS, NATHALIE DUQUESNOY, ROBERTA D'ANTONA and HENRI CAMBON Service Hospitalier FredericJoliot, Commissariat h l'EnergieAtomique, Dbpartement de Biologie, Hbpital d'Orsay, 91406 Orsay, France
Received 18 March 1985, revised MS received7 May 1985, accepted4 June 1985
B. MAZII~RE, C. LOC'H, J.-C. BARON, P. SGOUROPOULOS, N. DUQUESNOY, R. D'ANTONA and H. CAMBON, In riot quantitative imaging of dopamine receptors in human brain using positron emission tomography and [ZtBr]bromospiperone, European J. Pharmacol. 114 (1985) 267-272. The brain regional distribution and kinetics of [76Br]bromospiperone, a derivative of a neuroleptic (spiperone) labeled with the positron emitter bromine-76, were studied by time-of-flight tomography after i.v. injection in man. In a control subject the kinetic distribution study showed an accumulation of radioactivity which reached a maximum 3 h postinjection in the frontal cortex and cerebellum regions and 4-5 h postinjection in the basal ganglia. Thereafter the striatal activity remained essentially constant over a period of 25 h. In a group of 13 control subjects, the mean value for the striatum-to-cerebellum ratio, at 4.5 h postinjection, was 1.84 (S.D. 0.21). In two schizophrenics treated with high doses of haloperidol, this ratio was found to be only 1.22. These data indicate that radiolabeled bromospiperone is very suitable for human pharmacological or pathological investigations of the central dopaminergic system. Dopamine receptors
Positron tomography
[76Br]Bromospiperone
1. Introduction Post-mortem human studies have shown that changes in brain dopamine receptor densities and binding characteristics are correlated with various neurological diseases such as schizophrenia (Owen et al., 1978), Parkinson's disease (Reisine et al., 1977b; Lee et al., 1978) and Huntington's disease (Reisine et al., 1977a). Neuroleptic drugs such as spiperone, are known to bind extensively to dopamine and serotonin receptors in the rat brain (Laduron et al., 1978). In previous successful attempts to visualize these receptors in vivo by means of positron emission tomography (PET), spiperone and its methylated derivative were labeled with 11C (Fowler et al., 1982; Wagner et al., 1983; Burns et al., 1984) or 18F (Maeda et al., 1981; Kilbourn et al., 1984). Since it has been demon-
* To whom correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishers B.V.
strated that the substitution of bromine for hydrogen in the phenyl ring of the molecule introduces a minimal structural alteration without affecting pharmacological activity (Huang et al., 1980), spiperone has also been labeled with the bromine radioisotopes 76Br (Mazi&e et al., 1984) and 77Br (De Jesus et al., 1983). Using p-bromospiperone labeled with the positron emitter bromine-76 (T1/2 = 16.2 h), we have recently demonstrated in in vivo studies in rats and baboon that the striatal specific binding of this molecule [76Br]BSP mainly involved the dopamine receptors while the frontal cortical specific binding was due to a combination of dopamine and serotonin receptors (Mazitre et al., 1984). It was concluded from the baboon studies that [76Br]BSP was suitable for characterizing dopamine receptors in the basal ganglia by PET. Before using the method for pathophysiological investigations, the characteristics of its in vivo binding in normal subjects needed to be known.
268 We report here the first results of a study of the regional distribution and kinetics of [76Br]BSP in the human brain.
2. Materials and methods
2.1. Preparation of the labeled radiopharmaceutical 76Br was prepared by irradiating an arsenic target with a beam of 30 MeV helium-3 ions. The target was dissolved in an acidic medium and after oxidation the radioactive bromine was distilled and trapped as bromide in diluted ammonia which was then taken to dryness. 76Br2, generated in situ by an HEOE-acetic acid mixture, was allowed to react for 10 rain with an excess of spiperone by means of an electrophilic substitution reaction. The radioactive bromo compound, separated from the cold precursor by high-performance liquid chromatography, was dissolved in dilute acetic acid. The solution was sterilized by filtration through a Millipore membrane and brought to isotonicity using diluted N a O H (final pH 4).
