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It is well known that overproduction and accumulation of beta-amyloid (Abeta) plaques in the brain is a key event in the pathogenesis of Alzheimer's disease (AD). Previously it was demonstrated that [125I]TZDM, 2-(4'-dimethylaminophenyl)-6-iodobenzothiazole, a thioflavin derivative, was an effective ligand with good in vitro and in vivo binding characteristics. To further improve the initial uptake and washout rate from the brain, important properties for in vivo imaging agents, a novel radioiodinated ligand, 2-(4'-dimethylaminophenyl)-6-iodobenzoxazole ([125I]IBOX, 3), for detecting Abeta plaques in the brain, was synthesized and evaluated. The new iodinated ligand, IBOX, is based on an isosteric replacement of a sulfur atom of TZDM by an oxygen, by which the molecular weight is reduced while the lipophilicity of the iodinated ligand is increased. Partition coefficients (P.C.) of these two ligands were 70 and 124 for TZDM and IBOX, respectively. In vitro binding study indicated that the isosteric displacement yielded a new ligand with equal binding potency to Abeta(1-40) aggregates (K(i) = 1.9 and 0.8 nM for TZDM and IBOX, respectively). Autoradiography of postmortem brain sections of a confirmed AD patient by [125I]IBOX showed excellent labeling of plaques similar to that observed with [125I]TZDM. More importantly, in vivo biodistribution of [125I]IBOX in normal mice displayed superior peak brain uptake (2.08% at 30 min vs 1.57% at 60 min dose/brain for [125I]IBOX and [125I]TZDM, respectively). In addition, the washout from the brain was much faster for [125I]IBOX as compared to [125I]TZDM. Based on the data presented for [125I]IBOX, it is predicted that the brain trapping of this new radioiodinated ligand in the Abeta containing regions will be more favorable than that of the parent compound, [125I]TZDM. Further evaluation of [125I]IBOX is warranted to confirm the Abeta plaque labeling properties in vivo.

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UNLABELLED: We reported recently a highly selective radioligand, 2-([2-([dimethylamino]methyl)phenyl]thio)-5-[(123)I]iodophenylamine (ADAM), for SPECT imaging of serotonin transporters (SERT). In this article we describe the kinetic modeling of [(123)I]ADAM and its ability to quantitatively and reproducibly measure the concentrations of SERT in the nonhuman primate brain. We also investigate simplified models of tracer behavior that do not require invasive arterial blood sampling. METHODS: Three female baboons each underwent 3 [(123)I]ADAM SPECT studies. The studies consisted of a dynamic sequence of seventy-two 5-min scans after injection of 330 +/- 50 MBq (mean +/- SD) [(123)I]ADAM. Rapid arterial blood samples were obtained and corrected for the presence of labeled metabolites. Dynamic imaging and metabolite-corrected plasma data were analyzed using graphic analysis to give the distribution volumes (DVs) of different brain regions. DV ratios (DVRs) of target to cerebellum were derived and compared against a kinetic reference tissue model and simple target-to-background ratio. RESULTS: Averaged over all 9 scans, the mean DV in the midbrain was 4.86 +/- 1.06 mL/mL and the mean DV in the cerebellum was 2.25 +/- 0.48 mL/mL. The mean test-retest repeatability of the midbrain DV was 14.5%. The reference tissue model gave a mean midbrain DVR of 2.01 +/- 0.17 and correlated strongly with the DVR calculated from the full kinetic model (correlation coefficient [R(2)] = 0.94; P < 0.001), but with much improved repeatability (test-retest, 5.4%; intersubject variability, 5.2%). Similarly, the simple ratio method gave strong correlations with the full kinetic model (R(2) = 0.89; P < 0.001) and a test-retest of 7.6%. CONCLUSION: Accurate, repeatable quantification of SERT in the nonhuman primate brain is possible using kinetic modeling of dynamic [(123)I]ADAM SPECT scans. Simplified models, which do not require arterial blood sampling, gave accurate results that correlated strongly with the full kinetic model. The test-retest reliability of the simplified reference region models was excellent. Quantification of SERT is possible using full kinetic modeling and also with simpler reference region methods.

