Dewei Tang

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.

Multifunctional profiling of non-small cell lung cancer using 18F-FDG PET/CT and volume perfusion CT.

The aim of this study was to investigate correlations between glucose metabolism registered by 18F-FDG PET/CT and tumor perfusion quantified by volume perfusion CT and immunohistochemical markers Ki67 and microvessel density (MVD) in patients with non–small cell lung cancer (NSCLC). Methods: Between February 2010 and April 2011, 24 consecutive patients (21 women, 3 men; mean age ± SD, 67.6 ± 6.8 y; age range, 55.6–81.3 y) with histologically proven NSCLC (14 adenocarcinoma, 9 squamous cell lung carcinoma [SCC], and 1 mixed adenocarcinoma and SCC) underwent 18F-FDG PET/CT and additional volume perfusion CT. Maximum standardized uptake value (SUVmax), mean SUV, and the metabolic tumor volume were used for 18F-FDG uptake quantification. Blood flow (BF), blood volume (BV), flow extraction product (Ktrans), and standardized perfusion value (SPV) were determined as CT perfusion parameters. Both perfusion parameters and 18F-FDG uptake values were subsequently related to the histologic subtypes, proliferation marker Ki67, MVD according to CD34 staining, and total tumor volume. Results: Mean SUV, SUVmax, and the metabolic tumor volume (mL) were 5.8, 8.7, and 32.3, respectively, in adenocarcinoma and 8.5, 12.9, and 16.8, respectively, in SCC. Mean BF (mL/100 mL/min), mean BV (mL/100 mL), and Ktrans (mL/100 mL/min) were 35.4, 7.3, and 27.8, respectively, in adenocarcinoma and 35.5, 10.0, and 27.8, respectively, in SCC. Moderate correlations were found between the 18F-FDG PET/CT parameters and Ki67 as well as between CT perfusion parameters and MVD but not vice versa. For all tumors, the following correlations were found: between SUVmax and Ki67, r = 0.762 (P = 0.017); between SUVmax and MVD, r = −0.237 (P = 0.359); between mean BF and Ki67, r = −0.127 (P = 0.626); and between mean BF and MVD, r = 0.467 (P = 0.059). Interestingly, correlations between the BF–metabolic relationship and total tumor volume were higher in SCC (r = 0.762, P = 0.017) than in adenocarcinoma (r = −0.0791, P = 0.788). Conclusion: 18F-FDG uptake correlates with Ki67, whereas BF, BV, and Ktrans correlate with MVD. Therefore, 18F-FDG uptake and perfusion parameters provide complementary functional information. An improved tumor profiling will be beneficial for both prognosis and therapy response evaluation in these tumors.

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.

Preclinical imaging evaluation of novel TSPO-PET ligand 2-(5,7-Diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ([ (18)F]VUIIS1008) in glioma.

PurposeTranslocator protein (TSPO) concentrations are elevated in glioma, suggesting a role for TSPO positron emission tomography (PET) imaging in this setting. In preclinical PET studies, we evaluated a novel, high-affinity TSPO PET ligand, [18F]VUIIS1008, in healthy mice and glioma-bearing rats.ProceduresDynamic PET data were acquired simultaneously with [18F]VUIIS1008 injection, with binding reversibility and specificity evaluated in vivo by non-radioactive ligand displacement or blocking. Compartmental analysis of PET data was performed using metabolite-corrected arterial input functions. Imaging was validated with histology and immunohistochemistry.Results[18F]VUIIS1008 exhibited rapid uptake in TSPO-rich organs. PET ligand uptake was displaceable with non-radioactive VUIIS1008 or PBR06 in mice. Tumor accumulation of [18F]VUIIS1008 was blocked by pretreatment with VUIIS1008 in rats. [18F]VUIIS1008 exhibited improved tumor-to-background ratio and higher binding potential in tumors compared to a structurally similar pyrazolopyrimidine TSPO ligand, [18F]DPA-714.ConclusionsThe PET ligand [18F]VUIIS1008 exhibits promising characteristics as a tracer for imaging glioma. Further translational studies appear warranted.

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.

Facile synthesis of SSR180575 and discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide, a potent pyridazinoindole ligand for PET imaging of TSPO in cancer

A novel synthesis of the translocator protein (TSPO) ligand 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575, 3) was achieved in four steps from commercially available starting materials. Focused structure-activity relationship development about the pyridazinoindole ring at the N3 position led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (14), a novel ligand of comparable affinity. Radiolabeling with fluorine-18 ((18)F) yielded 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[(18)F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide ([(18)F]-14) in high radiochemical yield and specific activity. In vivo studies of [(18)F]-14 revealed this agent as a promising probe for molecular imaging of glioma.

Microwave-assisted, one-pot reaction of 7-azaindoles and aldehydes: a facile route to novel di-7-azaindolylmethanes

A novel and highly efficient synthetic method leveraging microwave-assisted organic synthesis (MAOS) to yield di-7-azaindolylmethanes (DAIMs) is reported. Under MAOS conditions, reaction of 7-azaindole with aldehydes resulted predominantly in DAIMs, as opposed to the expected 7-azaindole addition products that form at ambient temperature. Based upon studies of different indoles and azaindoles with various aromatic and aliphatic aldehydes, we herein propose a mechanism where rapid and efficient microwave heating promotes nucleophilicity of 7-azaindoles towards the corresponding alkylidene-azaindolene intermediate to form the DAIM. This sequence provides a versatile approach to efficiently synthesize novel DAIMs that may be useful pharmaceuticals.

