Development of a Potential Gallium-68-Labelled Radiotracer Based on DOTA-Curcumin for Colon-Rectal Carcinoma: From Synthesis to In Vivo Studies.

Colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women worldwide. We have recently reported that curcuminoid complexes labelled with gallium-68 have demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compared to normal human lymphocytes. In the present study, we report a new gallium-68-labelled curcumin derivative (68Ga-DOTA-C21) and its initial validation as marker for early detection of colorectal cancer. The precursor and non-radioactive complexes were synthesized and deeply characterized by analytical methods then the curcuminoid was radiolabelled with gallium-68. The in vitro stability, cell uptake, internalization and efflux properties of the probe were studied in HT29 cells, and the in vivo targeting ability and biodistribution were investigated in mice bearing HT29 subcutaneous tumour model. 68Ga-DOTA-C21 exhibits decent stability (57 ± 3% after 120 min of incubation) in physiological media and a curcumin-mediated cellular accumulation in colorectal cancer cell line (121 ± 4 KBq of radiotracer per mg of protein within 60 min of incubation). In HT29 tumour-bearing mice, the tumour uptake of 68Ga-DOTA-C21 is 3.57 ± 0.3% of the injected dose per gram of tissue after 90 min post injection with a tumour to muscle ratio of 2.2 ± 0.2. High amount of activity (12.73 ± 1.9% ID/g) is recorded in blood and significant uptake of the radiotracer occurs in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). Further studies are needed to understand the mechanism of accumulation and clearance; however, 68Ga-DOTA-C21 provides a productive base-structure to develop further radiotracers for imaging of colorectal cancer.

The 3S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Caspase-3 Substrates for Noninvasive Pharmacodynamic Imaging of Apoptosis by PET/CT.

Quantitative imaging of apoptosis in vivo could enable real-time monitoring of acute cell death pathologies such as traumatic brain injury, as well as the efficacy and safety of cancer therapy. Here, we describe the development and validation of F-18-labeled caspase-3 substrates for PET/CT imaging of apoptosis. Preliminary studies identified the O-benzylthreonine-containing substrate 2MP-TbD-AFC as a highly caspase 3-selective and cell-permeable fluorescent reporter. This lead compound was converted into the radiotracer [18F]-TBD, which was obtained at 10% decay-corrected yields with molar activities up to 149 GBq/µmol on an automated radiosynthesis platform. [18F]-TBD accumulated in ovarian cancer cells in a caspase- and cisplatin-dependent fashion. PET imaging of a Jo2-induced hepatotoxicity model showed a significant increase in [18F]-TBD signal in the livers of Jo2-treated mice compared to controls, driven through a reduction in hepatobiliary clearance. A chemical control tracer that could not be cleaved by caspase 3 showed no change in liver accumulation after induction of hepatocyte apoptosis. Our data demonstrate that [18F]-TBD provides an immediate pharmacodynamic readout of liver apoptosis in mice by dynamic PET/CT and suggest that [18F]-TBD could be used to interrogate apoptosis in other disease states.

SF2312 is a natural phosphonate inhibitor of enolase.

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers.

Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/µmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.

Positron emission tomographic imaging of CXCR4 in cancer: challenges and promises.

Molecular imaging is an attractive platform for noninvasive detection and assessment of cancer. In recent years, the targeted imaging of the C-X-C chemokine receptor 4 (CXCR4), a chemokine receptor that has been associated with tumor metastasis, has become an area of intensive research. This review article focuses on positron emission tomography (PET) and aims to provide useful and critical insights into the application of PET to characterize CXCR4 expression, including the chemical, radiosynthetic, and biological requirements for PET radiotracers. This discussion is informed by a summary of the different approaches taken so far and a comparison of their clinical translation. Finally, our expert opinions as to potential future advances in the field are expressed.

Preclinical evaluation of a CXCR4-specific (68)Ga-labelled TN14003 derivative for cancer PET imaging.

Molecular imaging is an ideal platform for non-invasive detection and assessment of cancer. In recent years, the targeted imaging of CXCR4, a chemokine receptor that has been associated with tumour metastasis, has become an area of intensive research. In our pursuit of a CXCR4-specific radiotracer, we designed and synthesised a novel derivative of the CXCR4 peptidic antagonist TN14003, CCIC16, which is amenable to radiolabelling by chelation with a range of PET and SPECT radiometals, such as (68)Ga, (64)Cu and (111)In as well as (18)F (Al(18)F). Potent in vitro binding affinity and inhibition of signalling-dependent cell migration by unlabelled CCIC16 were confirmed by a threefold uptake in CXCR4-over-expressing cells compared to their isogenic counterparts. Furthermore, in vivo experiments demonstrated the favourable pharmacokinetic properties of the (68)Ga-labelled tracer (68)Ga-CCIC16, along with its CXCR4-specific accumulation in tissues with desirable contrast (tumour-to-muscle ratio: 9.5). The specificity of our tracer was confirmed by blocking experiments. Taking into account the attractive intrinsic PET imaging properties of (68)Ga, the comprehensive preclinical evaluation presented here suggests that (68)Ga-CCIC16 is a promising PET tracer for the specific imaging of CXCR4-expressing tumours.

