Synthesis and Evaluation of 68Ga- and 177Lu-Labeled (R)- vs (S)-DOTAGA Prostate-Specific Membrane Antigen-Targeting Derivatives.

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells and therefore is an attractive target for prostate cancer diagnosis and radionuclide therapy. Recently, published results from clinical studies using a new PSMA-targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carboxymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for chelating 68Ga but is also amendable for complexing other radioactive metals for radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-[177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed high binding PSMA affinity (IC50 = 24-111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM). Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy agents in the future.

Synthesis and evaluation of indolinyl- and indolylphenylacetylenes as PET imaging agents for beta-amyloid plaques.

Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indoline 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting beta-amyloid (Abeta) plaques have been prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [(125)I]IMPY (6-iodo-2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K(i)=4.0 and 1.5nM for 8 and 14, respectively). Brain penetration of the corresponding radiofluorinated ligands, evaluated in the normal mice, showed good initial brain penetration (4.50 and 2.43% ID/g (injected dose/gram) for [(18)F]8 and [(18)F]14 at 2min after injection) with moderate to low washout rates from the brain (1.71% ID/g at 2h and 2.10% ID/g at 3h, respectively). Autoradiography and homogenate binding studies demonstrated the high specific binding of [(18)F]14 to the Abeta plaques; however, [(18)F]8 showed low specific binding. These preliminary results identified that indolylphenylacetylene, 14, may be a good lead for further structural modification to develop a useful Abeta plaque imaging agent.

2-(2'-((dimethylamino)methyl)-4'-(fluoroalkoxy)-phenylthio)benzenamine derivatives as serotonin transporter imaging agents.

A novel series of ligands with substitutions at the 5-position on phenyl ring A and at the 4'-position on phenyl ring B of 2-(2'-((dimethylamino)methyl)-4'-(fluoroalkoxy)phenylthio)benzenamine (4'-2-fluoroethoxy derivatives 28-31 and 4'-3-fluoropropoxy derivatives 40-42) were prepared and tested as serotonin transporter (SERT) imaging agents. The new ligands displayed high binding affinities to SERT (Ki ranging from 0.03 to 1.4 nM). The corresponding 18F labeled compounds, which can be prepared readily, showed excellent brain uptake and retention after iv injection in rats. The hypothalamus region showed high uptake values between 0.74% and 2.2% dose/g at 120 min after iv injection. Significantly, the hypothalamus to cerebellum ratios (target to nontarget ratios) at 120 min were 7.8 and 7.7 for [18F]28 and [18F]40, respectively. The selective uptake and retention in the hypothalamus, which has a high concentration of SERT binding sites, demonstrated that [18F]28 and [18F]40 are promising positron emission computed tomography imaging agents for mapping SERT binding sites in the brain.

5-Chloro-2-(2'-((dimethylamino)methyl)-4'-iodophenylthio)benzenamine: a new serotonin transporter ligand.

Two novel ligands with 4' substitution on the Phenyl Ring B of biphenylthiol, 5-chloro-2-(2'-((dimethylamino)methyl)-4'-iodophenylthio)benzenamine (7) and 2-(2'-((dimethylamino)methyl)-4'-methoxyphenylthio)-5-iodobenzenamine (8), were prepared and tested as potential serotonin transporter (SERT) imaging agents. The new ligands displayed extremely high binding affinities to SERT (K(i)=0.22+/-0.09 and 0.11+/-0.04 nM, respectively), with very low binding affinities to dopamine and norepinephrine transporters (K(i)>1000 nM). The corresponding [(125)I]7 and [(125)I]8 were successfully prepared from tri-n-butyltin derivatives. They showed good brain uptakes and prolonged retention after intravenous injection in rats (brain uptake was 1.77% and 0.98% dose/g for [(125)I]7, and 0.92% and 0.29% dose/g for [(125)I]8, at 2 and 120 min, respectively). Significantly, [(125)I]7 showed excellent uptake and prolonged retention in the hypothalamus, where SERT concentration was highest. The hypothalamus/cerebellum (HY/CB) ratios (target/background ratios) were 4.24, 7.10, 8.24 and 12.6 at 2, 4, 6 and 12 h, respectively. The HY/CB ratios for [(125)I]8 were 3.97, 5.57 and 5.06 at 1, 2 and 4 h, respectively. Adding the 4'-iodo group to the Phenyl Ring B of Compound (7) appeared to reduce the rate of clearance from the brain, and kinetics favored uptake and retention in the hypothalamus. The localization of [(125)I]7 in the hypothalamus region in the rat brain could be blocked by pretreatment with (+)McN5652, escitalopram and ADAM (2), which are all selective SERT ligands (at 2 mg/kg iv, 5 min pretreatment). Ex vivo autoradiograms of rat brain sections (at 4 h after intravenous injection of [(125)I]7) showed intense labeling in regions of the brain known to have high SERT density. The excellent selective uptake and retention in the hypothalamus region suggest that [(123)I]7 is a potential lead compound for developing new imaging agents targeting SERT-binding sites with single-photon emission computed tomography.