allows simultaneous acquisition of 7 slice images, 12 mm thick on average. The undetected space between slices is 3 mm. By means of bony landmarks and laser beams, the subject was carefully positioned so that the lowest slice, 1 cm above and parallel to the orbito-meatal (OM) plane, included the cerebellar hemispheres and the third slice (OM + 4 cm), the basal ganglia. After collection of transmission scan data via an external 68Ge source for subsequent correction of radiation attenuation effects, 1 mCi (37 MBq) of [76Br]BSP in isotonic acetate solution was injected in a brachial vein of the subject. For the initial kinetic distribution study, eight 30 to 60 min scans (accumulating a total of 300 000 counts typically for each brain level) were performed starting at the end of the injection and continuing for 26 h. For the subsequent brain distribution studies, two 30 min scans of each brain level were conducted 10 rain and 4.5 h after the injection of the radiopharmaceutical. Venous blood samples were collected 15 rain before the end of each scan and their radioactivity content assayed by means of a sodium iodide well counter.
2.2. Patients 2.4. Data analysis To determine the optimal time for quantitative uptake measurements a study of the variation with time of the regional distribution of [76Br]BSP in brain was performed on a 46-year-old healthy male volunteer. Brain biodistribution studies were subsequently performed on 12 control subjects and two schizophrenics under treatment with neuroleptics. The control subjects, 7 males and 5 females ranging from 39 to 87 years of age, were free from any known brain disease and were not taking medication suspected of interacting with dopamine receptors; the two schizophrenics, on the other hand, were stt~died 2 h after'intramuscular injection of a therapeutic dose (10 mg) of haloperidol. Informed consent was obtained from all patients.
2.3. P E T studies PET scans were performed by means of a timeof-flight tomograph (Soussaline et al., 1984) which
In the images with the highest total counts (that obtained 10 min postinjection for the slice at OM + 1 cm and that at 4.5 h post injection for the slice passing through the basal ganglia) the external border of the cerebellum and of the brain was taken empirically to be delineated by a contour representing, respectively, 60% and 30% of the maximum image intensity. With reference to a brain PET atlas (Mazziotta et al., 1981) a circular region of interest (ROI) was then placed visually over each cerebellar hemisphere (OM + 1 cm slice, ROI area = 28.6 cm 2), each striatal area (OM + 4 cm slice, ROI area = 3.7 cm 2) and over the frontal cortex (OM + 7 cm slice, ROI area = 7.1 cm2). Furthermore, a ROI was placed on the image displaying the basal ganglia structures (OM + 4 cm), corresponding to a contour equal to 80% of the maximum image intensity. When these structures were not visible (as with haloperidol-treated patients), the striatal ROIs placed on the images
269 from a control subject were copied on to those from the patient. The available software, which allowed automatic transfer of each ROI from one scan to another and correction for radioactive decay, produced values for a known time after injection of the fraction of the injected dose in each ROI. These data were used to calculate the frontal cortex-to-cerebellum radioactivity and the striatum-to-cerebellum radioactivity ratios for each study at each point in time.
3. Results
3.1. Radiopharmacetttical preparation Typically 5-6 mCi of 'no carrier added' [76Br]BSP were prepared under the above experimental conditions (radiochemical yield: 80%) with a specific activity varying from 0.5 to 2.0 Ci//~mol. Preliminary analytical and animal studies had shown that this radiopharmaceutical, prepared according to the protocol described, was sterile, pyrogen-free and free from any measurable chemical toxicity. The radionuclidic purity of [76Br]BSP was checked via 7-ray spectroscopy. At the time of injection the radioactive contaminants were 75Br and 77Br which amounted, respectively, to 0.5% and 1.6% of the total activity administered. Radiotoxicity was estimated by means of the results of a wholebody kinetic bio-distribution study performed on a baboon, showing that about 25% of the injected dose was concentrated in the liver and was eliminated with a biological half-life of 2.6 h. Using the classical model of the absorbed dose fractions (Smith et al., 1968) it was calculated that, for an i.v. injection of 1 mCi [76Br]BSP in man, the radiation dose absorbed by the liver and arising from positron and y-rays emissions and electron captures was 1.5 rad (1.5 cGy). The contribution of the radioactive contaminants [75Br] BSP and [77Br]BSP to this radiation dose was lower than 0.5%. Such a dose does not preclude PET studies in man.
