ORIGINAL 
Annals of Nuclear Medicine Vol. 7, No. 1, 29-38, 1993 
Dopanrine D2 receptor SPECT imaging : Basic in vivo characteristics and clinical applications of 123I-IBZM in humans 
Hiroshi TOYAMA,*t Masanori ICHISE,* James R. BALLINGER,* Luis FORNAZZARI** and Joel C. KIRSH* 
* Division of Nuclear Medicine, Department of Radiological Sciences, Mount Sinai Hospital ** Clarke Institute of Psychiatry , University of Toronto Toronto Canada 
Present address : Department of Radiology, Fujita Health University, Toyoake, Japan 
The purposes of this study are to evaluate the utility of kit formulation, the basic in vivo characteristics, and clinical usefulness of dopamine D2 receptor imaging with 123I-(S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrodinyl)methyl]-benzamide (123I-IBZM). We studied 22 normal controls, 3 early symptomatic Huntington's disease patients, and I patient with visual hallucination on and off neuroleptics. 123I-IBZM could be conveniently prepared with a high degree of purity from a kit, but with relatively low radiochemical yield. We demonstrated 123I-IBZM receptor binding equilibrium by performing serial SPECT scanning in a normal volunteer. The basal ganglia/frontal cortex (BG/FC) ratios plateaued after the specific binding reached equilibrium approximately 60 minutes after injection. The BG/FC ratio declined significantly with age. The ratios for the Huntington's disease patients were significantly lower than those for normal controls. The images of the patient off neuroleptic therapy showed dramatically increased BG activity compared with those obtained while on therapy. The BG/FC ratio provides an estimate of Bmax/Kd and hence the receptor density. It appears important to perform SPECT early in the equilibrium phase and at a fixed time after injection to obtain significantly high signal to noise ratios. 123I-IBZM is an ideal tracer for SPECT including a rotating gamma camera type which can provide estimates of the receptor density objectively by calculating the BG/FC ratio, and is a promising agent for the investigation of dopamine D2 receptors in clinical conditions. 
Key words : single photon emission computed tomography (SPECT), 123I-(S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrodinyl)methyl]-benzamide (123I-IBZM), dopamine D2 receptor, Huntington's disease, equilibrium analysis 
INTRODUCTION 
RECENTLY, SEVERAL KINDS Of neuroreceptor imaging tracers for single photon emission computed tomography (SPECT) have been designed.1 A major advantage of functional brain imaging over postmortem studies is that it provides insight into receptor changes at various stages of illness as well as follow-up data on and pharmacological modification of such changes.2 Although the resolution and sensitivity of positron emission tomography (PET) are superior to those of SPECT,3 SPECT is much more widely available. 
There has been much clinical and pharmacological interest in the use of dopamine D2 agonists to treat Parkinson's disease and dopamine D2 antagonists as antipsychotic drugs. Reflecting this, several basic in vitro and in vivo imaging studies of dopamine D2 receptors employing neuroleptic ligands have been recently reported.4-12 123I-IBZM is a specific dopamine D2 receptor antagonist with high affinity.4,5 Using 123I-IBZM, studies of neuropsychiatric diseases such as Huntington's disease,8,13 Parkinson's disease,8,14 as well as patients on and off neuroleptic therapy7,8 have shown the potential for clinical usefulness. We had an opportunity to use a kit formulation for preparation of 123I-IBZM developed recently by Kung et al.15 from the University of Pennsylvania. 
The purposes of this study are to 1) assess the utility of this 123I-IBZM kit as a clinical method, 2) study the basic in vivo characteristics in order to determine optimal imaging parameters and establish a normal range, and 3) evaluate its potential clinical usefulness. 
MATERIALS AND METHODS 
1) Synthesis of 123I-IBZM 
123I-IBZM was synthesized by a kit procedure, which involves direct labeling of the non-iodinated precursor, BZM. 
