Synthesis and Biological Evaluation of Fluorine-18 and Deuterium Labeled l-Fluoroalanines as Positron Emission Tomography Imaging Agents for Cancer Detection.

To fully explore the potential of 18F-labeled l-fluoroalanine for imaging cancer and other chronic diseases, a simple and mild radiosynthesis method has been established to produce optically pure l-3-[18F]fluoroalanine (l-[18F]FAla), using a serine-derivatized, five-membered-ring sulfamidate as the radiofluorination precursor. A deuterated analogue, l-3-[18F]fluoroalanine-d3 (l-[18F]FAla-d3), was also prepared to improve metabolic stability. Both l-[18F]FAla and l-[18F]FAla-d3 were rapidly taken up by 9L/lacZ, MIA PaCa-2, and U87MG cells and were shown to be substrates for the alanine-serine-cysteine (ASC) amino acid transporter. The ability of l-[18F]FAla, l-[18F]FAla-d3, and the d-enantiomer, d-[18F]FAla-d3, to image tumors was evaluated in U87MG tumor-bearing mice. Despite the significant bone uptake was observed for both l-[18F]FAla and l-[18F]FAla-d3, the latter had enhanced tumor uptake compared to l-[18F]FAla, and d-[18F]FAla-d3 was not specifically taken up by the tumors. The enhanced tumor uptake of l-[18F]FAla-d3 compared with its nondeuterated counterpart, l-[18F]FAla, warranted the further biological investigation of this radiotracer as a potential cancer imaging agent.

Evaluating the Impact of the Tyr158 pKa on the Mechanism and Inhibition of InhA, the Enoyl-ACP Reductase from Mycobacterium tuberculosis.

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F2Y) has no effect on kcatapp/KMapp nor on the binding of inhibitors to the open form of the enzyme (Kiapp), whereas both kcatapp/KMapp and Kiapp are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F3Y158 InhA). 19F NMR spectroscopy suggests that 2,3,5-F3Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, Ki*app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F2Y158 and 2,3,5-F3Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced ∼four-fold for 2,3,5-F3Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.

Synthesis and Preclinical Evaluation of a Novel Fluorine-18-Labeled Tracer for Positron Emission Tomography Imaging of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a target for treating B-cell malignancies and autoimmune diseases. To aid in the discovery and development of BTK inhibitors and improve clinical diagnoses, we have developed a positron emission tomography (PET) radiotracer based on a selective BTK inhibitor, remibrutinib. [18F]PTBTK3 is an aromatic, 18F-labeled tracer that was synthesized in 3 steps with a 14.8 ± 2.4% decay-corrected radiochemical yield and ≥99% radiochemical purity. The cellular uptake of [18F]PTBTK3 was blocked up to 97% in JeKo-1 cells using remibrutinib or non-radioactive PTBTK3. [18F]PTBTK3 exhibited renal and hepatobiliary clearance in NOD SCID (non-obese diabetic/severe combined immunodeficiency) mice, and the tumor uptake of [18F]PTBTK3 in BTK-positive JeKo-1 xenografts (1.23 ± 0.30% ID/cc) was significantly greater at 60 min post injection compared to the tumor uptake in BTK-negative U87MG xenografts (0.41 ± 0.11% ID/cc). In the JeKo-1 xenografts, tumor uptake was blocked up to 62% by remibrutinib, indicating the BTK-dependent uptake of [18F]PTBTK3 in tumors.

Evaluations of the performances of PET and MRI in a simultaneous PET/MRI instrument for pre-clinical imaging.

BACKGROUND: PET/MRI is an attractive imaging modality due to the complementary nature of MRI and PET. Obtaining high quality small animal PET/MRI results is key for the translation of novel PET/MRI agents and techniques to the radiology clinic. To obtain high quality imaging results, a hybrid PET/MRI system requires additional considerations beyond the standard issues with separate PET and MRI systems. In particular, researchers must understand how their PET system affects the MR acquisitions and vice versa. Depending on the application, some of these effects may substantially influence image quality. Therefore, the goal of this report is to provide guidance, recommendations, and practical experiments for implementing and using a small animal PET/MRI instrument. RESULTS: Various PET and MR image quality parameters were tested with their respective modality alone and in the presence of both systems to determine how the combination of PET/MRI affects image quality. Corrections and calibrations were developed for many of these effects. While not all image characteristics were affected, some characteristics such as PET quantification, PET SNR, PET spatial resolution, PET partial volume effects, and MRI SNR were altered by the presence of both systems. CONCLUSIONS: A full exploration of a new PET/MRI system before performing small animal PET/MRI studies is beneficial and necessary to ensure that the new instrument can produce highly accurate and precise PET/MR images.