3.2. Brain uptake kinetics Fig. 1 displays two tomographic images of the distribution of the radioactivity in brain slices at OM + 4 cm, taken at 10 min and 4.5 h after i.v. injection of 1 mCi [76Br]BSP in a healthy volunteer. As the preliminary baboon study led us to expect, the early image showed a perfusion-like distribution pattern with a higher activity in gray than in white matter. On the other hand, a clear delineation of the striatal structures was easily observed in the late image. At 4.5 h after the injection of 1 mCi [76Br]BSP about 350 000 counts were accumulated in 30 min in the section including the striatum and 220 000 and 260000 counts in 30 min in the sections displaying, respectively, the cerebellum and the frontal cortex. The root-mean-square percent uncertainty in the number of events in an average resolution cell (Budinger et al., 1978) was 6% for the striatum activity, and 9% and 10% respectively for the frontal cortex and cerebellum activities. W h a t e v e r form of ROI was used to determine the striatum activity, the radioactivity uptake data obtained did not differ significantly. The data (fig. 2) on radioactivity uptake per unit volume measured as a function of time in various brain structures during the kinetic distribution study show an accumulation which reached a maximum 3 h postinjection in the frontal cortex
Fig. 1. PET images of a human brain (OM + 4 cm) after i.v. injection of [76Br]BSP.Images obtained 10 to 25 min (A) and 4.5 to 5 h (B) after injection.
270
O
TABLE 1 Striatum-to-cerebellum (Str/Crb) and frontal cortex-to-cerebellum (Ctx/Crb) radioactivity concentration ratios in 13 control subjects and 2 haloperidol-treated shizophrenics, 4.5 h after the injection of 0.8 to 1.2 mCi [76Br]BSP (spec. act.: 0.5-2.0 Ci/t~ mol).
STRIATUM
3 T O~
_a
2
Subject Sex Age Str/Crb
a. ¢o
i
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Controls
x
1
-
o
;
WHOLE
A
BLOOD
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Fig. 2. Radioactivity uptake, expressed as a fraction of the dose injected (I.D.), measured in 1 cm3 of striatum (11), frontal cortex (0), cerebellum (e) and whole blood (A) as a function of time. Statistical uncertainties were calculated according to Budinger et al. (1978).
a n d c e r e b e l l u m regions a n d 4-5 h p o s t i n j e c t i o n in the b a s a l ganglia. T h e r e a f t e r the d e c a y - c o r r e c t e d activity in the s t r i a t u m r e m a i n e d essentially constant with time d u r i n g a p e r i o d of 25 h while the activity in the cortical region a n d in the c e r e b e l l u m d e c r e a s e d at a similar rate. T h e whole b l o o d radioactivity (corrected for decay) decreased d u r i n g the whole e x p e r i m e n t , b u t this decrease was slower t h a n in the cerebellum.
1 2 3 4 5 6 7 8 9 10 11 12 13 Treated 1 schizophrenics 2
M M M F M F M M M M F F F M M
39 46 58 59 59 63 67 72 72 76 79 81 87 49 33
2.10 2.23 1.78 1.85 1.96 1.63 1.75 1.85 2.03 1.73 1.45 1.72 1.80 1.24 1.21
Ctx/Crb 1.40 1.26 0.96 1.29 1.06 0.89 0.99 1.17 1.17 0.80 1.14 1.04 1.18 1.12 0.93
T h e m a x i m u m [76Br]BSP b r a i n uptake, which occurred 4-5 h after the a d m i n i s t r a t i o n of the r a d i o l i g a n d , was a b o u t 1.5% of the dose injected a n d the whole b l o o d c o n t r i b u t i o n to this q u a n t i t y was less than 1 × 1 0 - 2 % of the dose injected.