A 10uL aliquot of BZM solution in ethanol (0.4 mg/mL), equivalent to 4 ug BZM, was mixed with 100 uL of 0.5 M ammonium acetate, pH 4.0, and added to a reaction tube containing 10-60 mCi (370-2,220 MBq) sodium 123I-iodide in 40-100 uL dilute sodium hydroxide. The reaction was started by the addition of 10uL freshly diluted 1.5% peracetic acid. After 10 minutes at room temperature, the reaction was quenched by the addition of 100 uL sodium metabisulfite (300 mg/mL) and the mixture was neutralized by the careful addition of 700 uL sodium bicarbonate (100 mg/mL). The product was taken up on a C-18 extraction cartridge (which had been activated with absolute ethanol followed by sterile distilled water), washed with 5 mL sterile distilled water, and eluted with 1-2.5 mL absolute ethanol. To make the product suitable for injection into patients, the ethanol solution was diluted with 9-18 mL saline (i.e. final ethanol concentration 10-15 % v/v) and sterilized by passage through a 0.22-uM membrane filter. Radiochemical purity was determined by chromatography using 8 mm x 70 mm strips cut from solvent saturation pads (Gelman Sciences Inc.) and developed in ethyl acetate ; hydrophilic impurities remained near the origin while 123I-IBZM migrated near the solvent front. 
2) Imaging protocols and data analyses 
We studied 22 normal control subjects (15 males, 7 females, age range: 20-76, mean=39.7+-14.7 yr), 3 early symptomatic Huntington's disease patients with no significant caudate atrophy as determined by X-ray CT (2 males,1 female, 51.0+-10.4 yr), and one 22-year-old male patient with visual hallucination secondary to alcohol abuse twice, on and off neuroleptics. All Huntington's disease patients took no medication for at least 4 weeks before this study. The patient with visual hallucination had no history of neuroleptic medication before his admission. 
After informed consent was obtained, a dose of 5 mCi (185 MBq) of 123I-IBZM was injected intravenously into each subject. In order to reduce the radiation dose to the thyroid gland, potassium iodide (Lugol's solution) was given prior to and for 3 days after the study. Immediately after injection, 1 minute/frame lateral dynamic planar images of the head were acquired for 60 minutes. For brain wash-out analyses, square regions of interest (ROI) (1.8 x 1.8 cm) were placed over the basal ganglia, frontal cortex and cerebellum respectively. SPECT was then carried out for 40 minutes. In one normal volunteer, beginning immediately after injection, 6 serial SPECT scans were obtained, each with a 30 minute acquisition. 
SPECT imaging was performed with a truncated single head rotating gamma camera interfaced to a dedicated computer (Elscint 409 ECT). Sixty 36 second images were obtained through a 360 circular rotation with a low energy, medium resolution and sensitivity collimator. The full width at half maximum of the system was 15 mm at clinically relevant count levels. The radius of rotation was 14 cm or less with the subject's head securely positioned in an adjustable head holder. The data were acquired onto a 64 x 64 matrix. One pixel-thick (approximately 4 mm) transaxial slices were reconstructed with a modified Hanning back projection filter parallel to the canthomeatal (CM) line as determined from a lateral scout image with radioactive markers identifying the external auditory meatus and the outer canthus of the eye. The first order Chang method of attenuation correction (0.12 cm-1) was employed. These transaxial images were then compressed into nine slices. One slice was chosen from these in which the basal ganglia were best visualized, and another in which the cerebellum was seen. ROI were drawn manually in both right and left basal ganglia (BG), frontal cortex (FC) bilaterally, and both cerebellar hemispheres (CE). These ROIs were approximately 6 cm3 for each BG, 22 cm3 for FC, and 30 cm3 for CE in volume, respectively. Mean counts per pixel were calculated in these regions. For semi-quantitative evaluation, mean basal ganglia/frontal cortex count ratios and mean basal ganglia/cerebellum count ratios were calculated. 
RESULTS 
1) Radiopharmaceutical Preparation 
In ten preparations, the radiochemical yield was 50+-6% (range 39 to 57%) and the radiochemical purity was 94+-3% (range 90 to 98%)¥ Total preparation time, including quality control, was approximately 1 hour. Retrospective sterility testing was negative. 
2) Imaging and Kinetic study in the brain 
Figure 1 shows a set of summed lateral dynamic planar images, and Fig. 2 shows 60 minute time activity curves for the basal ganglia, frontal cortex, and cerebellum of a normal volunteer. In the phase soon after injection, the accumulation of 123I-IBZM was seen in the whole brain as well as in basal ganglia. This brain uptake washed out gradually thereafter. However, the basal ganglia uptake remained relatively higher than uptake in the other regions. The basal ganglia activity reached its maximum about 15 minutes post injection. 
