Radiolabeling Optimization and Preclinical Evaluation of the New PSMA Imaging Agent [18F]AlF-P16-093.

Prostate-specific membrane antigen (PSMA)-targeted radioligands have played an increasing role in the diagnosis of prostate cancer. [68Ga]Ga-P16-093 is a PSMA-targeting agent for positron emission tomography imaging, currently under a Phase 2 clinical trial. In the present study, P16-093 was labeled with 18F via [18F]AlF2+ complex formation, and the biological properties of [18F]AlF-P16-093 were evaluated. Optimization of radiolabeling efficiency was performed by testing a series of parameters, including the amount of free ligand; the amount of Al3+; and the influence of solvent, pH, temperature, reaction time, and reaction volume. Optimal labeling results were achieved at pH 5 by reacting at 60 °C for 15 min in a vial containing 74-370 MBq of [18F]fluoride, 46 nmol of P16-093, 40 nmol of AlCl3·6 H2O, and 50% EtOH. [18F]AlF-P16-093 was prepared with a non-decay-corrected radiochemical yield of 54.4 ± 4.4% (n = 9) within 30 min (final radiochemical purity ≥95%). In vitro, [18F]AlF-P16-093 showed PSMA-specific high uptakes in PIP-PC3 cells. The binding affinity of [18F]AlF-P16-093 to PSMA was determined as Kd of 12.4 ± 2.0 nM. The tumor uptake in mice with a xenografted PSMA-expressing PIP-PC3 tumor was high (18.8 ± 5.14% ID/g at 1 h postinjection) and retained without washout for 2 h. In addition, tumor uptake was almost completely blocked by coinjecting a PSMA inhibitor, 2-PMPA. The bone activity at 1 h post injection was higher with [18F]AlF-P16-093 (2.83 ± 0.49% ID/g) in comparison to that of [68Ga]Ga-P16-093 (0.26 ± 0.07% ID/g). In summary, an efficient and simple radiosynthesis of [18F]AlF-P16-093 was achieved. [18F]AlF-P16-093 showed desirable in vivo pharmacokinetics and excellent PSMA-targeting properties for imaging PSMA expression in prostate cancer.

A New [68Ga]Ga-HBED-CC-Bisphosphonate as a Bone Imaging Agent.

Positron emission tomography (PET) imaging using 68Ga-labeled bisphosphonates to target bone metastasis could be a valuable tool in cancer diagnosis and monitoring therapeutic treatment. A 68Ga labeled ligand, N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) containing one bisphosphonate group (HBED-CC-BP, 1) was prepared and evaluated. The new ligand, 1, reacted rapidly to form [68Ga]Ga-1, via complexing with [68Ga]GaCl3 eluted from a commercially available 68Ge/68Ga generator (in a sodium acetate buffer at pH 4, reaching >95% labeling yield at room temperature in 5 min). The resulting [68Ga]Ga-1 showed excellent stability in vitro and in vivo. [68Ga]Ga-1 displayed high binding affinity to hydroxyapatite and good uptake in the tibia and femur bone of normal mice. Biodistribution and MicroPET imaging studies of [68Ga]Ga-1 in normal mice and rats showed excellent bone uptake and retention comparable to that of Na[18F]F. The results suggested that [68Ga]Ga-1 might be suitable as a bone imaging agent in humans and it could be useful as a convenient alternative to the current bone imaging PET agent, Na[18F]F, without the need of a near-by cyclotron. Also, an automated synthesis module was developed to produce clinical doses of [68Ga]Ga-1 in a consistent and reproducible manner. Currently, the investigation new drug application (IND) for [68Ga]Ga-HBED-CC-BP, [68Ga]Ga-1, has received FDA approval, and it is currently under clinical trial (IND #129870).

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer.

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with 125I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC50 = 1-13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [125I]8 and [125I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [125I]19a/19b and [125I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [125I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2-3-fold lower uptake in the liver and blood than that observed for [125I]MIP-1095. [125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.

Deuterium-substituted 2-(2'-((dimethylamino)methyl)-4'-[18 F](fluoropropoxy)phenylthio)benzenamine as a serotonin transporter imaging agent.