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A novel in vivo imaging agent, 99mTc labeled [(N-[2-((3'-N'-propyl-[3,3,1]aza-bicyclononan-3alpha-yl)(2"-methoxy-5-methyl-phenylcarbamate)(2-mercaptoethyl)amino)acetyl]-2-aminoethanethiolato] technetium(V) oxide), [99mTc]2, displaying specific binding towards sigma-2 receptors was prepared and characterized. In vitro binding assays showed that the rhenium surrogate of [99mTc]2, Re-2, displayed excellent binding affinity and selectivity towards sigma-2 receptors (K(i) = 2,723 and 22 nM for sigma-1 and sigma-2 receptor, respectively). Preparation of [99mTc]2 was achieved by heating the S-protected starting material, 1, in the presence of acid, reducing agent (stannous glucoheptonate) and sodium [99mTc]pertechnetate. The lipophilic racemic mixture was successfully prepared in 10 to 50% yield and the radiochemical purity was >98%. Separation of the isomers, peak A and peak B, was successfully achieved by using a chiralpak AD column eluted with an isocratic solvent (n-hexane/isopropanol; 3:1; v/v). The peak A and peak B appear to co-elute with the isomers of the surrogate, Re-2, under the same HPLC condition. Biodistribution studies in tumor bearing mice (mouse mammary adenocarcinoma, cell line 66, which is known to over-express sigma-2 receptors) showed that the racemic [99mTc]2 localized in the tumor. Uptake in the tumor was 2.11, 1.30 and 1.11 %dose/gram at 1, 4 and 8 hr post iv injection, respectively, suggesting good uptake and retention in the tumor cells. The tumor uptake was significantly, but incompletely, blocked (about 25-30% blockage) by co-injection of "cold" (+)pentazocine or haloperidol (1 mg/Kg). A majority of the radioactivity localized in the tumor tissue was extractable (>60%), and the HPLC analysis showed that it is the original compound, racemic [99mTc]2 (>98% pure). The distribution of the purified peak A and peak B was determined in the same tumor bearing mice at 4 hr post iv injection. The tumor uptake was similar for both isomers, but the blood and peripheral tissue content for the isomer in peak B was higher than that for the isomer in peak A. It is evident that the isomer in peak A displayed significantly better tumor/blood and tumor/muscle ratios. The higher rate of in vivo metabolism was also confirmed by the higher thyroid uptake values for the isomer in peak B as compared to peak A. In summary, a 99mTc-labeled sigma receptor imaging agent, [99mTc]2, has demonstrated the feasibility of using a 99mTc-labeled agent for imaging sigma receptor expression in tumor cells. This is the first time a subtype-selective 99mTc-labeled agent for imaging sigma receptor sites is reported.

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We report for the first time that small molecule-based radiodiodinated ligands, showing selective binding to Abeta aggregates, cross the intact blood-brain barrier by simple diffusion. Four novel ligands showing preferential labeling of amyloid aggregates of Abeta(1-40) and Abeta(1-42) peptides, commonly associated with plaques in the brain of people with Alzheimer's disease (AD), were developed. Two 125I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene, 13 (IMSB), and two 125I-labeled thioflavins, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM), and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands showed excellent binding affinities with Kd values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates of Abeta(1-40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Abeta(1-42), respectively. Interestingly, under a competitive-binding assaying condition, different binding sites on Abeta(1-40) and Abeta(1-42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down's syndrome known to contain primarily Abeta(1-42) aggregates in the brain showed that both [(125)I]18a and [125I]18b labeled these brain sections, but [125I]13, selective for Abeta(1-40) aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies in normal mice after an iv injection showed that [125I]18a and [(125)I]18b exhibited excellent brain uptake and retention, the levels of which were much higher than those of [125I]12 and [125I]13. These findings strongly suggest that the new radioiodinated ligands, [125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), may be useful as biomarkers for studying Abeta(1-40) as well as Abeta(1-42) aggregates of amyloidogenesis in AD patients.

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In developing probes for detecting beta-amyloid (Abeta) plaques in the brain of Alzheimer's disease (AD), we have synthesized 1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (5, BSB). Due to the presence of two double bonds, formation of four different isomers is possible. Four isomers, E,E-5, E,Z-5, Z,E-5, and Z,Z-5, were prepared. Surprisingly, all showed strong fluorescent labeling of Abeta plaques in the brain of postmortem brain sections of patients with confirmed AD. In vitro binding assay also showed that all four isomers of BSB (E,E-5, E,Z-5, Z,E-5, and Z,Z-5) displayed a similar high binding affinity inhibiting the binding of [(125)I]E,E-6, 1-iodo-2,5-bis-(3-hydroxycarbonyl-4-methoxy)styrylbenzene (IMSB) to Abeta(1-40) aggregates. The inhibition constants (K(i)) of E,E-5, E,Z-5, Z,E-5, and Z,Z-5 were 0.11 +/- 0.01, 0.19 +/- 0.03, 0.27 +/- 0.06, and 0.13 +/- 0.02 nM, respectively. Due to the fact that geometric stability of these styrylbenzenes is unknown, and the conversion of Z,Z-5 to E,E-5 may occur automatically in the binding or labeling assaying conditions, we have investigated the kinetics of conversion of Z,Z-5 to E,E-5 by NMR in D(2)O/NaOD at elevated temperatures (70, 95, and 115 degrees C). The activation energy was determined to be 14.15 kcal/mol. The results strongly suggest that the isomeric conversion at room temperature in aqueous buffer solution is unlikely. All of the styrylbenzene isomers clearly showed potential as useful tools for studying Abeta aggregates in the brain. The data suggest that, despite the rigidity of this series of styrylbenzenes, the binding sites on Abeta aggregates may have certain flexibility and the binding pockets could be adaptable for binding to other smaller ligands. Such information could be exploited to develop new ligands for detecting amyloid plaques in AD.