Evaluation of TSPO PET Ligands [18F]VUIIS1009A and [18F]VUIIS1009B: Tracers for Cancer Imaging

PurposePositron emission tomography (PET) ligands targeting translocator protein (TSPO) are potential imaging diagnostics of cancer. In this study, we report two novel, high-affinity TSPO PET ligands that are 5,7 regioisomers, [18F]VUIIS1009A ([18F]3A) and [18F]VUIIS1009B ([18F]3B), and their initial in vitro and in vivo evaluation in healthy mice and glioma-bearing rats.ProceduresVUIIS1009A/B was synthesized and confirmed by X-ray crystallography. Interactions between TSPO binding pocket and novel ligands were evaluated and compared with contemporary TSPO ligands using 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy. In vivo biodistribution of [18F]VUIIS1009A and [18F]VUIIS1009B was carried out in healthy mice with and without radioligand displacement. Dynamic PET imaging data were acquired simultaneously with [18F]VUIIS1009A/B injections in glioma-bearing rats, with binding reversibility and specificity evaluated by radioligand displacement. In vivo radiometabolite analysis was performed using radio-TLC, and quantitative analysis of PET data was performed using metabolite-corrected arterial input functions. Imaging was validated with histology and immunohistochemistry.ResultsBoth VUIIS1009A (3A) and VUIIS1009B (3B) were found to exhibit exceptional binding affinity to TSPO, with observed IC50 values against PK11195 approximately 500-fold lower than DPA-714. However, HSQC NMR suggested that VUIIS1009A and VUIIS1009B share a common binding pocket within mammalian TSPO (mTSPO) as DPA-714 and to a lesser extent, PK11195. [18F]VUIIS1009A ([18F]3A) and [18F]VUIIS1009B ([18F]3B) exhibited similar biodistribution in healthy mice. In rats bearing C6 gliomas, both [18F]VUIIS1009A and [18F]VUIIS1009B exhibited greater binding potential (k3/k4)in tumor tissue compared to [18F]DPA-714. Interestingly, [18F]VUIIS1009B exhibited significantly greater tumor uptake (VT) than [18F]VUIIS1009A, which was attributed primarily to greater plasma-to-tumor extraction efficiency.ConclusionsThe novel PET ligand [18F]VUIIS1009B exhibits promising characteristics for imaging glioma; its superiority over [18F]VUIIS1009A, a regioisomer, appears to be primarily due to improved plasma extraction efficiency. Continued evaluation of [18F]VUIIS1009B as a high-affinity TSPO PET ligand for precision medicine appears warranted.

Abstract 110: Preclinical evaluation of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide: A novel pyridazinoindole ligand for PET imaging of TSPO in cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA There exists a continued need for the development of improved positron emission tomography (PET) biomarkers for glioma imaging to aid in tumor diagnosis, inform clinical outcome, and quantify response to therapeutic intervention. Translocator protein (TSPO) expression is elevated in cancer and has been linked with disease progression and diminished survival and tends to be a hallmark of aggressive tumors. Our laboratory has pursued development of TSPO PET ligands as candidates for molecular imaging of glioma, as TSPO ligand binding correlates with tumor grade in this setting. We report the first exploration of the structure-activity relationship (SAR) around the pyridazinoindole ring and subsequent radiofluorination of a potent and novel pyridazinoindole-based TSPO-selective ligand for cancer imaging. Library and SAR development around the N3 position of the pyridazinoindole scaffold led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (VUIIS8310), a novel TSPO ligand exhibiting a binding affinity comparable to SSR180575, yet bearing a fluorine atom for subsequent radiolabeling with fluorine-18 (18F) to give 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (18F-VUIIS8310). Initial production feasibility and radiochemical development were evaluated with microfluidics and subsequently translated to traditional box-based methods. Subsequently, quantitative, preclinical elevation of 18F-VUIIS8310 was conducted in a rat glioma model, given our previous experience in this setting. Glioma-bearing rats were imaged in a microPET system, with dynamic PET acquisitions acquired simultaneously upon tracer injection. PET ligand reversibility and specificity were evaluated by ligand displacement studies that utilized VUIIS8310. 18F-VUIIS8310 exhibited elevated uptake and specific, displaceable binding in tumor, in contrast with normal brain, which exhibited very low uptake. 18F-VUIIS8310 uptake in the tumor, relative to normal brain, reached a tumor-to-normal brain ratio of greater than 10:1. Ex vivo histological analysis of resected tissue correlated well with PET imaging data. These preclinical studies illuminate 18F-VUIIS8310 as a promising, novel TSPO PET ligand for imaging glioma and potentially other solid tumors. Importantly, the in vivo stability and high signal-to-noise achieved between tumor and surrounding normal brain suggest the potential of this PET ligand for early tumor detection within this setting. Future head-to-head comparisons between this tracer and emerging, potent TSPO ligands (J. Med. Chem. 2013, 56, 3429) are underway. Citation Format: Yiu-Yin Cheung, Jason R. Buck, Michael L. Nickels, Dewei Tang, H. Charles Manning. Preclinical evaluation of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[18F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide: A novel pyridazinoindole ligand for PET imaging of TSPO in 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 110. doi:10.1158/1538-7445.AM2014-110