Zhihao Zha

PET Imaging of Glutaminolysis in Tumors by 18F-(2S,4R)4-Fluoroglutamine

Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled 18F-FDG PET tumor imaging. However, of the millions of 18F-FDG PET scans conducted per year, a significant number of malignant tumors are 18F-FDG PET–negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, 18F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. Methods: A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of 18F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. Results: In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, ≈16% dose/100 μg of protein). The cell uptake of 18F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of 3H-l-glutamine but higher than that of 18F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that 18F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. Conclusion: The results support that 18F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.

[18F](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 [18F](2S,4R)- and [18F](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 18F and 19F (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-[3H]glutamine ([3H]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 [18F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [18F](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-[3H]glutamine and the new probe showed that the uptake of [3H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [18F](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). [18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

Multidentate 18F-Polypegylated Styrylpyridines As Imaging Agents for Aβ Plaques in Cerebral Amyloid Angiopathy (CAA)

β-Amyloid plaques (Aβ plaques) in the brain are associated with cerebral amyloid angiopathy (CAA). Imaging agents that could target the Aβ plaques in the living human brain would be potentially valuable as biomarkers in patients with CAA. A new series of (18)F styrylpyridine derivatives with high molecular weights for selectively targeting Aβ plaques in the blood vessels of the brain but excluded from the brain parenchyma is reported. The styrylpyridine derivatives, 8a-c, display high binding affinities and specificity to Aβ plaques (K(i) = 2.87, 3.24, and 7.71 nM, respectively). In vitro autoradiography of [(18)F]8a shows labeling of β-amyloid plaques associated with blood vessel walls in human brain sections of subjects with CAA and also in the tissue of AD brain sections. The results suggest that [(18)F]8a may be a useful PET imaging agent for selectively detecting Aβ plaques associated with cerebral vessels in the living human brain.

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.

68Ga-Bivalent Polypegylated Styrylpyridine Conjugates for Imaging Aβ Plaques in Cerebral Amyloid Angiopathy

Aβ plaques deposited on blood vessels are associated with cerebral amyloid angiopathy (CAA). In an effort to selectively map these Aβ plaques, we are reporting a new series of (68)Ga labeled styrylpyridine derivatives with high molecular weights. In vitro binding to Aβ plaques in post-mortem Alzheimers 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 Aβ 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.

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.

Synthesis and evaluation of novel tropane derivatives as potential PET imaging agents for the dopamine transporter.

A novel series of tropane derivatives containing a fluorinated tertiary amino or amide at the 2β position was synthesized, labeled with the positron-emitter fluorine-18 (t(1/2)=109.8 min), and tested as potential in vivo dopamine transporter (DAT) imaging agents. The corresponding chlorinated analogs were prepared and employed as precursors for radiolabeling leading to the fluorine-18-labeled derivatives via a one-step nucleophilic aliphatic substitution reaction. In vitro binding results showed that the 2β-amino compounds 6b, 6d and 7b displayed moderately high affinities to DAT (K(i)<10nM). Biodistribution studies of [(18)F]6b and [(18)F]6d showed that the brain uptakes in rats were low. This is likely due to their low lipophilicities. Further structural modifications of these tropane derivatives will be needed to improve their in vivo properties as DAT imaging agents.

Deuterated 18F-9-O-hexadeutero-3-fluoropropoxyl-(+)-dihydrotetrabenazine (D6-FP-(+)-DTBZ): a vesicular monoamine transporter 2 (VMAT2) imaging agent

INTRODUCTION Vesicular monoamine transporters 2 (VMAT2) in the brain serve as transporter for packaging monoamine in vesicles for normal CNS neurotransmission. Several VMAT2 imaging agents, [11C]-(+)-DTBZ, dihydrotetrabenazine and [18F]FP-(+)-DTBZ (9-O-fluoropropyl-(+)-dihydro tetrabenazine, a.k.a. [18F]AV-133), are useful for studying the changes in brain function related to monoamine transmission by in vivo imaging. Deuterated analogs have been reported targeting VMAT2 binding sites. METHODS A novel deuterated [18F]9-O-hexaduterofluoropropyl-(+)-dihydrotetrabenazine, [18F]D6-FP-(+)-DTBZ, [18F]1, was prepared as a VMAT2 imaging agent. This 18F agent which targeted VMAT2 was evaluated by in vitro binding, in vivo biodistribution and microPET imaging studies in rodents. RESULTS The one step radiolabeling reaction led to the desired [18F]D6-FP-(+)-DTBZ, [18F]1, which showed excellent binding affinity to VMAT2 (Ki=0.32±0.07nM) comparable to that of FP-(+)-DTBZ (Ki=0.33±0.02nM) using [18F]FP-(+)-DTBZ and rat striatum membrane homogenates. In vivo biodistribution in normal rats showed that 1, exhibited excellent brain uptake and comparable high ratio of striatum to cerebellum (target/background) ratio at 1h after injection (ratio of 6.05±0.43 vs 5.66±0.72 for [18F]FP-(+)-DTBZ vs [18F]1, respectively). MicroPET imaging studies in rats further confirm that the striatum with high VMAT2 concentration was clearly delineated in normal rat brain after iv injection of [18F]1. We observed minor changes of metabolism in rat plasma between these two agents; however, the changes showed little effect on regional brain uptake and retention. CONCLUSIONS The results reported here lend support for using [18F]D6-FP-(+)-DTBZ, [18F]1, as in vivo PET imaging agent for VMAT2 binding in the brain.

Alanine and glycine conjugates of [ 18 F](2 S, 4 R )4-fluoroglutamine for tumor imaging

INTRODUCTION Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[18F]fluoroglutamine (4F-Gln) was reported as a glutamine-related metabolic imaging agent. To improve the in vivo kinetics of this radiotracer, two new dipeptides, [18F]Gly-(2S,4R)4-fluoroglutamine (Gly-4F-Gln) and [18F]Ala-(2S,4R)4-fluoroglutamine (Ala-4F-Gln) were investigated. METHODS Radiolabeling was performed via 2-steps 18F-fluorination. Cell uptake studies of Gly-4F-Gln and Ala-4F-Gln were investigated in 9 L cell lines. In vitro and in vivo metabolism studies were carried out in Fisher 344 rats. Biodistribution and microPET imaging studies were performed in 9 L tumor-bearing rats. RESULTS In vitro incubation of these [18F]dipeptides in rat and human blood showed a rapid conversion to (2S,4R)-4-[18F]fluoroglutamine (t1/2 = 2.3 and 0.2 min for [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, respectively for human blood). Biodistribution and PET imaging in Fisher 344 rats bearing 9 L tumor xenografts showed that these dipeptides rapidly localized in the tumors, comparable to that of (2S,4R)-4-[18F]fluoroglutamine (4F-Gln). CONCLUSIONS The results support that these dipeptides, [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, are prodrugs, which hydrolyze in the blood after an iv injection. They appear to be selectively taken up and trapped by tumor tissue in vivo. The dipeptide, [18F]Ala-4F-Gln, may be suitable as a PET tracer for imaging glutaminolysis in tumors.