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Quantification of dopamine transporters (DAT) using [99mTc]TRODAT-1 and single-photon emission tomography (SPET) requires full kinetic modeling of the data, using complex and invasive arterial blood sampling to provide an input function to the model. We have shown previously that a simpler reference tissue model provides accurate quantitative results, using a reference region devoid of DAT as the input to the model and thereby obviating the need for blood sampling. We now extend this work into humans, and develop further simplifications to make the imaging protocol much more practical as a routine procedure. Fourteen healthy subjects (age 29.8 +/- 8.4 years, range 18.7-45.5 years) underwent dynamic SPET for 6 h following injection of 752 +/- 28 MBq [99mTc]TRODAT-1. The kinetic data were analyzed using nonlinear regression analysis (NLRA) and Logan-Patlak graphical analysis. In addition, simple average ratios of striatal-to-background counts were obtained for three 1-h periods (3-4 h, 4-5 h, 5-6 h), and compared against the kinetic models. All methods gave an index of specific binding, proportional to the binding potential, known as the distribution volume ratio (DVR). The reference tissue NLRA gave mean values of k3=0.013 +/- 0.003 min(-1), k4=0.011 +/- 0.002 min(-1), and DVR=2.29 +/- 0.17. Graphical analysis gave a value of DVR=2.28 +/- 0. 16, and the three ratio values of DVR were: 3-4 h, 2.18 +/- 0.15; 4-5 h, 2.34 +/- 0.13; and 5-6 h, 2.46 +/- 0.19. Graphical analysis was highly correlated with NLRA (R2=0.91, slope=0.90 +/- 0.08). The ratio methods correlated well with NLRA (3-4 h, R2=0.71, slope= 0.73 +/- 0.13; 4-5 h, R2=0.86, slope=0.73 +/- 0.09; 5-6 h, R2=0.80, slope=1.00 +/- 0.15), and also with graphical analysis (3-4 h, R2=0.65, slope=0.74 +/- 0.16; 4-5 h, R2=0.85, slope=0.78 +/- 0.09; 5-6 h, R2=0.88, slope=1.11 +/- 0.12). The optimum equilibrium time point for obtaining a simple ratio was approximately 4.5-5.5 h. In conclusion, the simple ratio techniques for obtaining a quantitative measure of specific binding correlated well with the reference tissue kinetic models, using both NLRA and graphical analysis. The optimum time for obtaining a ratio appeared to be in the range 4.5-5.5 h. Earlier time points, while still relatively accurate, had a lower sensitivity and may not be optimized for measuring small changes in DAT concentrations.

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UNLABELLED: [99mTc]TRODAT-1 is a radiolabeled tropane that binds dopamine transporters. The primary goal of this study was to determine whether its regional cerebral distribution could differentiate between patients with Parkinson's disease and healthy human volunteers. METHODS: The sample consisted of 42 patients with Parkinson's disease, 23 age-matched controls, and 38 healthy adults younger than 40 y old. SPECT scans of the brain were acquired on a triple-head gamma camera 3-4 h after the intravenous injection of 740 MBq (20 mCi) [99mTc]TRODAT-1. Mean counts per pixel were measured manually in subregions of the basal ganglia and normalized to the mean background counts to give specific uptake values ([SUVs] approximately k3/k4). Patient and control groups were also compared with automated statistical parametric mapping techniques. Logistic discriminant analyses were performed to determine the optimum uptake values for differentiating patients from age-matched controls. RESULTS: Quantitative image analysis showed that the group mean SUVs in patients were less than the mean values in controls for all regions (all Ps < 0.000001). There was overlap in the caudate as well as in the anterior-most portion of the putamen, but not in the posterior putamen, even when the asymptomatic sides of 5 patients with clinically defined hemi-Parkinson's disease were factored in. CONCLUSION: The findings indicate that Parkinson's disease can be detected with [99mTc]TRODAT by simply inspecting the images for uptake in the posterior putamen. Appropriate asymmetries seem to be visible with quantification in patients with clinically defined hemi-Parkinson's disease, even though changes in the putamen contralateral to the clinically unaffected side in these patients appear to precede the development of symptoms.