Positron emission tomography imaging of serotonin transporter (SERT) is useful for studying brain diseases with altered serotonergic function. A deuterated imaging agent, ([18 F]2-((2-((bis(methyl-d3 )amino)methyl)-4-(3-fluoropropoxy-1,1,2,2,3,3-d6 )phenyl)thio)aniline, [18 F]D12FPBM, [18 F]1), was prepared as a new chemical entity. The deuterated agent, 1, showed excellent binding affinity to SERT; Ki was 0.086 nM, comparable with the undeuterated FPBM. In vivo biodistribution studies in rats with [18 F]1 showed good brain uptake (1.09% dose/g at 2 min post injection) and high specific uptake into the hypothalamus (HY) as compared with cerebellum (CB) (HY/CB = 7.55 at 120 min), suggesting a specific localization to SERT binding sites. Regional brain distribution in rats provided clear indication that [18 F]1 concentrated in the hypothalamus, hippocampus, and striatum, areas with a high SERT density. Results indicate that very little D to H substitution effect was found; [18 F]FPBM and [18 F]1 showed very similar SERT binding. [18 F]1 might be an excellent candidate for SERT imaging.

(68)Ga-Bivalent Polypegylated Styrylpyridine Conjugates for Imaging Aß Plaques in Cerebral Amyloid Angiopathy.

Aß plaques deposited on blood vessels are associated with cerebral amyloid angiopathy (CAA). In an effort to selectively map these Aß plaques, we are reporting a new series of (68)Ga labeled styrylpyridine derivatives with high molecular weights. In vitro binding to Aß plaques in post-mortem Alzheimer's disease (AD) brain tissue showed that these (68)Ga labeled bivalent styrylpyridines displayed good affinities and specificity (Ki < 30 nM). In vitro autoradiography using post-mortem AD brain sections showed specific binding of these (68)Ga complexes to Aß plaques. Biodistribution studies in normal mice showed very low initial brain uptakes (<0.3% dose/g) indicating a low blood-brain barrier (BBB) penetration. The preliminary results suggest that (68)Ga labeled bivalent styrylpyridines may be promising candidates as PET imaging radiotracers for detecting CAA.

[(18)F](2S,4S)-4-(3-Fluoropropyl)glutamine as a tumor imaging agent.

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [(18)F](2S,4R)- and [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with (18)F and (19)F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (≥95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of l-[(3)H]glutamine ([(3)H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [(18)F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-isotope experiment using l-[(3)H]glutamine and the new probe showed that the uptake of [(3)H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L xenographs with an excellent tumor to muscle ratio (maximum of ∼8 at 40 min). [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

Optimization and scale up of production of the PSMA imaging agent [18F]AlF-P16-093 on a custom automated radiosynthesis platform.

BACKGROUND: Recent advancements in positron emission tomograph (PET) using prostate specific membrane antigen (PSMA)-targeted radiopharmaceuticals have changed the standard of care for prostate cancer patients by providing more accurate information during staging of primary and recurrent disease. [68Ga]Ga-P16-093 is a new PSMA-PET radiopharmaceutical that demonstrated superior imaging performance in recent head-to-head studies with [68Ga]Ga-PSMA-11. To improve the availability of this new PSMA PET imaging agent, [18F]AlF-P16-093 was developed. The 18F-analog [18F]AlF-P16-093 has been synthesized manually at low activity levels using [18F]AlF2+ and validated in pre-clinical models. This work reports the optimization of the production of > 15 GBq of [18F]AlF-P16-093 using a custom automated synthesis platform. RESULTS: The sensitivity of the radiochemical yield of [18F]AlF-P16-093 to reaction parameters of time, temperature and reagent amounts was investigated using a custom automated system. The automated system is a low-cost, cassette-based system designed for 1-pot syntheses with flow-controlled solid phase extraction (SPE) workup and is based on the Raspberry Pi Zero 2 microcomputer/Python3 ecosystem. The optimized none-decay-corrected yield was 52 ± 4% (N = 3; 17.5 ± 2.2 GBq) with a molar activity of 109 ± 14 GBq/µmole and a radiochemical purity of 98.6 ± 0.6%. Run time was 30 min. A two-step sequence was used: SPE-purified [18F]F- was reacted with 80 nmoles of freeze-dried AlCl3·6H2O at 65 °C for 5 min followed by reaction with 160 nmoles of P16-093 ligand at 40 °C for 4 min in a 1:1 mixture of ethanol:0.5 M pH 4.5 NaOAc buffer. The mixture was purified by SPE (> 97% recovery). The final product formulation (5 mM pH 7 phosphate buffer with saline) exhibited a rate of decline in radiochemical purity of ~ 1.4%/h which was slowed to ~ 0.4%/h when stored at 4 °C. CONCLUSION: The optimized method using a custom automated system enabled the efficient (> 50% none-decay-corrected yield) production of [18F]AlF-P16-093 with high radiochemical purity (> 95%). The method and automation system are simple and robust, facilitating further clinical studies with [18F]AlF-P16-093.

