Karl Plössl

Imaging of dopamine transporters in humans with technetium-99m TRODAT-1.

Technetium-99m TRODAT-1, a tropane derivative, has shown promise as a tracer for the imaging of dopamine transporters in preliminary studies in rats and baboons. The present report concerns the first study of the use of [99mTc]TRODAT 1 for the same purpose in humans. The specific uptake of [99mTc]TRODAT1 in dopamine transporter sites located in the basal ganglia area was confirmed: the best contrast between the basal ganglia and the occipital area, which is devoid of dopamine transporters, was achieved at 120–140 min following injection. The development of a99mTc-based agent bypasses the need for cyclotron-produced radionuclides, which will be of benefit for routine clinical studies.

[99mTc]TRODAT-1 : a novel technetium-99m complex as a dopamine transporter imaging agent

Technetium-99m is the most commonly used radionuclide in routine nuclear medicine imaging procedures. Development of99mTc-labeled receptor-specific imaging agents for studying the central nervous system is potentially useful for evaluation of brain function in normal and disease states. A novel99mTc-labeled tropane derivative, [99mTc]TRODAT 1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. After i.v. injection into rats, [99mTc]TRODAT-1 displayed specific brain uptake in the rat striatal region (striatum-cerebellum/cerebellum ratio 1.8 at 60 min), where dopamine neurons are concentrated. The specific striatal uptake could be blocked by pretreating rats with a dose of competing dopamine transporter ligand, ß-CIT (or RTI-55, i.v., 1 mg/kg). However, the specific striatal uptake of [99mTc]TRODAT-] was not affected by co-injection of excess free ligand (TRODAT-1, up to 200 μg per rat) or by pretreating the rats with haloperidol (i.v., 1 mg/kg). The specific uptake in striatal regions of rats that had prior 6-hydroxydopamine lesion in the substantia nigra area showed a dramatic reduction. The radioactive material recovered from the rat striatal homogenates at 60 min after i.v. injection of [99mTc]TRODAT-1 showed primarily the original compound (>95%), a good indication of in vivo stability in brain tissue. Similar and comparable organ distribution patterns and brain regional uptakes of [99mTc]TRODAT-1 were obtained for male and female rats. Ex vivo autoradiography results of rat brain sections further confirmed the high uptake and retention of [99mTc]TRODAT-1 in the striatal region. In vitro binding studies measuring the affinity to dopamine transporters for the free ligand, TRODAT-1, and a nonradioactive rhenium derivative, Re-TRODAT-1, showed Ki values of 9.7 nM and 14.1 nM, respectively. Behavioral studies in rats using the free ligand, TRODAT-1 and Re-TRODAT-1 indicated that, unlike other tropane derivatives, they displayed no effect on locomotor activity, suggesting low toxicity. These results strongly support the conclusions that this novel99mTc radioligand binds selectively to dopamine transporters in the brain and that is is potentially useful for in vivo assessment of the loss of dopamine neurons in Parkinsons and other neurodegeneralive diseases.

Small and Neutral TcvO BAT, Bisaminoethanethiol (N2S2) Complexes for Developing New Brain Imaging Agents

Bisaminoethanethiol (BAT) ligands with various gem-dimethyl and amide groups were prepared, and the corresponding neutral Tc-99m complexes were prepared and evaluated for their relative stabilities by ligand-exchange reactions. It was demonstrated that technetium complexes containing gem-dimethyl substituents have higher lipophilicities, whereas those with an amide group possess greater stability, which enhances ligand-exchange reaction. The most interesting observation was that the brain uptake in rats is not determined only by lipophilicity. Apparently, Tc-99m complexes with an amide functional group display lower brain uptakes in rats compared to those without an amide group. The brain uptake was strongly influenced by substituents on the BAT ligand. These factors are critically important and should be taken into consideration when designing Tc-99m-labeled agents for CNS receptor imaging.

Single-photon emission tomography imaging of serotonin transporters in the non-human primate brain with the selective radioligand [(123)I]IDAM.

Abstract.A new radioligand, 5-iodo-2-[[2–2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([123I]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 (Ki=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET Ki= 234 nM and DAT Ki>10 µM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [123I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [123I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90–120 min after injection of [123I]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, koff=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 [123I]IDAM in the brain. These results suggest that [123I]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.

In Vivo Quantitative Noninvasive Imaging of Gene Transfer by Single-Photon Emission Computerized Tomography

Systems aimed at detecting gene expression noninvasively in vivo are desirable for evaluating the outcome of gene transfer in clinical trials. Several approaches have been exploited using magnetic resonance imaging and spectroscopy ((31)P MRS), positron emission tomography (PET), single-photon emission tomography (SPECT), and detection of bioluminescent signals. An ideal system is based on transfer of a marker gene, the activity of which can be detected against a background from the target tissue without interfering with normal physiology or eliciting an immune response. The majority of approaches described to date use genes encoding a nonmammalian protein that can elicit immune responses or a transmembrane receptor as a marker gene whose ectopic expression may cause aberrant signaling in the target cell through binding to endogenous ligands. The dopamine transporter (DAT) is normally expressed at high levels, mainly in the dopaminergic neurons of the central nervous system. We previously synthesized a radioactive ligand, [(99m)Tc]TRODAT-1, that binds with high affinity to the dopamine transporter, allowing for SPECT imaging of the striatum in normal control subjects and individuals affected with Parkinsons disease. Here we describe a strategy to monitor gene transfer based on adeno-associated viral vector (AAV)-mediated transduction of DAT in murine muscle followed by [(99m)Tc]TRODAT-1 imaging by SPECT of cells expressing the transgene. We show that quantitative, noninvasive imaging of gene transfer is successfully achieved in vivo, using a single-photon computed tomography camera. This system employs a reporter gene encoding a mammalian protein that is absent in most tissues, has no enzymatic activity, and does not activate intracellular pathways. This should be useful to monitor gene transfer in the settings of gene therapy.