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We have described previously a selective serotonin transporter (SERT) radioligand, [(123)I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([(123)I]ODAM). The imaging characteristics of this radioligand were studied and compared against [(123)I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [(123)I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [(123)I]ODAM. Total integrated brain uptake of [(123)I]ODAM was about 30% higher than [(123)I]IDAM. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT. Peak specific binding in the midbrain occurred 120 min after injection, with an equilibrium midbrain to cerebellar ratio of 1. 50+/-0.08, which was slightly lower than the value for [(123)I]IDAM (1.80+/- 0.13). Both the binding kinetics and the metabolism of [(123)I]ODAM were slower than those of [(123)I]IDAM. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited washout from areas with high concentrations of SERT, with a dissociation kinetic rate constant k(off)=0.0085+/-0.0028 min(-1) in the midbrain. Similar studies using nisoxetine and methylphenidate showed no displacement, consistent with its low binding affinity to norepinephrine and dopamine transporters, respectively. These results suggest that [(123)I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [(123)I]IDAM, but also a slightly lower selectivity for SERT.

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UNLABELLED: 99mTc-TRODAT-1 is a new radiopharmaceutical that selectively binds the dopamine transporters. This study characterized the effects of aging on its regional cerebral distribution in healthy human volunteers. METHODS: The sample consisted of 27 men and 28 women with a mean age of 41.1 +/- 17.1 y (age range 18.7-73.8 y). Dynamic SPECT scans of the brain were obtained with a standardized acquisition and processing protocol on a triple-head camera. Mean counts per pixel were measured in multiple regions of interest within each basal ganglia. Regression analyses were used to relate the specific uptake values at 3-4 h after administration to age. Both linear and nonlinear models of aging were tested. RESULTS: The relative concentration of radioactivity in most subregions of the basal ganglia decreased significantly with age (all P values < 0.0001). Nonlinear models of aging fit the data significantly better than a straight line. The rate of decline was significantly faster in young adults than in older volunteers (P < 0.001). The break-point age at which the rate of change slowed down and became more stable was 36 y old for the whole striatum and ranged from 32 to 44 y old depending on subregion. CONCLUSION: The effects of aging on central nervous system dopamine transporters do not appear to be linear. Most effects seem to occur during young adulthood before people reach their 40s. The distribution then appears to remain relatively stable until late in life. The findings suggest that the adult life cycle is better characterized as a series of phases than as a continuum.

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A new radioligand, 5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([(123)I]IDAM), has been developed for selective single-photon emission tomography (SPET) imaging of SERT. In vitro binding studies suggest a high selectivity of IDAM for SERT (K(i)=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET K(i)= 234 nM and DAT K(i)>10 microM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [(123)I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [(123)I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90-120 min after injection of [(123)I]IDAM. Similar studies were performed using a NET inhibitor, nisoxetine, and a DAT blocker, methylphenidate. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT, with the highest uptake in the midbrain area (hypothalamus, raphe nucleus, substantia nigra), and the lowest uptake in the cerebellum (an area presumed free of SERT). Peak specific binding in the midbrain occurred at 120 min, with a ratio to the cerebellum of 1.80+/-0.13. At 30 min, 85% of the radioactivity in the blood was metabolite. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited rapid washout from areas with high concentrations of SERT (dissociation rate constant in the midbrain, averaged over three baboons, k(off)=0. 025+/-0.002 min(-1)), while the cerebellar activity distribution was undisturbed (washout rate 0.0059+/- 0.0003 min(-1)). Calculation of tracer washout rate pixel-by-pixel enabled the generation of parametric images of the dissociation rate constant. Similar studies using nisoxetine and methylphenidate had no effect on the distribution of [(123)I]IDAM in the brain. These results suggest that [(123)I]IDAM is suitable for selective SPET imaging of SERT in the primate brain, with high contrast, favorable kinetics, and negligible binding to either NET or DAT.