Theranostic Agent Targeting Bone Metastasis: A Novel [68Ga]Ga/[177Lu]Lu-DOTA-HBED-bisphosphonate.

Bone metastasis in cancer patients is a major disease advancement for various types of cancer. Previously, [68Ga]Ga-HBED-CC-bisphosphonate ([68Ga]Ga-P15-041) showed excellent bone uptake and efficient detection of bone metastasis in patients. To accommodate different α- or ß--emitting metals for radionuclide therapy, a novel DOTA-HBED-CC-bisphosphonate (P15-073, 1) was prepared and the corresponding [68Ga]Ga-1 and [177Lu]Lu-1 were successfully synthesized in high yields and purity. Gallium-68 conjugation to HBED-CC at room temperature and lutetium-177 conjugation to DOTA at 95 °C were verified in model compounds through secondary mass confirmation. These bisphosphonates, [68Ga]Ga-1 and [177Lu]Lu-1, displayed high binding affinity to hydroxyapatite in vitro. After an iv injection, it showed excellent uptake in the spine of normal mice, and micro-PET/CT imaging of nude mice model of bone metastasis showed high bone uptake in tumor tissue. The results indicated that [68Ga]Ga/[177Lu]Lu-1 holds promise as a theranostic radioligand agent for managing cancer bone metastases.

Lu-177-Labeled Hetero-Bivalent Agents Targeting PSMA and Bone Metastases for Radionuclide Therapy.

Prostate-specific membrane antigen (PSMA) is an excellent target for imaging and radionuclide therapy of prostate cancer. Recently, [177Lu]Lu-PSMA-617 (Pluvicto) was approved by the FDA for radionuclide therapy. To develop hetero-bivalent agents targeting both PSMA and bone metastasis, [177Lu]Lu-P17-079 ([177Lu]Lu-1) and [177Lu]Lu-P17-081 ([177Lu]Lu-2) were prepared. In vivo biodistribution studies of [177Lu]Lu-PSMA-617, [177Lu]Lu-1, and [177Lu]Lu-2 in mice bearing PC3-PIP (PSMA positive) tumor showed high uptake in PSMA-positive tumor (14.5, 14.7, and 11.3% ID/g at 1 h, respectively) and distinctively different bone uptakes (0.52, 6.52, and 5.82% ID/g at 1 h, respectively). PET imaging using [68Ga]Ga-P17-079 ([68Ga]Ga-1) in the same mouse model displayed excellent images confirming the expected dual-targeting to PSMA-positive tumor and bone. Results suggest that [177Lu]Lu-P17-079 ([177Lu]Lu-1) is a promising candidate for further development as a hetero-bivalent radionuclide therapy agent targeting both PSMA expression and bone metastases for the treatment of prostate cancer.

Correlation of amyloid PET ligand florbetapir F 18 binding with Aß aggregation and neuritic plaque deposition in postmortem brain tissue.

BACKGROUND: Florbetapir F 18 (F-AV-45) is a positron emission tomography imaging ligand for the detection of amyloid aggregation associated with Alzheimer disease. Earlier data showed that florbetapir F 18 binds with high affinity to ß-amyloid (Aß) plaques in human brain homogenates (Kd=3.7 nM) and has favorable imaging pharmacokinetic properties, including rapid brain penetration and washout. This study used human autopsy brain tissue to evaluate the correlation between in vitro florbetapir F 18 binding and Aß density measured by established neuropathologic methods. METHODS: The localization and density of florbetapir F 18 binding in frozen and formalin-fixed paraffin-embedded sections of postmortem brain tissue from 40 patients with a varying degree of neurodegenerative pathology was assessed by standard florbetapir F 18 autoradiography and correlated with the localization and density of Aß identified by silver staining, thioflavin S staining, and immunohistochemistry. RESULTS: There were strong quantitative correlations between florbetapir F 18 tissue binding and both Aß plaques identified by light microscopy (Silver staining and thioflavin S fluorescence) and by immunohistochemical measurements of Aß using 3 antibodies recognizing different epitopes of the Aß peptide. Florbetapir F 18 did not bind to neurofibrillary tangles. CONCLUSIONS: Florbetapir F 18 selectively binds Aß in human brain tissue. The binding intensity was quantitatively correlated with the density of Aß plaques identified by standard neuropathologic techniques and correlated with the density of Aß measured by immunohistochemistry. As Aß plaques are a defining neuropathologic feature for Alzheimer disease, these results support the use of florbetapir F 18 as an amyloid positron emission tomography ligand to identify the presence of Alzheimer disease pathology in patients with signs and symptoms of progressive late-life cognitive impairment.