Synthesis of a new fluorine-18 glycosylated 'click' cyanoquinoline for the imaging of epidermal growth factor receptor.

This study reports the radiosynthesis of a new fluorine-18 glycosylated 'click' cyanoquinoline [(18) F]5 for positron emission tomography imaging of epidermal growth factor receptor (EGFR). The tracer was obtained in 47.7 ± 7.5% (n = 3) decay-corrected radiochemical yield from 2-[(18) F]fluoro-2-deoxy-ß-d-glucopyranosyl azide, and the overall nondecay-corrected radiochemical yield from aqueous fluoride was 8.6 ± 2.3% (n = 3). An in vitro preliminary cellular uptake study showed selectivity of the tracer for EGFR-positive A431 cell lines versus EGFR-negative MCF-7 cell lines. [(18) F]5 tracer uptake in A431 cells was significantly reduced by addition of the cold isotope analogue compound 5.

Hypoxia-activated prodrug and antiangiogenic therapies cooperatively treat pancreatic cancer but elicit immunosuppressive G-MDSC infiltration.

We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12D TP53+/R172H Pdx1-Cre-derived (KPC-derived) model of pancreatic adenocarcinoma to examine the tumor response and adaptive resistance mechanisms involved in response to 2 established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast with prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8+ T cell response. Bulk tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid-derived suppressor cells (G-MDSCs) through CCL9 secretion. Blockade of the CCL9/CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302, anti-VEGFR-2, and ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.

Scavenging strategy for specific activity improvement: application to a new CXCR4-specific cyclopentapeptide positron emission tomography tracer.

Huisgen cycloaddition is attractive to label peptide because of its rapidity and bioorthogonality. However, for larger tracers, the physico-chemical differences between the precursor and the tracer are usually insufficient to allow their separation by HPLC, reducing the specific activity. This is of importance for peptidic tracers because the combination of their high-affinity receptor with low specific activity results in the precursor saturating the receptors, causing non-specific tracer binding. Here, we report a fast, one-pot, general strategy to circumvent this issue, yielding a tracer of improved specific activity. It consists in adding a lipophilic azide after the labeling step to scavenge unreacted precursor into a more lipophilic species that does not co-elute with the tracer. We applied this strategy to a new fluorinated cyclopentapeptidic CXCR4 antagonist for the PET imaging of cancer, CCIC15, for which we managed to reduce the apparent peptide concentration by a factor of 34 in 10 min. This tracer was radiolabeled by click chemistry with 2-[(18) F]fluoroethylazide, yielding the tracer in 18 ± 6% (n = 5) end-of-synthesis radiochemical yields (EOS-RCY) in 1.5 h from [(18) F]fluoride with a specific activity of 19.4 GBq µmol(-1) . Preliminary biological evaluation of the probe confirmed potency and specificity for CXCR4; further biological evaluation is underway.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

Production, isolation, and shipment of clinically relevant quantities of astatine-211: A simple and efficient approach to increasing supply.

The alpha emitter astatine-211 (211At) is a promising candidate for cancer treatment based on Targeted Alpha (α) Therapy (TAT). A small number of facilities, distributed across the United States, are capable of accelerating α-particle beams to produce 211At. However, challenges remain regarding strategic methods for shipping 211At in a form adaptable to advanced radiochemistry reactions and other uses of the radioisotope. PURPOSE: Our method allows shipment of 211At in various quantities in a form convenient for further radiochemistry. PROCEDURES: For this study, a 3-octanone impregnated Amberchrom CG300M resin bed in a column cartridge was used to separate 211At from the bismuth matrix on site at the production accelerator (Texas A&M) in preparation for shipping. Aliquots of 6 M HNO3 containing up to ≈2.22 GBq of 211At from the dissolved target were successfully loaded and retained on columns. Exempt packages (<370 MBq) were shipped to a destination radiochemistry facility, University of Texas MD Anderson Cancer Center, in the form of a convenient air-dried column. Type A packages have been shipped overnight to University of Alabama at Birmingham. MAIN FINDINGS: Air-dried column hold times of various lengths did not inhibit simple and efficient recovery of 211At. Solution eluted from the column was sufficiently high in specific activity to successfully radiolabel a model compound, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1), with 211At. The method to prepare and ship 211At described in this manuscript has also been used to ship larger quantities of 211At a greater distance to University of Alabama at Birmingham. PRINCIPAL CONCLUSIONS: The successful proof of this method paves the way for the distribution of 211At from Texas A&M University to research institutions and clinical oncology centers in Texas and elsewhere. Use of this simple method at other facilities has the potential increase the overall availability of 211At for preclinical and clinical studies.