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Accurate quantification of neuroreceptors requires full kinetic modeling of the dynamic single-photon emission tomography (SPET) or positron emission tomography (PET) images, with highly invasive arterial blood sampling. This study investigated the application of a reference region kinetic model to the dynamics of [99mTc]TRODAT-1 in nonhuman primates, obviating the need for blood sampling. A series of dynamic SPET scans were performed on two baboons following the injection of approximately 700 MBq of [99mTc]TRODAT-1. Rapid arterial blood samples were taken automatically during scanning. Reconstructed SPET images were coregistered with magnetic resonance imaging (MRI) scans of the baboons, and regions of interest (ROIs) placed on the striatum, cerebellum and cerebral hemispheres. The ROI data were combined with metabolite-corrected blood data, and fitted to a three-compartment kinetic model using nonlinear least squares techniques. The same data were also used in a simplified reference region model, in which the input function was derived from the nondisplaceable tissue compartment. In addition, semiquantitative blinded analysis was performed by three raters to determine the point of transient equilibrium in the specific binding curves. All methods generated values for the ratio of the kinetic rate constants k3/k4, which gives an estimate of the binding potential, BP. These were compared with the full kinetic model. The mean values of k3/k4 for the three different analysis techniques for each baboon were: 1.17 +/- 0.21 and 1.12 +/- 0.13 (full kinetic model), 0.93 +/- 0.13 and 0.90 +/- 0.07 (reference region model), and 0.97 +/- 0.18 and 0.92 +/- 0.08 (equilibrium method). The reference region method gave significantly lower results than the full kinetic model (P = 0.01), but it also produced a much smaller spread and better quality fits to the kinetic data. The reference region model results for k3/k4 correlated very strongly with the full kinetic analysis (r2 = 0.992, P < 0.001), and with the equilibrium model (r2 = 0.88, P = 0.002). The subjectivity inherent in the equilibrium method produces inferior results compared with both kinetic analyses. It is suggested that the self-consistency of the reference region model, which requires no arterial blood sampling, provides a more precise and reliable estimate of the binding of [99mTc]TRODAT-1 to dopamine transporters than full kinetic modeling. The reference region method is also better suited to a routine clinical environment, and would be able to distinguish smaller differences in dopaminergic function between patient groups.

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Recently, [Tc-99m]TRODAT-1, the first Tc-99m-labeled tracer for imaging CNS dopamine transporters in humans, was reported. This tracer displayed excellent specific binding to dopamine transporters in the basal ganglia region of the brain, thus it is potentially useful for the diagnosis of deficit of dopamine transporters in neurodegenerative diseases, such as Parkinson's disease. Preparation of [Tc-99m]TRODAT-1 was previously achieved by a multistep kit formulation. It is highly desirable to further improve the preparation by developing a simplified one-vial formulation with a reduced amount of TRODAT-1 ligand for routine clinical use. To achieve this goal, a series of studies to optimize labeling efficiency by varying a combination of factors (amount of free ligand, reaction reagents, and reaction pH) was carried out. [Tc-99m]TRODAT-1 prepared by this new kit formulation was evaluated by assessing the brain uptake and target (striatum) versus nontarget (cerebellum) ratios in rats. Appropriate amounts of various ingredients for a one-vial kit formulation providing > or =90% radiolabeling yields were identified. The most consistent and reliable formulation contained 10 microg of TRODAT-1 (a reduction of free ligand from 200 microg to 10 microg), 32 microg of SnCl2, 10 mg of sodium glucoheptonate, and 840 microg of disodium EDTA in one vial as a lyophilized kit. It is feasible to reconstitute the vial with [Tc-99m]pertechnetate (0.5-2 mL, < or =1110 MBq, 30 mCi), resulting in a final solution with a pH value of 4.5-5.0. [Tc-99m]TRODAT-1, prepared by this new kit, was stable at room temperature for 6 h. Biodistribution studies of this agent in rats with the new formulation showed similar regional brain distribution as compared with those obtained with the previous preparation (high striatum-to-cerebellum ratio). In conclusion, using this lyophilized one-vial kit formulation, [Tc-99m]TRODAT-1 can be prepared with greater than 90% radiochemical purity. This simplified kit will significantly improve the reliability of preparation of this agent for routine clinical use.

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Technetium-99m-labeled radiopharmaceuticals are currently the most commonly used agents in nuclear medicine. To prepare binding site-specific small molecules containing a Tc-99m complexing core, it is important to consider a ligand system, which selectively forms only one stereoisomer. A novel series of bisaminoethanethiol (BAT) derivatives as a model system were prepared. Stereoisomers of N-benzyl-3,4-di(N-2-mercaptoethyl)-amino pyrrolidines (P-BAT): (3R,4R)-P-BAT (R,R-4) and (3,4)meso-P-BAT (8), the trans and meso isomer, respectively, as a chelating group were prepared successfully. The desired Tc-99m P-BAT complexes were obtained by using Sn(II)/glucoheptonate as the reducing agent for [99mTc]pertechnetate. As predicted, after complexation with [99mTc]Tc'O, the trans isomer, (3R,4R)-P-BAT (R,R-4), showed only one isomer; whereas the corresponding meso isomer, (3,4)meso-P-BAT (8), produced two distinctive complexes isolated readily by high performance liquid chromatography (HPLC). The [99mTc](R,S) meso-P-BAT (8) isomers showed a different lipophilicity (partition coefficient [P.C.] = 54.3 and 55.4 for peak A and peak B, respectively), as compared with that of the corresponding [99mTc](3R,4R)-P-BAT (R,R-4), trans isomer (P.C. = 163). Results of the biodistribution study in rats of these isomers show different heart and brain uptake, suggesting that the intrinsic differences in biodistribution are due to structural and stereospecific factors. Examples in this report confirm that it is possible to design stereospecific Tc-99m complexes based on the bisaminoethanethiol (N2S2, BAT) ligand system. Consideration on stereoselectivity of site-specific agents labeled with Tc-99m is likely an essential requirement on developing binding-site specific radiopharmaceuticals.