New PSMA-Targeting Ligands: Transformation from Diagnosis (Ga-68) to Radionuclide Therapy (Lu-177).

Prostate-specific membrane antigen (PSMA) is a promising target for the diagnosis and radionuclide therapy of prostate cancer. This study reports conversion of a previously reported 68Ga-imaging agent, [68Ga]Ga-P16-093, to a Lu-177 radionuclide therapeutic agent. Substitution of the HBED-CC metal chelating group with DOTA(GA)2 led to P17-087 (4) and P17-088 (7). Both agents showed excellent PSMA binding affinity (IC50 = 10-30 nM) comparable to that of recently FDA-approved [177Lu]Lu-PSMA-617 (Pluvicto). Biodistribution studies in PSMA expressing tumor bearing mice showed that [177Lu]Lu-4 exhibited very high tumor uptake and a fast blood clearance similar to those of [177Lu]Lu-PSMA-617. Conversely, [177Lu]Lu-7, containing an albumin binder, extended its blood half-life and exhibited significantly higher uptake and longer tumor residence time than [177Lu]Lu-4 and [177Lu]Lu-PSMA-617. The switch from chelator HBED-CC to DOTA(GA)2 and the switch from the imaging isotope gallium-68 to the therapeutic isotope lutetium-177 have successfully transformed a PSMA-targeting agent from diagnosis to promising radionuclide therapeutic agents.

Synthesis and biological evaluation of 3-alkyl-dihydrotetrabenazine derivatives as vesicular monoamine transporter-2 (VMAT2) ligands.

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [(125)I]-iodovinyl-TBZ or [(18)F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 (K(i)=5.98 nM) and may be a useful lead compound for future structure-activity studies.

Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of ß-amyloid in the brain.

INTRODUCTION: Since the approval of three 18F labeled ß-amyloid-targeting PET imaging agents, Amyvid (florbetapir f18, AV-45), Neuraceq (florbetaben f18, AV-1) and Vizamyl (flutemetamol f18, F-PIB), they have increasingly been employed to assist differential diagnosis of Alzheimer's disease in patients with dementia. Also, they are frequently used in selecting patients participating drug trials aiming to reduce ß-amyloid (Aß) plaques in the brain. The first approved tracer in this class was [18F]AV-45, which is metabolized rapidly in blood and some of its N-demethylated metabolites cross the blood brain barrier and resulted in lowering the image contrast. To improve metabolic stability of [18F]AV-45, we hypothesized that substituting N-CH3 with N-CD3 at the metabolically labile position, creating [18F]D3FSP, may reduce in vivo N-demethylation. We report the preclinical evaluation of [18F]D3FSP as an Aß imaging agent. METHODS: Preclinical pharmacology of [18F]D3FSP was evaluated using in vitro autoradiography and competitive binding assay. Biodistribution of [18F]D3FSP was evaluated in wild-type CD-1 mice. In vivo metabolism in mice and in vitro microsomal metabolism were analyzed by HPLC. Single dose acute toxicity of D3FSP was also performed in rats. RESULTS: [18F]D3FSP showed high binding affinity to ß-amyloid plaques (Ki = 3.44 ± 1.22 nM, a value similar as AV-45 (Ki = 4.02 ± 0.22 nM)), and displayed excellent ß-amyloid binding in AD brain sections consistent with known Aß regional distribution. After an iv injection it exhibited good initial brain uptake and fast washout in wild-type CD-1 mice. In vitro microsomal metabolism and in vivo metabolism in mice did not result in any significant differences between [18F]D3FSP and [18F]AV-45. No treatment-related mortality or any adverse effects were observed in single dose acute toxicity studies administered at hundred-folds of maximum human dose. CONCLUSION: A new small molecule, [18F]D3FSP, was prepared and tested as an alternative to [18F]AV-45 to reduce N-demethylation in vivo. This strategy did not lead to better in vivo stability. However, [18F]D3FSP displayed very similar Aß targeting property comparable to [18F]AV-45. Preclinical studies suggest that [18F]D3FSP is useful as a ß-amyloid-targeting PET imaging agent.