In another normal volunteer, beginning immediately after injection, six serial SPECT scans were obtained, each with a 30 minute acquisition. Figure 3 shows a set of 6 serial SPECT images, and Fig. 4 shows 180 minute time activity curves after decay correction for the basal ganglia, frontal cortex, and cerebellum. The wash-out from the cerebellum appeared slightly more marked than that from the frontal cortex. The specific binding of 123I-IBZM in the basal ganglia was calculated as the difference between the basal ganglia and frontal cortex. Equilibrium was seen from 60 to 120 minutes, indicating the binding reversibility of 123I-IBZM. Basal ganglia/frontal cortex ratios increased until 100 minutes, and then plateaued as the specific binding reached equilibrium (Fig. 5). In contrast, basal ganglia/cerebellum ratios increased until 180 minutes. 
According to the theoretical saturation analysis model16-18, the basal ganglia/frontal cortex ratio or (Bound+Free)/Free ratio appears to best provide a semiquantitative index for Bmax/Kd when measurements are made under conditions near equilibrium (See APPENDIX). 
3) Norma/ control value 
Two-five million counts/study were obtained. The mean basal ganglia/frontal cortex ratio for 22 normal controls was 1.58+-0.06 (range: 1.47-1.67). 
4) Effect of age 
There was an age dependency of 123I-IBZM uptake in the the basal ganglia. The basal ganglia/frontal cortex ratio declined significantly with increasing age (r=-0.58, p<0.01) (Fig. 6). 
5 Clinical applications 
a) Huntington's disease 
The basal ganglia/frontal cortex ratios of Huntington's disease patients (mean=51.0+-10.4 yr) were significantly lower than those of the 11 age matched normal controls (mean = 49.4+-ll.8 yr) (Fig. 7, Fig. 8). 
b) Patient on and off neuroleptics 
The patient with visual hallucination secondary to alcohol abuse on an amount of loxapine equivalent to 600 mg/day of chlorpromazine for 2 weeks showed decreased basal ganglia activity, but the uptake dramatically recovered when the patient had been off therapy for 4 weeks prior to re-imaging (Fig. 9). 
DISCUSSION 
Dopamine receptors are classified into D1 and D2 types according to their pharmacological and biochemical characteristics.19 Stimulation of dopamine D1 receptors increases adenylate cyclase while D2 receptor stimulation reduces it. Recently, following cloning of cDNA, two subtypes of dopamine D2 receptor (D2A and D2B20-23), D3,24, D4,25 and D526 receptors have been identified. The dopamine receptor is localized on pre- and postsynaptic nerve terminals. The presynaptic receptor is also called an autoreceptor, which has characteristics similar to those of a D2 receptor.27 The functional role of the D2 receptor is related to such behavior such as stereotypy, locomotion, vomiting, psychotic activity and normalization of motor activities.27 Because the D2 receptor density correlates with the pathophysiology and treatment status of movement disorders such as Parkinson's disease, and psychiatric disorder such as schizophrenia, the characterization of the D2 receptors has been the first step in basic and clinical investigations.20,23,27 
Wagner et al. reported for the first time the dopamine receptor imaging using 11C-labeled 3-N-methylspiperone by PET,28 and Crawley et al. showed SPECT images with 77Br-p-bromo-spiperone.29 Since spiperone and its derivatives have relatively low specificity (and also bind to serotonin receptors) and are slow to reach equilibrium, benzamide derivatives have been designed.16,28 11C-raclopride, which is one of the derivatives of benzamide, has a high specificity and affinity, and dissociates rapidly from the receptors, allowing binding equilibrium to be established in vivo within the time span of a PET experiment.16,17 
123I-IBZM is also a benzamide derivative, and a close analog of raclopride. In vivo binding studies with 123I-IBZM showed both high specificity and high affinity for dopamine D2 receptors. 4,5 An in vivo normal volunteer study with 123I-IBZM showed high uptake in the brain (3.72 %), a low radiation dose, and pharmacological safety.6 
123I-IBZM can be conveniently prepared with a kit procedure which does not require special equipment.15 The non-iodinated precursor BZM is labeled with 123I by peracetic acid oxidation, then the lipophilic products are separated on an extraction cartridge. This purification does not separate 123I-IBZM from the residual precursor BZM ; but the quantity of BZM is small, its binding affinity is much lower than IBZM, and the resultant effective specific activity (-500 Ci/mmol) is suitable for use in patients. The main disadvantage of the kit procedure is its relatively low radiochemical yield (-50 %) which makes inefficient use of expensive 123I. 