Single-photon emission tomography imaging of serotonin transporters in the nonhuman primate brain with [123I]ODAM

Abstract. We have described previously a selective serotonin transporter (SERT) radioligand, [123I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([123I]ODAM). The imaging characteristics of this radioligand were studied and compared against [123I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [123I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [123I]ODAM. Total integrated brain uptake of [123I]ODAM was about 30% higher than [123I]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 [123I]IDAM (1.80± 0.13). Both the binding kinetics and the metabolism of [123I]ODAM were slower than those of [123I]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 koff=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 [123I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [123I]IDAM, but also a slightly lower selectivity for SERT.

An improved kit formulation of a dopamine transporter imaging agent: [Tc-99m]TRODAT-1

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 Parkinsons 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.

An improved radiosynthesis of [18F]AV-133: a PET imaging agent for vesicular monoamine transporter 2.

INTRODUCTION Recently, a PET tracer, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system has been reported. It is currently under Phase II clinical trials to establish its usefulness in the diagnosis of neurodegenerative diseases including dementia with Lewy bodies and Parkinsons disease. The radiolabeling of [(18)F]AV-133, nucleophilic fluorination reaction and potential effects of pseudo-carrier were evaluated by in vivo biodistribution. METHODS The preparation of [(18)F]AV-133 was evaluated under different conditions, specifically by employing different precursors (-OTs or -Br as the leaving group at the 9-propoxy position), reagents (K222/K(2)CO(3) vs. tributylammonium bicarbonate) and solvents (acetonitrile vs. DMSO), reaction temperature and reaction time. With optimized conditions from these experiments, radiosynthesis and purification with solid-phase extraction (SPE) of [(18)F]AV-133 were performed by an automated nucleophilic [(18)F]fluorination module. In vivo biodistribution in mice on [(18)F]AV-133 purified by either HPLC (no-carrier-added) or the SPE method (containing a pseudo-carrier) was performed and the results compared. RESULTS Under a mild fluorination condition (heating at 115 degrees C for 5 min in dimethyl sulfoxide), [(18)F]AV-133 was obtained in a high yield using either -OTs or -Br as the leaving group. However, the -OTs precursor gave better radiochemical yields (>70%, thin layer chromatography analysis) compared to those of the -Br precursor. The optimized reaction conditions were successfully implemented to an automated nucleophilic fluorination module. Labeling and purification of [(18)F]AV133 were readily achieved via this automatic module in good radiochemical yield of 21-41% (n=10) in 40 min. The radiochemical purity was larger than 95%. Biodistribution of SPE-purified product (containing a pseudo-carrier) in mice showed a high striatum/cerebellum ratio (4.18+/-0.51), which was comparable to that of HPLC-purified [(18)F]AV-133 (4.51+/-0.10). CONCLUSIONS The formation of [(18)F]AV-133 was evaluated under different labeling conditions. These improved labeling conditions and SPE purification were successfully implemented into an automated synthesis module. This offers a short preparation time (about 40 min), simplicity in operation and ready applicability for routine clinical operation.

Comparison of region-of-interest analysis and human observers in the diagnosis of Parkinson's disease using [99mTc]TRODAT-1 and SPECT

This study determined the relative accuracy of diagnosis of Parkinsons disease (PD) using SPECT imaging data, comparing a semi-quantitative region-of-interest (ROI) approach and human observers. A set of patients with PD and normal healthy control subjects were studied using the dopamine transporter tracer [(99m)Tc]TRODAT-1 and SPECT. The sample comprised 81 patients (mean age +/- SD, 63.4 +/- 10.4 years; age range, 39.0-84.2 years) and 94 healthy controls (mean age +/- SD, 61.8 +/- 11.0 years; age range, 40.9-83.3 years). A standardized template containing six ROIs was transposed onto subregions of the brain, and the ratio of striatal to background ROI values was used as a semi-quantitative outcome measure. All images were used in a human observer study, with four experienced investigators. The data from the observer and ROI studies were analysed using a receiver operating characteristic (ROC) analysis, where the area under the ROC curve (AUC) indicated the diagnostic accuracy. ROI analysis and human observers gave similar diagnostic performance (mean observer AUC = 0.89, best ROI AUC = 0.90). This suggested that the human observers are visually acquiring similar information from the images that are contained in the semi-quantitative striatal uptake.

Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents

INTRODUCTION This paper reports the synthesis and labeling of (18)F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. METHODS Three new (18)F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labeling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[(18)F]fluoromethyl)-L-alanine (L-[(18)F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). RESULTS New (18)F alanine derivatives were prepared with 7%-34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[(18)F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than that observed for the other two alanine derivatives and [(18)F]FDG in the first 1h. Inhibition of cell uptake studies suggested that L-[(18)F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[(18)F]FMA remained stable and was not incorporated into protein within 2h. In vivo biodistribution studies demonstrated that L-[(18)F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[(18)F]FMA in both 9L rat and transgenic mouse. CONCLUSION L-[(18)F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor proliferation.