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UNLABELLED: The central nervous system dopamine transporters (DATs) and dopamine D2/D3 receptors are implicated in a variety of neurological disorders. Both sites are also targets for drug treatment. With the successful development of [99mTc]TRODAT-1, single-isotope imaging studies using this ligand for DAT imaging can be complemented by additional use of 123I-labeled D2/D3 receptor ligand co-injected to assess both pre- and postsynaptic sites of the dopaminergic system simultaneously. METHODS: Twelve SPECT scans of the brain were obtained in two baboons after intravenous administration of 740 MBq (20 mCi) [99mTc]-TRODAT-1 (technetium, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3,2,1]oct-2-yl]methyl ](2-mercaptoethyl) amino]ethyl]-amino]ethanethiolato (3-)]- oxo-[1R-(exo-exo)]) and 185 MBq (5 mCi) [123I]iodobenzamide or [123I]iodobenzofuran. SPECT data were acquired by a triple-head gamma camera equipped with ultra-high-resolution fanbeam collimators (scan duration = 210 min). Two sets of SPECT data were obtained using energy windows of 15% centered on 140 keV for 99mTc and 10% asymmetric with a lower bound at 159 keV for 123I. After coregistration with MRI, region-of-interest analysis was performed using predefined templates from coregistered MRI. In blocking studies, baboons were pretreated with N-methyl-2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT, 14 mg) or raclopride (14 mg) to block DAT or D2/D3 binding site, respectively. RESULTS: Image quality of dual-isotope studies was similar to that obtained from single-isotope studies. When one site was blocked with CFT or raclopride, the binding of the respective ligand to the other site was not affected. CONCLUSION: This is the first example that clearly demonstrates the feasibility of simultaneous imaging of both pre- and postsynaptic sites of the dopaminergic system in baboons with dual-isotope SPECT studies. With or without corrections for cross-contamination of 123I into the 99mTc window, striatum-to-cerebellum ratios (target-to-nontarget) of dual-isotope experiments did not differ significantly from single-isotope experiments. This method may be a valuable and cost-effective tool for gaining comprehensive information about the dopaminergic system in one SPECT imaging session.

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[99mTc]TRODAT-1 was the first 99mTc-labeled imaging agent to show specific binding to dopamine transporters (DAT) in the striatum (STR) of human brain. Additionally, in vitro binding and autoradiographic experiments demonstrated that this tracer also binds to serotonin transporters (SERT) in the midbrain/hypothalamus (MB) area. In this study, [99mTc]TRODAT-1 was investigated as a potentially useful ligand to image SERT in the MB of living brain. A total of eight single-photon emission tomography (SPET) scans were performed in two baboons (Papio anubis) after intravenous (i.v.) injection of 740 MBq (20 mCi) of [99mTc]TRODAT-1 using a triple-head gamma camera equipped with ultra-high-resolution fan-beam collimators (scan time: 0-210 min). In four blocking studies, baboons were pretreated with (+)McN5652 (1 mg/kg, i.v.) or methylphenidate (1 mg/kg, i.v.) to specifically block SERT or DAT, respectively. After co-registration with magnetic resonance images of the same baboon, a region of interest analysis was performed using predefined templates to calculate specific uptake in the midbrain area and the striatum, with the cerebellum as the background region [(MB-CB)/CB, (STR-CB)/CB]. Additionally, two PET scans of the same baboons were performed after i.v. injections of 74-111 MBq (2-3 mCi) of [11C](+)McN5652 to identify the SERT sites. In [99mTc]TRODAT-1/SPET scans, the SERT sites in the MB region were clearly visualized. Semiquantitative analysis revealed a specific uptake in MB ([MB-CB]/CB) of 0.30+/-0.02, which was decreased to 0. 040+/-0.005 after pretreatment with nonradioactive (+)McN5652, a selective SERT ligand. Pretreatment with methylphenidate reduced the specific binding of [99mTc]TRODAT-1 to DAT sites [(STR-CB)/CB] from 2.45+/-0.13 to 0.32+/-0.04 without any effect on its binding to SERT sites [(MB-CB)/CB], which was confirmed by the co-registration of the [11C](+)McN5652/PET scans. This preliminary study suggests that specific binding of [99mTc]TRODAT-1 to SERT sites can be detected by in vivo SPET imaging despite the low target to background ratio. These findings provide impetus for further development of similar compounds with improved binding affinity and selectivity to SERT sites.