According to our kinetic analyses with 6 serial SPECT scans over 180 minutes, the specific binding in the basal ganglia reached equilibrium, and washed out thereafter. These findings are consistent with the characteristics of benzamide derivatives which dissociate rapidly from the receptors.8,9,16,17 There was a tendency towards higher wash-out from the cerebellum than the frontal cortex. For reference regions to evaluate free and nonspecific binding, cerebellum in PET with 11C-raclopride,16 and frontal cortex in SPECT with 123I-IBZM7-9 have been chosen. Seeman et al. pointed out that the baseline amount of nonspecific binding of radioactive raclopride to the human striatum is considerably higher than that to the cerebellum from the same human brain.30 For this reason, Brucke et al. reported that the cerebellum might not be an ideal region for reference, because its nonspecific binding might be lower than that in the striatum.8 Our preliminary data from one subject could suggest their hypo-thesis. Dynamic SPECT analyses of several subjects might be helpful in this regard. 
The equilibrium basal ganglia/frontal cortex ratio provides an estimate of Bmax/Kd (Eq. A7) and hence the receptor density, provided that Kd is constant (Eq. A8). Measurements of affinity (Kd) are relatively constant in the rat,31,32 and no appreciable changes in Kd in postmortem humans whether with age or diseased states such as Parkinson's disease, have been reported.33-35 Therefore, reductions in the basal ganglia/frontal cortex ratio will reflect the decline in receptor density (Bmax). This may be due either to degenerative loss of receptors or as a result of D2 receptor down-regulation. Similarly, rises in the basal ganglia/frontal cortex ratio will reflect the increase in Bmax as a result of D2 receptor upregulation secondary to deafferentation.36 According to the equation (Eq.A7), the basal ganglia/frontal cortex ratio will become constant after equilibrium has been reached. This is in keeping with our results where the ratio plateaued after specific binding equilibrium was reached. A similar result has been reported with 11C-raclopride PET,36 but conflicting results have also been reported with 123I-IBZM SPECT.7,9 Although theoretically this ratio should remain constant under conditions after equilibrium, actual regional counts wash out with time. Therefore, measurements made in the later phase may be more subject to statistical error because of the decreased signal to noise ratio. It is, therefore, important to perform SPECT imaging early in the equilibrium phase to obtain sufficient counts. In addition, although imaging at a fixed time after injection may not be required for equilibrium analysis, it may reduce statistical error in comparisons among subjects. Based on our serial SPECT data for one subject and previously reported papers,8,9 the ideal data collection time is estimated to be between 60 and 120 minutes. The dopamine receptor density can be evaluated objectively with the ratio based on the data of a single SPECT scan of the rotating gamma camera type. Once the imaging timing has been established, serial or continuous dynamic SPECT imaging is no longer required. In terms of a mean basal ganglia/frontal cortex ratio in normal controls, there are slight differences between our data (1.58+-0.06) and those of other investigators {1.74+-0.11,^8 1.55+-0.05^14} . This may be due to the differences in the SPECT acquisition timing, choice of reconstruction filter, attenuation correction method, size of ROI used, and SPECT system resolution.14 Kung et al. reported that one major difference between the no-carrier-added preparation and the kit formulation method is the presence of a small amount of uniodinated BZM in the latter preparation. Because the difference between IBZM and BZM in binding affinity for D2 receptors is high, the small amount of uniodinated BZM together with the radioactive IBZM will not affect the binding or imaging studies of the labeled IBZM.12 Actually, regional brain ratios in rats were similar values to the no-carrier-added preparation and the kit formulation method.12 The basal ganglia/cerebellum ratio of in vitro experiments of a monkey was 4.93,4 and the ratio of in vitro autoradiography of mice was 7.3.5 The main reason for this underestimation of this ratio by means of the SPECT technique is likely to be related to a partial volume effect.1 In order to reduce statistical sampling error, the smallest volume of ROI in the present study was approximately 6 cm3 which is roughly 2 x (FWHM)3 of the system used. As the value for Kd increases, the ratio is expected to decrease. However, with PET and 11C-raclopride with a larger value for Kd than that for IBZM, the mean basal ganglia/cerebellum ratio for the normal control was found to be 3.7,36 which is considerably higher than that for 123I-IBZM SPECT. This can also be explained in terms of the better spatial resolution of the PET system. 