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.

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.

Simultaneous Evaluations of pH and Enzyme Activity with a CEST MRI Contrast Agent.

The extracellular tumor microenvironment of many solid tumors has high acidosis and high protease activity. Simultaneously assessing both characteristics may improve diagnostic evaluations of aggressive tumors and the effects of anticancer treatments. Noninvasive imaging methods have previously been developed that measure extracellular pH or can detect enzyme activity using chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). Herein, we developed a single-hybrid CEST agent that can simultaneously measure pH and evaluate protease activity using a combination of dual-power acidoCEST MRI and catalyCEST MRI. Our agent showed CEST signals at 9.2 ppm from a salicylic acid moiety and at 5.0 ppm from an aryl amide. The CEST signal at 9.2 ppm could be measured after selective saturation was applied at 1 and 4 µT, and these measurements could be used with a ratiometric analysis to determine pH. The CEST signal at 5.0 ppm from the aryl amide disappeared after the agent was treated with cathepsin B, while the CEST signal at 9.2 ppm remained, indicating that the agent could detect protease activity through the amide bond cleavage. Michaelis-Menten kinetics studies with catalyCEST MRI demonstrated that the binding affinity (as shown with the Michaelis constant KM), the catalytic turnover rate (kcat), and catalytic efficiency (kcat/KM) were each higher for cathepsin B at lower pH. The kcat rates measured with catalyCEST MRI were lower than the comparable rates measured with liquid chromatography-mass spectrometry (LC-MS), which reflected a limitation of inherently noisy and relatively insensitive CEST MRI analyses. Although this level of precision limited catalyCEST MRI to semiquantitative evaluations, these semiquantitative assessments of high and low protease activity still had value by demonstrating that high acidosis and high protease activity can be used as synergistic, multiparametric biomarkers.

Evaluation of the Biodistribution of Serinolamide-Derivatized C60 Fullerene.

Carbon nanoparticles have consistently been of great interest in medicine. However, there are currently no clinical materials based on carbon nanoparticles, due to inconsistent biodistribution and excretion data. In this work, we have synthesized a novel C60 derivative with a metal chelating agent (1,4,7-Triazacyclononane-1,4,7-triacetic acid; NOTA) covalently bound to the C60 cage and radiolabeled with copper-64 (t1/2 = 12.7 h). Biodistribution of the material was assessed in vivo using positron emission tomography (PET). Bingel-Hirsch chemistry was employed to functionalize the fullerene cage with highly water-soluble serinolamide groups allowing this new C60 conjugate to clear quickly from mice almost exclusively through the kidneys. Comparing the present results to the larger context of reports of biocompatible fullerene derivatives, this work offers an important evaluation of the in vivo biodistribution, using experimental evidence to establish functionalization guidelines for future C60-based biomedical platforms.

Summary of Imaging in 2020: Visualizing the Future of Healthcare with MR Imaging.

The Imaging in 2020 meeting convenes biannually to discuss innovations in medical imaging. The 2018 meeting, titled "Visualizing the Future of Healthcare with MR Imaging," sought to encourage discussions of the future goals of MRI research, feature important discoveries, and foster scientific discourse between scientists from a variety of fields of expertise. Here, we highlight presented research and resulting discussions of the meeting.

Clinical applications of chemical exchange saturation transfer (CEST) MRI.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) has been developed and employed in multiple clinical imaging research centers worldwide. Selective radiofrequency (RF) saturation pulses with standard 2D and 3D MRI acquisition schemes are now routinely performed, and CEST MRI can produce semiquantitative results using magnetization transfer ratio asymmetry (MTRasym ) analysis while accounting for B0 inhomogeneity. Faster clinical CEST MRI acquisition methods and more quantitative acquisition and analysis routines are under development. Endogenous biomolecules with amide, amine, and hydroxyl groups have been detected during clinical CEST MRI studies, and exogenous CEST agents have also been administered to patients. These CEST MRI tools show promise for contributing to assessments of cerebral ischemia, neurological disorders, lymphedema, osteoarthritis, muscle physiology, and solid tumors. This review summarizes the salient features of clinical CEST MRI protocols and critically evaluates the utility of CEST MRI for these clinical imaging applications. LEVEL OF EVIDENCE: 5 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:11-27.