Matthew R. Hight

Quantitative Preclinical Imaging of TSPO Expression in Glioma Using N,N-Diethyl-2-(2-(4-(2-18F-Fluoroethoxy)Phenyl)-5,7-Dimethylpyrazolo[1,5-a]Pyrimidin-3-yl)Acetamide

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-18F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (18F-DPA-714), as a translational probe for quantification of TSPO levels in glioma. Methods: Glioma-bearing rats were imaged with 18F-DPA-714 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-DPA-714 (130–200 MBq/0.2 mL). Blood was collected to derive the arterial input function (AIF), with high-performance liquid chromatography radiometabolite analysis performed on selected samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of 3H-PK 11195 with DPA-714 in vitro and displacement of 18F-DPA-714 with an excess of DPA-714 in vivo. Immediately after imaging, tumor and healthy brain tissues were harvested for validation by Western blotting and immunohistochemistry. Results: 18F-DPA-714 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced 18F-DPA-714 binding by greater than 60% on average. Tumor uptake of 18F-DPA-714 was similar to another high-affinity TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline, and agreed with ex vivo assay of TSPO levels in tumor and healthy brain. Conclusion: These studies illustrate the feasibility of using 18F-DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, 18F-DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of 18F-DPA-714 PET to serve as a novel predictive cancer imaging modality.

Quantitative, Preclinical PET of Translocator Protein Expression in Glioma Using 18F-N-Fluoroacetyl-N-(2,5-Dimethoxybenzyl)-2-Phenoxyaniline

Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. Methods: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70–100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. Results: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. Conclusion: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.

Synthesis and Structure-Activity Relationships of 5,6,7-substituted Pyrazolopyrimidines: Discovery of a novel TSPO PET Ligand for Cancer Imaging

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.

Multispectral fluorescence imaging to assess pH in biological specimens.

Simple, quantitative assays to measure pH in tissue could improve the study of complicated biological processes and diseases such as cancer. We evaluated multispectral fluorescence imaging (MSFI) to quantify extracellular pH (pHe) in dye-perfused, surgically-resected tumor specimens with commercially available instrumentation. Utilizing a water-soluble organic dye with pH-dependent fluorescence emission (SNARF-4F), we used standard fluorimetry to quantitatively assess the emission properties of the dye as a function of pH. By conducting these studies within the spectroscopic constraints imposed by the appropriate imaging filter set supplied with the imaging system, we determined that correction of the fluorescence emission of deprotonated dye was necessary for accurate determination of pH due to suboptimal excitation. Subsequently, employing a fluorimetry-derived correction factor (CF), MSFI data sets of aqueous dye solutions and tissuelike phantoms could be spectrally unmixed to accurately quantify equilibrium concentrations of protonated (HA) and deprotonated (A-) dye and thus determine solution pH. Finally, we explored the feasibility of MSFI for high-resolution pHe mapping of human colorectal cancer cell-line xenografts. Data presented suggest that MSFI is suitable for quantitative determination of pHe in ex vivo dye-perfused tissue, potentially enabling measurement of pH across a variety of preclinical models of disease.

A Peptide-Based Positron Emission Tomography Probe for In Vivo Detection of Caspase Activity in Apoptotic Cells

Purpose: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid–specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [18F]4-fluorobenzylcarbonyl–Val–Ala–Asp(OMe)–fluoromethylketone ([18F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET). Experimental Design: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [18F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [18F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer. Results: Accumulation of [18F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in V600EBRAF colon cancer. In the latter setting, [18F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size. Conclusions: These studies illuminate [18F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine. Clin Cancer Res; 20(8); 2126–35. ©2014 AACR.

Abstract 2058: Molecular imaging of glutaminolysis as a tool for evaluating therapeutic response in preclinical models of colorectal cancer