A New Highly Deuterated [18F]AV-45, [18F]D15FSP, for Imaging ß-Amyloid Plaques in the Brain.

[18F]AV-45 (florbetapir f18, Amyvid) is an FDA-approved PET imaging agent targeting Aß plaques in the brain for diagnosis of Alzheimer's disease (AD). Its metabolites led to a high background in the brain and large bone uptake of [18F]F-, produced from dealkylation of the PEG chain. To slow down the in vivo metabolism, we report the design, synthesis, and evaluation of a highly deuterated derivative, [18F]D15FSP, and compared it with N-methyl-deuterated [18F]D3FSP and nondeuterated [18F]AV-45. D15FSP displayed excellent binding affinity (K i = 7.52 nM) to Aß aggregates. In vitro autoradiography of [18F]D15FSP, [18F]D3FSP, and [18F]AV-45 showed excellent binding to Aß plaques in human AD brain sections. Biodistribution studies displayed lower bone uptake at 120 min for [18F]D15FSP compared to that for [18F]D3FSP and [18F]AV-45 (1.44 vs 4.23 and 4.03%ID/g, respectively). As the highly deuterated [18F]D15FSP displayed excellent Aß binding affinity, high initial brain penetration, and lower bone retention, it might be suitable for PET imaging in detecting Aß plaques.

[68Ga]Ga-HBED-CC-DiAsp: A new renal function imaging agent.

INTRODUCTION: [68Ga]Ga-EDTA ([68Ga]Ga-ethylenediaminetetraacetic acid) was previously reported as a renal imaging agent for measuring GFR (glomerular filtration rate). In an effort to provide new agents with better in vivo characteristics for renal imaging, [68Ga]Ga-HBED-CC-DiAsp (Di-Aspartic acid derivative of N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl]-ethylenediamine-N,N'-diacetic acid) was prepared and tested. METHOD: HBED-CC-DiAsp was synthesized and labeled with [68Ga]GaCl4- at room temperature. Plasma protein and red blood cells (RBC) binding were also evaluated. Biodistribution and dynamic PET imaging studies were performed in mice and rats, respectively. RESULTS: [68Ga]Ga-HBED-CC-DiAsp was radiolabeled at room temperature by a one-step kit formulation in high purity without any purification (radiochemical purity >98%). Previous reports suggested that Ga-HBED-CC exhibited a higher stability constant and rapid chelating formation rate than that of Ga-EDTA (logKGaL = 38.5 vs 22.1, respectively). In vitro stability studies indicated that it was stable up to 120 min. The log DOW value, partition coefficient between n-octanol and water, was found to be -2.52 ± 0.08. Plasma protein and RBC binding was similar to that observed for [68Ga]Ga-EDTA. Biodistribution and dynamic PET/CT imaging studies in rats revealed a rapid clearance primarily through the renal-urinary pathway. The PET-derived [68Ga]Ga-HBED-CC-DiAsp renograms in rats showed an average time-to-peak of 3.6 ± 0.7 min which was similar to that observed for [68Ga]Ga-EDTA (3.1 ± 0.5 min). The time-to-half-maximal activity was also comparable to that of [68Ga]Ga-EDTA (8.8 vs 8.2 min, respectively). Pretreatment of probenecid, a renal tubular excretion inhibitor, showed no significant effect on renal excretion. CONCLUSIONS: [68Ga]Ga-HBED-CC-DiAsp could be prepared quickly at room temperature in high yield and purity. Results of in vitro studies and in vivo biodistribution in mice and rats suggested that [68Ga]Ga-HBED-CC-DiAsp might be useful as a PET imaging agent for measurement of GFR.

VMAT2 imaging agent, D6-[18F]FP-(+)-DTBZ: Improved radiosynthesis, purification by solid-phase extraction and characterization.