In vivo detection of stem cells grafted in infarcted rat myocardium.

UNLABELLED: The evaluation of stem cell-mediated cardiomyoplasty by noninvasive in vivo imaging is critical for its clinical application. We hypothesized that dual-tracer small-animal SPECT would allow simultaneous imaging of (99m)Tc-sestamibi to assess myocardial perfusion and of (111)In-labeled stem cells to delineate stem cell engraftment. METHODS: Three to 4 million rat embryonic cardiomyoblasts (H9c2 cells) were labeled with 11.1-14.8 MBq (0.3-0.4 mCi) of (111)In-oxyquinoline and then injected into the border zones of infarcted myocardium of rats. (111)In images were acquired with a SPECT scanner 2, 24, 48, 72, and 96 h after the stem cells were injected into the infarcted myocardium. To visualize the perfusion deficit in the infarcted myocardium, we injected 74 MBq (2 mCi) of (99m)Tc-sestamibi (Cardiolite) intravenously 48 h after grafting. Dual-isotope pinhole SPECT was used to image (99m)Tc-sestamibi uptake simultaneously with (111)In to delineate retention of (111)In-labeled stem cells. The presence of labeled stem cells was confirmed by autoradiography and histology. RESULTS: SPECT of (99m)Tc-sestamibi was used to delineate perfusion deficits and infarcted myocardium. Bull's-eye plots indicated that the (111)In signal from the labeled stem cells overlapped the perfusion deficits identified from the (99m)Tc-sestamibi images. The (111)In signal associated with the radiolabeled stem cells could be detected with SPECT of the heart for 96 h after engraftment. CONCLUSION: This study demonstrated the feasibility of using dual-isotope pinhole SPECT for high-resolution detection of perfusion deficits with (99m)Tc-sestamibi and with (111)In-labeled stem cells grafted into the region of the infarct.

2-(2-(dimethylaminomethyl)phenoxy)-5-iodophenylamine: an improved serotonin transporter imaging agent.

Imaging serotonin transporters (SERT) is an emerging research tool potentially useful to cast light on the mechanisms of drug action as well as to monitor the treatment of depressed patients. We have prepared two new derivatives of 3, 2-(2-(dimethylaminomethyl)phenoxy)-5-iodophenylamine (4) and 2-(2-(dimethylaminomethyl)benzyl)-5-iodophenylamine (5) (K(i) for SERT = 0.37 and 48.6 nM, respectively). Both [(125)I]4 and [(125)I]5 displayed excellent brain uptakes in rats, and they showed a highest uptake in hypothalamus (between 60 and 240 min), a region populated with the highest density of SERT. The specific uptake of [(125)I]4 in the hypothalamus resulted in a target to nontarget ratio ([hypothalamus-cerebellum]/cerebellum) of 4.3 at 2 h. Autoradiography of rat brain sections (ex vivo at 2 h) of [(125)I]4 showed an excellent regional distribution pattern consistent with known SERT localization. These data suggest that [(123)I]4 may be useful for imaging SERT binding sites in the brain by single photon emission computed tomography (SPECT).

N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)-benzylamine (4-(18)F-ADAM): an improved PET radioligand for serotonin transporters.

UNLABELLED: There has been considerable interest in the development of PET radioligands that are useful for imaging serotonin transporter (SERT) in the living human brain. For the last decade, (11)C-(+)McN5652 has been the most promising PET agent for studying SERT in humans. However, this agent has some limitations. Recently, a new promising SERT PET radioligand, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile, has been reported. We recently reported the synthesis of a new (18)F-labeled SERT PET radioligand, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-(18)F-ADAM), which may have advantages over (11)C-labeled radioligands. The purpose of this study was to evaluate this newly developed (18)F-labeled PET radioligand as a promising agent for studying SERT in the living human brain. METHODS: This agent was evaluated by studying its in vitro binding to different monoamine transporters, its in vivo biodistributions in rats, its integrity and pharmacologic profiles in rat brain, and its distribution in a female baboon brain. RESULTS: In vitro binding assays showed that 4-F-ADAM displayed high affinity to SERT sites (inhibition constant = 0.081 nmol/L, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1,000- and 28,000-fold selectivity for SERT over norepinephrine transporter and dopamine transporter, respectively. Biodistribution of 4-(18)F-ADAM in rats showed a high initial uptake and slow clearance in the brain (2.13%, 1.90%, and 0.95% injected dose per organ at 2, 30, and 60 min after intravenous injection, respectively), with the specific binding peaking at 2 h after injection (hypothalamus/cerebellum = 12.49). The uptake in blood, muscle, lung, kidney, and liver was also initially high but cleared rapidly. The radioactivity in the femur increases with time for 4-(18)F-ADAM, indicating that in vivo defluorination may occur. In vivo metabolism studies in rats showed that 4-(18)F-ADAM was not metabolized in rat brain (>96% of radioactivity was recovered as parent compound at 1 h after injection). However, it metabolized rapidly in the blood. Less than 7% of the radioactivity recovered from plasma was the parent compound, with the majority of radioactivity in the plasma not extractable by ethyl acetate. Blocking studies showed significant decreases in the uptake of 4-(18)F-ADAM in the brain regions (hypothalamus, hippocampus, and striatum) where SERT concentrations are high when rats were pretreated with (+)McN5652 (2 mg/kg 5 min before intravenous injection of 4-(18)F-ADAM). However, changes in the uptake of 4-(18)F-ADAM in these brain regions were less significant when rats were pretreated with either methylphenidate or nisoxetine. The baboon study showed that uptake of 4-(18)F-ADAM in the midbrain peaked at approximately 1 h after injection and then declined slowly. The ratios of the radioactivity in the midbrain to that in the cerebellum (where the concentration of SERT is low) at 2 and 3 h after injection were 3.2 and 4.2, respectively. CONCLUSION: 4-(18)F-ADAM is suitable as a PET radioligand for studying SERT in the living brain. Further characterization of this new radioligand in humans is warranted.