The metabolic repertoire of cancer cells diverge significantly from that of normal cells. Energy production in cancer cells tends to depend on aerobic glycolysis, a feature that is routinely monitored by positron emission tomography (PET) imaging using 2-[ 18 F]fluoro-2-deoxy-D-glucose ([ 18 F]FDG). In addition to predilection for glycolysis, cancer cells may possess other unique metabolic characteristics, such as increased consumption of glutamine. As with glucose, glutamine also serves as a key carbon source for ATP production and biosynthesis. Given this, quantitative measures of glutaminolysis may reflect critical processes in oncology. Accordingly, PET agents targeting glutamine uptake, such as 4-[ 18 F]fluoro-glutamine ([ 18 F]4F-GLN), have been reported and used in preclinical models of cancer. The goal of this study was to elucidate the feasibility of using [ 18 F]4F-GLN PET to predict response to targeted therapy within the context of colorectal cancer (CRC). Initially we validated expression of SLC1A5, the primary plasma membrane transporter of glutamine, in tumor and normal colon specimens for 58 patients with primary and advanced CRC. We found that SLC1A5 expression was elevated in over 80%of primary tumor specimens but did not correlate with grade or gender. Furthermore, elevated SLC1A5 expression correlated strongly with elevated Ki67, a molecular marker of proliferation. Given this, we evaluated [ 18 F]4F-GLN PET in preclinical models of V600E BRAF-expressing CRC as a means of detecting anti-proliferation responses to targeted therapeutics. Simulating a clinical study within the context of the Vanderbilt GI SPORE, the regimen included an inhibitor of mutant BRAF, a PI3K/mTOR inhibitor, and a combination there of. Strikingly, [ 18 F]4F-GLN PET was found to correlate more closely to markers of anti-proliferative responses in vivo than analogously performed [ 18 F]FDG PET imaging studies. We believe that these findings not only provide a greater understanding of the role that glutaminolysis plays in CRC but also illuminate the potential impact that glutaminolysis derived PET could have towards guiding drug development clinical trials as an imaging metric of early therapeutic response detection. Citation Format: Matthew R. Hight, Michael L. Schulte, Samir Saleh, Gregory D. Ayers, Frank L. Revetta, M. Kay Washington, Robert J. Coffey, H. Charles Manning. Molecular imaging of glutaminolysis as a tool for evaluating therapeutic response in preclinical models of colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2014-2058

Abstract 4545: Positron emission tomography (PET) of TSPO expression in preclinical models of human cancer

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of human cancer. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we have evaluated a high-affinity pyrazolopyrimidinyl TSPO imaging ligand ([ 18 F]DPA-714) and an aryloxyanilide-based TSPO imaging ligand ([ 18 F]PBR06), as translational probes for quantification of TSPO levels in glioma, breast cancer, and colorectal cancer. In these studies, glioma-bearing rats or genetically engineered mice bearing spontaneously arrising breast or colorectal cancers were imaged with TSPO PET ligands in a microPET system. Dynamic images were acquired simultaneously upon injection of radioactive TSPO PET ligands. Arterial blood was collected to derive the input function (AIF), with HPLC radiometabolite analysis performed upon select samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Immediately following imaging, tumor and healthy surrounding tissues were harvested for validation by western blotting and immunohistochemistry. Our results illustrate the feasibility of using TSPO PET ligands for visualization of TSPO-expressing tumors of the brain, breast and colon. Importantly, both [ 18 F]DPA-714 and [ 18 F]PBR06 appear suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of these imaging agents to serve as a novel predictive cancer imaging biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4545. doi:1538-7445.AM2012-4545

Abstract 5743: Positron emission tomography (PET) imaging analogs of glutamine: Development and evaluation as probes for cancer imaging

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Emerging evidence suggests that nutrient uptake in cancer cells is controlled by oncogenic signaling pathways. The natural amino acid glutamine is an essential nutrient for cell growth and proliferation. Glutamine also participates in protein synthesis and is a nitrogen source for the production of select nucleotides and amino acids. Consequently, quantitative measures of glutamine uptake may reflect critical processes in oncology. Recently, a fluorinated glutamine analog for positron emission tomography (PET) was shown to possess both highly specific cellular uptake in glioma cell lines and in vivo tumor localization (J Am Chem Soc 2011;133:1122-1133). Synthetic methodology leading to this agent, an optically pure 4-fluoro-glutamine derivative, was also reported. Though effective, this convergent synthesis requires multiple steps, extensive chromatographic separation, and nearly a week of reaction time. Employing a chiral auxiliary Schiff base and microwave-assisted organic synthesis, we have significantly improved the synthesis of this and similar agents, drastically reducing the number of synthetic steps (from ten to four) and the overall reaction time (from seven days to two hours). While the radiochemistry relies upon traditional box methods, we are also currently employing high-throughput microfluidic radiolabeling strategies to expedite probe production and labeling efficiency. Finally, using optically pure [18F]4-FGln (2S,4R) synthesized by our improved synthesis, we report preclinical PET images of models of human colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5743. doi:1538-7445.AM2012-5743