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UNLABELLED: [99mTc]Technetium[2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1] oct-2-yl]-methyl] (2-mercaptoethyl) amino] ethyl] amino] ethane-thiolato(3-)-N2,N2',S2,S2']oxo-[1R-(exo-exo)] ([99mTc] TRODAT-1) is a useful imaging agent for central nervous system dopamine transporters. The purpose of this study was to characterize the in vivo binding potential and kinetic rate constants of this agent in nonhuman primates. METHODS: A series of four SPECT scans were performed on each of two female baboons with a bolus injection of [99mTc]TRODAT-1 (717+/-78 MBq; 19.38+/-2.12 mCi). Dynamic images of the brain were acquired over 4 h using a triple-head camera equipped with fan-beam collimators. Arterial and venous blood were sampled frequently using a peristaltic pump throughout the duration of the study. Regions of interest were drawn on the corresponding MRI scan to which each functional image was coregistered. Using analytical solutions to the three-compartment model with the Levenberg-Marquardt minimization technique, each study was individually fitted to a kinetic parameter vector (method I). Additionally, within each subject, three corresponding intrasubject studies were fitted simultaneously to a single parameter vector by constraining the binding potential, distribution volume and dissociation rate constant to improve the identifiability of the parameter estimates (method II). RESULTS: The results clearly indicated that [99mTc] TRODAT-1 localized in the striatum with slower washout rate than other brain regions. A maximal target/nontarget ratio of 3.5 between striatum and cerebellum was obtained. SPECT image analysis of the striatum yielded unconstrained k3/k4 values of 3.4+/-1.4, 2.4+/-0.7, 3.0+/-1.5, and 4.0+/-10.3, with respective constrained (fixed k4) values of 2.9 +/- 0.4, 2.4 +/- 0.4, 1.7+/-0.4 and 1.8+/-0.4 in one baboon using method I. With method II, the corresponding simultaneously fitted values were 2.1+/-0.3 using no constraints and 2.2+/-0.2 using a fixed k4. The second baboon had similar results. CONCLUSION: These findings suggest that the binding potential and corresponding kinetic rate constants can be reliably estimated in nonhuman primates with dynamic SPECT imaging of the dopamine transporter using a technetium-based tropane analogue. Furthermore, method II parameter vectors compare favorably to those produced using method I based on SEEs.

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[99mTc]TRODAT-1 is a useful imaging agent in evaluating changes in presynaptic dopamine transporters (DAT) for Parkinson's disease and other central nervous system (CNS) neurodegenerative diseases, for which a reduction of dopamine neurons is indicated. As part of an effort to establish a quantitative single-photon emission tomography (SPECT) procedure for imaging CNS DAT, measurement of nonmetabolized [99mTc]TRODAT-1 in human plasma was investigated. After an intravenous injection of [99mTc]TRODAT-1, there are three possible radioactive components in human plasma: hydrophilic compounds (pertechnetate, etc.), lipophilic metabolite(s), and unchanged [99mTc]TRODAT-1. Based on the differences in lipophilicity of [99mTc]TRODAT-1 and its lipophilic metabolite [99mTc]BAT, a new quantitative method for measuring [99mTc]TRODAT-1 with a simple solvent extraction method was developed. Various organic solvents or mixtures of solvents were tested, among which cyclohexane gave the best extraction yield for [99mTc]TRODAT-1 (76.06 +/- 3.32%) with a low extraction for [99mTc]BAT (2.43 +/- 0.82%). Extractions of [99mTc]TRODAT-1 and [99mTc]BAT mixtures in different predetermined ratios to simulate the actual human plasma samples with cyclohexane from phosphate buffer (5 mM, pH 8.0) were evaluated. The experimentally obtained ratios were in good agreement with the theoretical ratios. To investigate further the possibility of replacing the previously established high performance liquid chromatography (HPLC) method with the new solvent extraction method for the clinical application, both HPLC and extraction methods were used side by side to determine the unchanged [99mTc]TRODAT-1 in human plasma samples during [99mTc]TRODAT-1/SPECT imaging studies. The results from four human subjects showed that both methods consistently produced similar values for unchanged [99mTc]TRODAT-1 in the plasma samples. This improved solvent extraction method provides an easy and reliable technique to quantify unchanged [99mTc]TRODAT-1 in human plasma, thus making the clinical application of this agent readily available for quantitation of the DAT binding sites in the brain by SPECT imaging.