OBJECTIVES: Recently, a deuterated tracer, D6-[18F]FP-(+)-DTBZ, 9-O-hexadeutero-3-[18F]fluoropropoxyl-(+)-dihydrotetrabenazine ([18F]9), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system, was reported as a useful imaging agent for the diagnosis of Parkinson's disease (PD). The production of [18F]9 was optimized and simplified by using solid-phase extraction (SPE) purification. METHODS: Three major nonradioactive impurities were synthesized and characterized. The preparation of [18F]9 was optimized by using different labeling conditions, and an SPE purification method was evaluated. The influence of chemical impurities in the final dose of [18F]9 was assessed by an in vitro binding assay, an assay of the in vivo biodistribution in mice, and ex vivo and in vitro autoradiography of brain sections. RESULTS: Optimized fluorination conditions for [18F]9 were found - heating at 130 °C for 10 min in DMSO, and a high radiochemical yield and three major chemical impurities were observed. An SPE method involving a Sep-Pak® tC18 Plus Light cartridge with a two-step elution process was successfully implemented. This process gave a good radiochemical yield (38.7 ±â€¯10.5%, decay corrected; radiochemical purity >99%) and low chemical impurities. An in vivo biodistribution study and autoradiography of brain sections showed that there was no significant difference between HPLC-purified and SPE-purified [18F]9. CONCLUSION: A VMAT2 targeting imaging agent, D6-[18F]FP-(+)-DTBZ, [18F]9, was prepared by optimized labeling conditions and an easy SPE purification. This method offers a short preparation time and operational simplicity. In conjunction with PET imaging, this new VMAT2 agent might be a useful clinical tool for diagnosing PD.

Optimization of solid-phase extraction (SPE) in the preparation of [18F]D3FSP: A new PET imaging agent for mapping Aß plaques.

INTRODUCTION: Alzheimer's disease is a common neurodegenerative disease that is characterized by the presence of Aß plaques in the brain. The FDA has approved the use of Amyvid (florbetapir f18, AV-45) as a PET imaging agent for detecting Aß plaques in the living human brain. In an attempt to reduce N-demethylation in vivo by taking advantage of more stable C-D bonds, an analog of AV-45, [18F]D3FSP ([18F]7), was synthesized to improve image contrast for detecting and monitoring the Aß plaques. A convenient and improved preparation of [18F]D3FSP ([18F]7) is needed for widespread clinical application. We report herein the optimization of the radiosynthesis and solid-phase extraction (SPE) procedure. METHODS: Radiosyntheses of [18F]D3FSP ([18F]7) under different fluorination conditions were evaluated, and the intermediate, containing an N-Boc protecting group, was deprotected using different acids. One of the major objectives was to simplify the final purification step via SPE to avoid the commonly employed HPLC purification and maximize the radiochemical yields of [18F]D3FSP ([18F]7) while simultaneously removing several chemical impurities (pseudocarriers). Washing various solid-phase cartridges with different combinations of ethanol/water and acetonitrile/water was explored to optimize the purification step. To evaluate the potential interference in Aß plaques imaging from the presence of pseudocarriers, each chemical was identified and quantified by LC/MS and HPLC. An in vitro binding assay was employed to evaluate the binding affinities of [18F]D3FSP ([18F]7) and the pseudocarriers to Aß plaques using postmortem AD brain tissue. RESULTS: Using the optimized radiosynthesis method and SPE purification, the final dose of [18F]D3FSP ([18F]7) was obtained in 50 min with a very low content of pseudocarriers (21.7 ±â€¯5.5 µg). The radiochemical yield was 44.4 ±â€¯5.7% (decay corrected), and the radiochemical purity was >95%. SPE-purified doses of [18F]D3FSP ([18F]7) displayed excellent binding affinity and specificity for Aß plaques as measured in an in vitro binding assay using AD brain homogenates, and the OH-pseudocarrier, 8 (Ki = 19.5 ±â€¯0.5 nM), and the Cl-pseudocarrier, 10 (Ki = 18.6 ±â€¯3.9 nM), showed lower binding affinities for Aß plaques than those of AV-45 (Ki = 8.6 ±â€¯0.5 nM) and D3FSP, 7 (Ki = 9.8 ±â€¯0.5 nM). CONCLUSIONS: An optimized radiosynthesis and fast SPE purification method suitable for the preparation of clinical doses of [18F]D3FSP ([18F]7) was accomplished. The results of quality control tests and binding studies suggested that the SPE-purified doses of [18F]D3FSP ([18F]7) are appropriate for imaging Aß plaques in the human brain.

