Delphine L. Chen

[18F]- and [11C]-Labeled N-benzyl-isatin sulfonamide analogues as PET tracers for Apoptosis: synthesis, radiolabeling mechanism, and in vivo imaging study of apoptosis in Fas-treated mice using [11C]WC-98

The radiolabeled isatin sulfonamide caspase-3 inhibitor, [18F] 2 (WC-II-89), is a potential PET radiotracer for noninvasive imaging of apoptosis. The radiolabeling mechanism was studied by 13C NMR, ESI/MS, and computational calculations. It was found that the high electrophilicity of the C3 carbonyl group in the isatin ring, which served as a trap for [18F]fluoride, was responsible for the failure of the radiolabeling via nucleophilic substitution of the mesylate group in 7a by [18F]fluoride. Once treated with a strong base, 7a opened the isatin ring completely to form an isatinate intermediate 16, which lost the ability to trap [18F]fluoride, thereby allowing the displacement of the mesylate group to afford the 18F-labeled isatinate 17. [18F] 17 can be converted to isatin [18F] 2 efficiently under acidic conditions. The ring-opening and re-closure of the isatin ring under basic and acidic conditions were confirmed by reversed phase HPLC analysis, ESI/MS and 13C NMR studies. Computational studies of model compounds also support the above proposed mechanism. Similarly, the ring-opening and re-closure method was used successfully in the synthesis of the 11C labeled isatin sulfonamide analogue [11C] 4 (WC-98). A microPET imaging study using [11C] 4 in the Fas liver apoptosis model demonstrated retained activity in the target organ (liver) of the treated mice. Increased caspase-3 activation in the liver was verified by the fluorometric caspase-3 enzyme assay. Therefore, this study provides a useful method for radio-synthesis of isatin derivative radiotracers for PET and SPECT studies, and [11C] 4 is a potential PET radiotracer for noninvasive imaging of apoptosis.

[18F]Fluorodeoxyglucose Positron Emission Tomography for Lung Antiinflammatory Response Evaluation

RATIONALE Few noninvasive biomarkers for pulmonary inflammation are currently available that can assess the lung-specific response to antiinflammatory treatments. Positron emission tomography with [(18)F]fluorodeoxyglucose (FDG-PET) is a promising new method that can be used to quantify pulmonary neutrophilic inflammation. OBJECTIVES To evaluate the ability of FDG-PET to measure the pulmonary antiinflammatory effects of hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and recombinant human activated protein C (rhAPC) in a human model of experimentally-induced lung inflammation. METHODS Eighteen healthy volunteers were randomized to receive placebo, lovastatin, or rhAPC before intrabronchial segmental endotoxin challenge. FDG-PET imaging was performed before and after endotoxin instillation. The rate of [(18)F]FDG uptake was calculated as the influx constant K(i) by Patlak graphical analysis. Bronchoalveolar lavage (BAL) was performed to determine leukocyte concentrations for correlation with the PET imaging results. MEASUREMENTS AND MAIN RESULTS There was a statistically significant decrease in K(i) in the lovastatin-treated group that was not seen in the placebo-treated group, suggesting attenuation of inflammation by lovastatin treatment despite a small decrease in BAL total leukocyte and neutrophil counts that was not statistically significant. No significant decrease in K(i) was observed in the rhAPC-treated group, correlating with a lack of change in BAL parameters and indicating no significant antiinflammatory effect with rhAPC. CONCLUSIONS FDG-PET imaging is a sensitive method for quantifying the lung-specific response to antiinflammatory therapies and may serve as an attractive platform for assessing the efficacy of novel antiinflammatory therapies at early phases in the drug development process. Clinical trial registered with www.clinicaltrials.gov (NCT00741013).

Comparison of radiolabeled isatin analogs for imaging apoptosis with positron emission tomography

INTRODUCTION Caspase-3 is one of the executioner caspases activated as a result of apoptosis. Radiolabeled isatins bind to caspase-3 with high affinity and are potential tracers for use with positron emission tomography to image apoptosis. We compared the ability of two novel radiolabeled isatins, [18F]WC-IV-3 and [11C]WC-98, to detect caspase-3 activation in a rat model of cycloheximide-induced liver injury. METHODS Male Sprague-Dawley rats were treated with cycloheximide and then imaged with microPET 3 h later with [18F]WC-IV-3 and [11C]WC-98. Biodistribution studies were also performed simultaneously, with caspase-3 activation verified by fluorometric enzyme assay and Western blots. RESULTS MicroPET imaging studies demonstrated similar behavior of both tracers but with a lower maximum peak with [11C]WC-98 than with [18F]WC-IV-3. Biodistribution studies demonstrated increased uptake of both tracers in the liver and spleen, but this was statistically significant only in the liver with both compounds. The level of [18F]WC-IV-3 uptake appeared to correlate roughly with rates of caspase-3 activation by the enzyme assay, but the magnitude of difference between treated and control groups was lower than that observed in previously published data with [18F]WC-II-89, another radiolabeled isatin analog. Activation was also confirmed in the liver and spleen but not in fat by Western blot. CONCLUSION [18F]WC-IV-3 uptake appears to correlate with increased caspase-3 enzyme activity, but the dynamic range of uptake of these two tracers appears to be less than that seen with [18F]WC-II-89. Studies are ongoing to verify these results in other animal models of apoptosis.