Development and Validation of a PET/SPECT Radiopharmaceutical in Oncology.

In oncology, biomarker research aimed to provide insights on cancer biology via positron emission tomography (PET) and single photon emission tomography (SPECT) imaging has seen an incredible growth in the past two decades. Despite the increased number of publications on PET/SPECT radiopharmaceuticals, the field lacked standardization of in vitro and in vivo parameters necessary for the characterization of any radiotracer. Through the efforts of the World Molecular Imaging Society Education Committee, this white paper lays down validation studies that are essential to chemically and biologically characterize new radiopharmaceuticals derived from small molecules, peptides or proteins. Finally, a brief overview of the steps toward translation is also presented.Herein, we discuss the following: Chemistry and radiochemistry metrics to establish the identity of the imaging agent. In vitro and in vivo studies to examine the radiotracer's mechanism of action, which includes target specificity, pharmacokinetics and in vivo metabolism.

Imaging of innate immunity activation in vivo with a redox-tuned PET reporter.

High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus H2O2, but not H2O2 alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [18F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [18F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb-/- (Nox2-/-) gene deletion strongly abrogated [18F]4FN retention in vivo. Thus, [18F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT.

Radiometal-Based PET/MRI Contrast Agents for Sensing Tumor Extracellular pH.

Acidosis is a useful biomarker for tumor diagnoses and for evaluating early response to anti-cancer treatments. Despite these useful applications, there are few methods for non-invasively measuring tumor extracellular pH, and none are routinely used in clinics. Responsive MRI contrast agents have been developed, and they undergo a change in MRI signal with pH. However, these signal changes are concentration-dependent, and it is difficult to accurately measure the concentration of an MRI contrast agent in vivo. PET/MRI provides a unique opportunity to overcome this concentration dependence issue by using the PET component to report on the concentration of the pH-responsive MRI agent. Herein, we synthesized PET/MRI co-agents based on the design of a pH-dependent MRI agent, and we have correlated pH with the r1 relaxivity of the MRI co-agent. We have also developed a procedure that uses PET radioactivity measurements and MRI R1 relaxation rate measurements to determine the r1 relaxivity of the MRI co-agent, which can then be used to estimate pH. This simultaneous PET/MRI procedure accurately measured pH in solution, with a precision that depended on the concentration of the MRI co-agent. We used our procedure to measure extracellular pH in a subcutaneous flank model of MIA PaCa-2 pancreatic cancer. Although the PET co-agents were stable in serum, post-imaging studies showed evidence that the PET co-agents were degraded in vivo. These results showed that tumor acidosis can be evaluated with simultaneous PET/MRI, although improvements are needed to more precisely measure MRI R1 relaxation rates, and ensure the in vivo stability of the agents.

RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging.

PURPOSE: Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. PROCEDURES: Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. RESULTS: Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. CONCLUSIONS: These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.

Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization.

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma.

PET/MR Imaging of a Lung Metastasis Model of Clear Cell Renal Cell Carcinoma with (2S,4R)-4-[18F]Fluoroglutamine.

PURPOSE: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. PROCEDURES: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. RESULTS: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60-90% in the presence of excess glutamine in the media and by 20-50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. CONCLUSIONS: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Development of a new epidermal growth factor receptor positron emission tomography imaging agent based on the 3-cyanoquinoline core: synthesis and biological evaluation.

The epidermal growth factor receptor (EGFR/c-ErbB1/HER1) is overexpressed in many cancers including breast, ovarian, endometrial, and non-small cell lung cancer. An EGFR specific imaging agent could facilitate clinical evaluation of primary tumors and/or metastases. To achieve this goal we designed and synthesized a small array of fluorine containing compounds based on a 3-cyanoquinoline core. A lead compound, 16, incorporating 2'-fluoroethyl-1,2,3-triazole was selected for evaluation as a radioligand based on its high affinity for EGFR kinase (IC50=1.81+/-0.18 nM), good cellular potency (IC50=21.97+/-9.06 nM), low lipophilicity and good metabolic stability. 'Click' labeling afforded [18F]16 in 37.0+/-3.6% decay corrected radiochemical yield based on azide [18F]14 and 7% end of synthesis (EOS) yield from aqueous fluoride. Compound [18F]16 was obtained with >99% radiochemical purity in a total synthesis time of 3 h. The compound showed good stability in vivo and a fourfold higher uptake in high EGFR expressing A431 tumor xenografts compared to low EGFR expressing HCT116 tumor xenografts. Furthermore, the radiotracer could be visualized in A431 tumor bearing mice by small animal PET imaging. Compound [18F]16 therefore constitutes a promising radiotracer for further evaluation for imaging of EGFR status.