Selective binding of 2-[125I]iodo-nisoxetine to norepinephrine transporters in the brain.

A radioiodinated ligand, (R)-N-methyl-(2-[(125)I]iodo-phenoxy)-3-phenylpropylamine, [(125)I]2-INXT, targeting norepinephrine transporters (NET), was successfully prepared. A no-carrier-added product, [(125)I]2-INXT, displayed a saturable binding with a high affinity (K(d)=0.06 nM) in the homogenates prepared from rat cortical tissues as well as from LLC-PK(1) cells expressing NET. A relatively low number of binding sties (B(max)=55 fmol/mg protein) measured with [(125)I]2-INXT in rat cortical homogenates is consistent with the value reported for a known NET ligand, [(3)H]nisoxetine. Competition studies with various compounds on [(125)I]2-INXT binding clearly confirmed the pharmacological specificity and selectivity for NET binding sites. Following a tail-vein injection of [(125)I]2-INXT in rats, a good initial brain uptake was observed (0.56% dose at 2 min) followed by a slow washout from the brain (0.2% remained at 3 hours post-injection). The hypothalamus (a NET-rich region) to striatum (a region devoid of NET) ratio was 1.5 at 3 hours post-i.v. injection. Pretreatment of rats with nisoxetine significantly inhibited the uptake of [(125)I]2-INXT (70-100% inhibition) in locus coeruleus, hypothalamus and raphe nuclei, regions known to have a high density of NET; whereas escitalopram, a serotonin transporter ligand, did not show a similar effect. Ex vivo autoradiography of rat brain sections of [(125)I]2-INXT (at 3 hours after an i.v. injection) displayed an excellent regional brain localization pattern corroborated to the specific NET distribution in the brain. The specific brain localization was significantly reduced by a dose of nisoxetine pretreatment. Taken together, the data suggest that [(123)I]2-INXT may be useful for mapping NET binding sites in the brain.

Quantification of dopamine transporters in the mouse brain using ultra-high resolution single-photon emission tomography.

Functional imaging of small animals, such as mice and rats, using ultra-high resolution positron emission tomography (PET) and single-photon emission tomography (SPET), is becoming a valuable tool for studying animal models of human disease. While several studies have shown the utility of PET imaging in small animals, few have used SPET in real research applications. In this study we aimed to demonstrate the feasibility of using ultra-high resolution SPET in quantitative studies of dopamine transporters (DAT) in the mouse brain. Four healthy ICR male mice were injected with (mean+/-SD) 704+/-154 MBq [(99m)Tc]TRODAT-1, and scanned using an ultra-high resolution SPET system equipped with pinhole collimators (spatial resolution 0.83 mm at 3 cm radius of rotation). Each mouse had two studies, to provide an indication of test-retest reliability. Reference tissue kinetic modeling analysis of the time-activity data in the striatum and cerebellum was used to quantitate the availability of DAT. A simple equilibrium ratio of striatum to cerebellum provided another measure of DAT binding. The SPET imaging results were compared against ex vivo biodistribution data from the striatum and cerebellum. The mean distribution volume ratio (DVR) from the reference tissue kinetic model was 2.17+/-0.34, with a test-retest reliability of 2.63%+/-1.67%. The ratio technique gave similar results (DVR=2.03+/-0.38, test-retest reliability=6.64%+/-3.86%), and the ex vivo analysis gave DVR=2.32+/-0.20. Correlations between the kinetic model and the ratio technique ( R(2)=0.86, P<0.001) and the ex vivo data ( R(2)=0.92, P=0.04) were both excellent. This study demonstrated clearly that ultra-high resolution SPET of small animals is capable of accurate, repeatable, and quantitative measures of DAT binding, and should open up the possibility of further studies of cerebral binding sites in mice using pinhole SPET.