3.3. Brain activity distribution o <£
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FRONTAL
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T h e s t r i a t u m - t o - c e r e b e l l u m a n d cortex-to-cereb e l l u m r a d i o a c t i v i t y ratios m e a s u r e d in the g r o u p o f c o n t r o l subjects a n d in the two h a l o p e r i d o l t r e a t e d p a t i e n t s are listed on table 1. In the c o n t r o l g r o u p the m e a n value for the striatum-to-cerebell u m ratio at 4.5 h p o s t injection was 1.84 (S.D. + 0.21). In the two t r e a t e d schizophrenics, these ratios were only 1.21 a n d 1.24 (values statistically different from the control, P < 0.05).
<(
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4. Discussion
2b HOURS
Fig. 3. Stfiatum-to-ccrcbellum (O) and frontal cortex-to-cerebellum radioactivity concentration ratios (ll) as a function of
time. Root-mean-square uncertainties o n these ratios were calculated according to Budinger et al. (1978).
T o resolve in vivo specific a n d non-specific b i n d i n g of a d o p a m i n e r g i c radioligand, it is generally a s s u m e d that r a d i o a c t i v i t y in the c e r e b e l l u m m e a s u r e s non-specific b i n d i n g while the activity in
271 other brain structures reflects regional total binding (i.e. specific plus non-specific). Thus the specific binding for each brain structure can be expressed as the ratio of the concentration of the radioactivity for that structure to that of the cerebellum. Due to the steady washout of the non-specifically bound radioactivity (e.g. in the cerebellum), this ratio increases continuously for the striatum (fig. 3) and the highest value (4.8), corresponding to a specific binding of 75% of the total binding was observed 25 h post injection. However, due to the decay of 76Br, the statistical uncertainty of the measurement of this ratio was at a minimum 4-5 h after the administration of the radioligand, and this imaging time was thus chosen for subsequent brain distribution studies. A comparison of the' striatal (dopamine receptors) and frontal cortical (dopamine and serotonin receptors) radioactivity curves (fig. 3) suggest a significantly lower affinity of [76Br]BSP a n d / o r density of receptors for serotonin than for dopamine receptors. Partial volume effects are known to lower considerably the fraction of the true isotope concentration recovered in small structures (e.g. striatum) (Mazziotta et al., 1981). As these errors were not corrected for in our calculations, the real values of the striatum or frontal cortex-to-cerebellum activity ratios were notably underestimated. However, assuming that size and shape of striatum were roughly similar from subject to subject, the extent of this underestimation was essentially constant. Among the control group, a significant difference was observed (t-test, P < 0.05) between the mean values of the striatum-to-cerebellum activity ratios in the male (1.93) and female (1.69) subgroups. Due to differences in mean age and to the small number of subjects in each of these subgroups, it was not possible to establish whether this difference is, or is not due to different age-related specific binding decreases in the male and female subgroups (Wong et al., 1984). The low values for the ratios found in the two haloperidol-treated schizophrenics had been anticipated. There was a 70% decrease in specific binding in striatum (compared to that in controls) which is presumably due to partial occupation of
the dopamine receptors by the previously administrated haloperidol. From our PET radioactivity measurements, it can be calculated that i.v. injection of 1 mCi [76Br]BSP (spec. act. 1 C i / # m o l ) leads to a neuroleptic concentration of about 3 × 10-2 pmol per g striatum 4.5 h post administration. Human postmortem studies (Bokobza et al., 1984) have shown that the striatal dopamine receptor concentration, measured using [3H]spiperone, is about 6.0 pmol per g tissue. Since [3H]spiperone and [76Br]BSP have very similar in vitro specific binding characteristics for striatal membranes (Mazi6re et al., 1985) this indicates that, under our experimental conditions, only a small fraction (about 0.5%) of the striatal receptors was occupied by the radioligand and that non-specific binding of BSP was kept minimal. Dosimetric calculations have shown that [76Br]BSP can be used safely in man. However for clinical applications, 75Br, another positron emitter bromine isotope should be preferred over 76Br as label. Indeed as 75Br has more suitable emission characteristics and a shorter half-life (T1/2 = 1.6 h), the irradiation of the patient would be divided by a factor of 2. This study, which confirms the results of our previous animal experiments clearly indicates that radiolabeled bromospiperone is suitable for human pharmacological and pathological investigations of the central dopaminergic system.
Acknowledgements We wish to thank the SHFJ staff for its collaboration, N. De Blecker for typing the manuscript and Dr. B. Pate for language corrections.
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