Design and synthesis of 2-amino-4-methylpyridine analogues as inhibitors for inducible nitric oxide synthase and in vivo evaluation of [18F]6-(2-fluoropropyl)-4-methyl-pyridin-2-amine as a potential PET tracer for inducible nitric oxide synthase.

A series of position-6 substituted 2-amino-4-methylpyridine analogues was synthesized and compounds 9, 18, and 20 were identified as the inhibitors with the greatest potential to serve as PET tracers for imaging inducible nitric oxide synthase (iNOS). [(18)F]9 was synthesized and evaluated in a mouse model of lipopolysaccharide (LPS)-induced iNOS activation. In vivo biodistribution studies of [(18)F]9 indicate higher tracer uptake in the lungs of the LPS-treated mice when compared to control mice. Tracer uptake at 60 min postinjection was reduced in a blocking study using a known inhibitor of iNOS. The expression of iNOS was confirmed by Western blot analysis of lung samples from the LPS-treated mice. MicroPET studies also demonstrated accumulation of radiotracer in the lungs of the LPS-treated mice. Taken collectively, these data suggest that [(18)F]9 shows favorable properties as a PET tracer to image iNOS activation with PET.

Synthesis, [18F] radiolabeling, and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be (18)F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC50=6.3 nM). [(18)F]12 was synthesized under conventional conditions in high specific activity with 40-50% decay-corrected yield. MicroPET studies using [(18)F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [(18)F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [(18)F]12 in the tumor is specific to PARP-1 expression.

Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody

INTRODUCTION Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice. METHODS The behavior of three different radiolabeled isatins ([(18)F]WC-II-89, [(18)F]WC-IV-3 and [(11)C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [(18)F]WC-II-89 was also compared with [(99m)Tc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [(18)F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay. RESULTS All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [(18)F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [(99m)Tc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [(18)F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver. CONCLUSIONS The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.

Increased T Cell Glucose Uptake Reflects Acute Rejection in Lung Grafts

Although T cells are required for acute lung rejection, other graft–infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [18F]fluorodeoxyglucose ([18F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [18F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [18F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2‐NBDG revealed that T cells, and in particular CD8+ T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen‐presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.

Imaging Pulmonary Inducible Nitric Oxide Synthase Expression with PET

Inducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, 18F-NOS. In this study, we tested whether 18F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers. Methods: Healthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic 18F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. 18F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. 18F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes. Results: Seven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection. Conclusion: 18F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

PET of Poly (ADP-Ribose) Polymerase Activity in Cancer: Preclinical Assessment and First In-Human Studies

Purpose To demonstrate that positron emission tomography (PET) with fluorine 18 (18F) fluorthanatrace (FTT) depicts activated poly (adenosine diphosphate-ribose)polymerase (PARP) expression and is feasible for clinical trial evaluation. Materials and Methods All studies were conducted prospectively from February 2012 through July 2015 under protocols approved by the local animal studies committee and institutional review board. The area under the receiver operating characteristic curve (AUC, in g/mL· min) for 18F-FTT was assessed in normal mouse organs before and after treatment with olaparib (n = 14), a PARP inhibitor, or iniparib (n = 11), which has no PARP inhibitory activity. Murine biodistribution studies were performed to support human translational studies. Eight human subjects with cancer and eight healthy volunteers underwent imaging to verify the human radiation dosimetry of 18F-FTT. The Wilcoxon signed rank test was used to assess for differences among treatment groups for the mouse studies. Results In mice, olaparib, but not iniparib, significantly reduced the 18F-FTT AUC in the spine (median difference before and after treatment and interquartile range [IQR]: -17 g/mL· min and 10 g/mL · min, respectively [P = .0001], for olaparib and -3 g/mL · min and 13 g/mL · min [P = .70] for iniparib) and in nodes (median difference and interquartile range [IQR] before and after treatment: -23 g/mL · min and 13 g/mL · min [P = .0001] for olaparib; -9 g/mL · min and 17 g/mL · min [P = .05] for iniparib). The effective dose was estimated at 6.9 mSv for a 370-MBq 18F-FTT dose in humans. In humans, the organs with the highest uptake on images were the spleen and pancreas. Among five subjects with measurable tumors, increased 18F-FTT uptake was seen in one subject with pancreatic adenocarcinoma and another with liver cancer. Conclusion The results suggest that 18F-FTT uptake reflects PARP expression and that its radiation dosimetry profile is compatible with those of agents currently in clinical use.

Response Assessment Criteria and their Applications in Lymphoma: Part 1

The effectiveness of cancer therapy, both in individual patients and across populations, requires a systematic and reproducible method for evaluating response to treatment. Early efforts to meet this need resulted in the creation of numerous guidelines for quantifying posttherapy changes in disease extent, both anatomically and metabolically. Over the past few years, criteria for disease response classification have been developed for specific cancer histologies. To date, the spectrum of disease broadly referred to as lymphoma is perhaps the most common for which disease response classification is used. This review article provides an overview of the existing response assessment criteria for lymphoma and highlights their respective methodologies and validities. Concerns over the technical complexity and arbitrary thresholds of many of these criteria, which have impeded the long-standing endeavor of standardizing response assessment, are also discussed.