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UNLABELLED: Technetium-99m TRODAT-1 is an analog of cocaine that selectively binds the presynaptic dopamine transporters. The primary purpose of this study was to measure its whole-body biokinetics and radiation dosimetry in healthy human volunteers. The study was conducted within a regulatory framework that required its pharmacological safety to be assessed simultaneously. METHODS: The sample included 4 men and 6 women ranging in age from 22-54 yr. An average of 20 whole-body scans were acquired sequentially on a dual-head camera for up to 46 hr after the intravenous administration of 370+/-16 MBq (10.0+/-0.42 mCi) 99mTc TRODAT. The renal excretion fractions were measured from 12-24 discrete urine specimens. The fraction of the administered dose in 17 regions of interest and each urine specimen was quantified from the attenuation and background corrected geometric mean counts in conjugate views. Multiexponential functions were iteratively fit to each time-activity curve using a nonlinear, least squares regression algorithm. These curves were numerically integrated to yield source organ residence times. Gender-specific radiation doses were then estimated with the Medical Internal Radiation Dose technique for each subject individually before any results were averaged. RESULTS: There were no pharmacological effects of the radiotracer on any of the subjects. The early planar images showed differentially increased activity in the nose, pudendum and stomach. SPECT images demonstrated that the radiopharmaceutical localized in the basal ganglia in a distribution that was consistent with selective transporter binding. Image analysis showed that the kidneys excreted between 20% and 32% of the injected dose during the first 22-28 hr postadministration, after which no more activity could be recovered in the urine. The dose limiting organ in both men and women was the liver, which received an average of 0.046 mGy/MBq (0.17 rads/mCi, range 0.14-0.22 rad/mCi). In the worst case, which was clearly an over-estimation, it would have taken 22.7 mCi to deliver 5 rad to the liver. CONCLUSION: TRODAT may be a safe and effective radiotracer for imaging dopamine transporters in the brain and the body.

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The purpose of this study was to investigate the influence of drugs competing for the dopamine transporter (DAT) or changing intra- and/or extracellular dopamine levels on the binding of a novel technetium-99m labeled tropane derivative, technetium, [2-[[2-[[[3-(4-chloro- phenyl)-8-methyl-8-azabicyclo[3, 2, 1]oct-2-yl]methyl] (2-mercaptoethyl)amino]ethyl]amino]ethanethiolato(3)]-oxo-[1R-(exo -exo)]-, [99mTc]TRODAT-1, to DAT. This paper describes the further characterization of [99mTc]TRODAT-1 binding sites in rats under conditions which may exist in patients receiving various drug treatments. All experiments were carried out using an i.v. injection of [99mTc]TRODAT-1 into male Sprague-Dawley rats. Measurements of % dose/gram ratio of (striatum-cerebellum)/cerebellum at 1 h post injection were used as an indicator for specific DAT binding. The biodistribution studies were performed in the presence of drugs which compete for the binding site, such as CFT (WIN 35,428) and methylphenidate, drugs which influence dopamine levels, such as L-DOPA, gamma-hydroxybutyrolactone, and alpha-methyl-p-tyrosine, and d-amphetamine, which both acts as a competitor for DAT binding and increases dopamine levels. Additionally, the influence of dopamine receptor agonists, such as apomorphine and (+)bromocriptine, on biodistribution was tested. Binding of [99mTc]TRODAT-1 to DAT was found to be inhibited by CFT, methylphenidate, and d-amphetamine in a dose-dependent manner. The specific binding of [99mTc]TRODAT-1 was not altered by dopamine receptor agonists or by drugs which cause minor changes in dopamine levels. When administered in high doses (634 micromol/kg), L-DOPA also decreased the binding of [99mTc]TRODAT-1. It is likely that a low dose of L-DOPA (normally needed in the treatment of Parkinson's disease) will not affect the results on [99mTc]TRODAT-1 single-photon emission tomographic (SPET) imaging studies. In conclusion, the results clearly demonstrate the specificity of [99mTc]TRODAT-1 binding to DAT in vivo. Competition for [99mTc]TRODAT-1 binding was observed only with drug treatment that significantly increases dopamine levels or actively competes for binding at DAT. The results suggest that prior knowledge of whether patients are receiving various drug treatments may assist in the interpretation of DAT status as assessed by SPET imaging studies using [99mTc]TRODAT-1.

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