In vivo imaging of vesicular monoamine transporter 2 in pancreas using an (18)F epoxide derivative of tetrabenazine.

OBJECTIVES: Development of imaging agents for pancreatic beta cell mass may provide tools for studying insulin-secreting beta cells and their relationship with diabetes mellitus. In this paper, a new imaging agent, [(18)F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline [(18)F](+)4, which displays properties targeting vesicular monoamine transporter 2 (VMAT2) binding sites of beta cells in the pancreas, was evaluated as a positron emission tomography (PET) agent for estimating beta cell mass in vivo. The hydrolyzable epoxide group of (+)4 may provide a mechanism for shifting biodistribution from liver to kidney, thus reducing the background signal. METHODS: Both (18)F- and (19)F-labeled (+) and (-) isomers of 4 were synthesized and evaluated. Organ distribution was carried out in normal rats. Uptake of [(18)F](+)4 in pancreas of normal rats was measured and correlated with blocking studies using competing drugs, (+)dihydrotetrabenazine [(+)-DTBZ] or 9-fluoropropyl-(+)dihydro tetrabenazine [FP-(+)-DTBZ, (+)2]. RESULTS: In vitro binding study of VMAT2 using rat brain striatum showed a K(i) value of 0.08 and 0.15 nM for the (+)4 and (+/-)4, respectively. The in vivo biodistribution of [(18)F](+)4 in rats showed the highest uptake in the pancreas (2.68 %ID/g at 60 min postinjection). In vivo competition experiments with cold FP-(+)-DTBZ, (+)2, (3.5 mg/kg, 5 min iv pretreatment) led to a significant reduction of pancreas uptake (85% blockade at 60 min). The inactive isomer [(18)F](-)4 showed significantly lower pancreas uptake (0.22 %ID/g at 30 min postinjection). Animal PET imaging studies of [(18)F](+)4 in normal rats demonstrated an avid pancreatic uptake in rats. CONCLUSION: The preliminary results suggest that the epoxide, [(18)F](+)4, is highly selective in binding to VMAT2 and it has an excellent uptake in the pancreas of rats. The liver uptake was significantly reduced through the use of the epoxide group. Therefore, it may be potentially useful for imaging beta cell mass in the pancreas.

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.

Synthesis and evaluation of a novel urea-based 68Ga-complex for imaging PSMA binding in tumor.

INTRODUCTION: Prostate specific membrane antigen (PSMA) is a well-established target for diagnostic and therapeutic applications for prostate cancer. It is know that [68Ga]PSMA 11 ([68Ga]Glu-NH-CO-NH-Lys(Ahx)-HBED-CC) is the most well studied PET imaging agent for detecting over expressed PSMA binding sites of tumors in humans. In an effort to provide new agents with improved characteristics for PET imaging, we report a novel [68Ga]-Glu-NH-CO-NH-Lys(Ahx)-linker-HBED-CC conjugate with a novel O-(carboxymethyl)-L-tyrosine, as the linker group. METHODS: Radiosynthesis was performed by a direct method. In vitro binding and cell internalization of [68Ga]10 was investigated in PSMA positive LNCaP cell lines. Biodistribution and MicroPET imaging studies were performed in LNCaP tumor bearing mice. RESULTS: In vitro binding to LNCaP cells showed that natGa labeled O-(carboxymethyl)-L-tyrosine conjugate, [natGa]10, displayed excellent affinity and specificity (IC50 = 16.5 nM) a value comparable to that of PSMA 11. In vitro cell binding and internalization showed excellent uptake and retention; [68Ga]10 displayed significantly higher cellular internalization than [68Ga]PSMA 11 (12.5 vs 7.4% ID/106 cells at 1 h). Biodistribution studies in LNCaP tumor-bearing mice exhibited a high specific uptake in PSMA expressing tumors and fast clearance in normal organs (19.7 tumor/blood; 20.7 tumor/muscle at 1 h after iv injection). MicroPET imaging studies in mice confirmed that [68Ga]10 displayed excellent uptake and distinctive tumor localization, which was blocked by iv injection of a competing drug, 2-PMPA. CONCLUSIONS: The preliminary results strongly suggest that [68Ga]10 may be promising candidates as a PET imaging radiotracer for detecting PSMA expression in prostate cancer.