It has been observed that endogenous dopamine lowers the apparent density of D2 receptors detected by the derivatives of benzamide {raclopride,30,37,38 IBZM39}. Seeman et al. reported that the basis for this non competitive behavior between dopamine and raclopride may be related to dopamine's high affinity for the high-affinity state of D2 receptors. It might be necessary to consider the effect of endogenous dopamine upon the D2 receptor density measurements with 123I-IBZM. Premedication with reserpine to reduce endogenous dopamine might be useful.30,39 On the other hand, in vivo imaging of D2: receptor with benzamide derivatives might be useful in estimating the synaptic levels of endogenous dopamine.39 Further studies appear to be needed. 
The basal ganglia/frontal cortex ratio in this study showed an age dependency as shown by PET2,40,41 as well as SPECT.8 In vitro animal studies also showed a decrease in dopamine receptors with aging.31,42 Although decreased dopamine D2 receptor binding with age in the caudate nucleus has been shown, no such age changes were detected in the putamen in postmortem studies.33,34 Unfortunately it appears difficult to distinguish the caudate nucleus from putamen accurately by means of SPECT, or even PET.3 It is suggested that age reduces the functional reserve and adaptive capacity of dopaminergic mechanisms in the basal ganglia.33 Age matched control subjects must be chosen for comparison with each patient by means of the same equipment.1,2,40 
The receptor density in early symptomatic Huntington's disease patients showed a significant decrease, presumably because of degenerative loss of postsynaptic D2 receptors. Our data are consistent with those obtained in PET43 and SPECT8 studies. The dopamine D2 receptor imaging might be promising for early and preclinical detection of Huntington's disease.13 
The SPECT images of the patient with visual hallucination secondary to alcohol abuse on neuroleptic therapy were significantly different from those obtained when off therapy. These findings show the importance of the timing of imaging to evaluate the receptor status accurately after withdrawal from neuroleptic therapy.1,7,8 
APPENDIX 
From the hyperbolic function equation (16-18), 
(Eq. A1) 
where Bmax is the maximal number of receptor sites that specifically bind the ligand, Kd {equilibrium dissociation constant; the dissociation rate constant (koff)/the association rate constant (kon)} is the ligand concentration at which ligand binding to 50% of the receptors occurs,17,18 
Multipling through by (Kd +free) produces, 
(Eq. A2) 
(Eq. A3) 
(Eq. A4) 
(Eq. A5) 
A dose of 5 mCi (185 MBq) 123I-IBZM at no-carrier added level =20 pmol IBZM 
If the brain uptake is 5 % of the dose, 1 pmol+-IBZM is in the brain. Bmax is -15 pmol/ml. Because the total volume of basal ganglia is assumed to be 20 ml, the total D2 receptor number in the brain is 300 mol.6 
.'. The entire dose on receptors would occupy 1/300 or < 1% of the receptors, which is negligible and not significantly affected by the dose. Even if we take into account the presence of residual BZM with its 50-fold lower affinity, receptor occupancy is only 3%
From (Eq. A5), Bound/Bmax is fraction saturated.
 .'. at low saturation, 
(Eq. A6) 
The activity of basal ganglia (BG) means the (Bound +free), and the uptake of frontal cortex (FC) means the free radioligand. 
(Eq. A7) 
Rearrange 
(Eq. A8) 
ACKNOWLEDGMENTS 
The authors gratefully acknowledge Dr. Hank F. Kung, Department of Radiology, University of Pennsylvania for valuable cooperation in providing us with the precursor BZM, Nordion International Inc., Vancouver for providing us with part of the sodium 123I-iodide, and Douglas C. Vines, B.A.Sc., R.T.N.M. for technical support. 
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