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(89)Zr immuno-PET continues to be assessed in numerous clinical trials. This report evaluates the use of (89)Zr-chloride in the radiolabeling of monoclonal antibodies conjugated with desferrioxamine B (DFO), describes its effects on radiopharmaceutical reactivity toward antigen, and offers guidance on how to ensure long-term stability and purity. Methods: (89)Zr-DFO-trastuzumab and (89)Zr-DFO-cetuximab were prepared using (89)ZrCl(4). The stability of each was evaluated for 7 d in 20 mM histidine/240 mM sucrose buffer, 0.25 M sodium acetate (NaOAc) buffer containing 5 mg·mL(−1) n-acetyl-l-cysteine (NAC), or 0.25 M NaOAc containing 5 mg·mL(−1) l-methionine (L-MET). To assess antigen reactivity, (89)Zr-DFO-trastuzumab was evaluated using the Lindmo method and tested in PET/CT imaging of mouse models of human epidermal growth factor receptor 2–positive or –negative lung cancer. Results: Using (89)ZrCl(4), (89)Zr-DFO-trastuzumab and (89)Zr-DFO-cetuximab were prepared with increased specific activity and retained purities of 95% after 3 d when formulated in NaOAc buffer containing L-MET. Based on Lindmo analysis and small-animal PET/CT imaging, (89)Zr-DFO-trastuzumab remained reactive toward antigen after being prepared with (89)ZrCl(4). Conclusion: (89)ZrCl(4) facilitated the radiosynthesis of (89)Zr immuno-PET agents with increased specific activity. L-MET enhanced long-term solution stability better than all other formulations examined, and (89)Zr-DFO-trastuzumab remained reactive toward antigen. Although further evaluation is necessary, these initial results suggest that (89)ZrCl(4) may be useful in immuno-PET radiochemistry as radiolabeled monoclonal antibodies are increasingly integrated into precision medicine strategies.

// Anirudh Sattiraju 1, 2, * , Kiran Kumar Solingapuram Sai 1, 2, * , Ang Xuan 4, * , Darpan N. Pandya 2 , Frankis G. Almaguel 1 , Thaddeus J. Wadas 1, 3 , Denise M. Herpai 2, 3 , Waldemar Debinski 2, 3 and Akiva Mintz 1, 2 1 Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 2 Brain Tumor Center of Excellence, Wake Forest University Comprehensive Cancer Center, Winston-Salem, NC 27157, USA 3 Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA 4 Department of Nuclear Medicine and Radiology, The People’s Hospital of Zhengzhou University, Zhengzhou, Henan 450003, China * These authors contributed equally to this work Correspondence to: Akiva Mintz, email: amintz@wakehealth.edu Keywords: glioblastoma, IL13RA2, alpha particle therapy, Actinium-225, Copper-64 Received: January 10, 2017 Accepted: April 15, 2017 Published: May 11, 2017 ABSTRACT Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [ 64 Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [ 225 Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups ( p < 0.05). GBM tissue extracted from mice treated with [ 225 Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [ 225 Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Peptides that target cancer cell surface receptors are promising platforms to deliver diagnostic and therapeutic payloads specifically to cancer but not normal tissue. IL13RA2 is a tumor-restricted receptor found to be present in several aggressive malignancies, including in the vast majority of high-grade gliomas and malignant melanoma. This receptor has been successfully targeted for diagnostic and therapeutic purposes using modified IL-13 ligand and more recently using a specific peptide, Pep-1L. In the current work, we establish the in vitro and in vivo tumor binding properties of radiolabeled Pep-1L, designed for tumor imaging. We radiolabeled Pep-1L with Copper-64 and demonstrated specific cell uptake in the IL13RA2-over expressing G48 glioblastoma cell line having abundant IL13RA2 expression. [64Cu]Pep-1L binding was blocked by unlabeled ligand, demonstrating specificity. To demonstrate in vivo tumor uptake, we intravenously injected into tumor-bearing mice and demonstrated that [64Cu]Pep-1L specifically bound tumors at 24 hours, which was significantly blocked (3-fold) by pre-injecting unlabeled peptide. To further demonstrate specificity of Pep-1L towards IL13RA2 in vivo, we exploited an IL13RA2-inducible melanoma tumor model that does not express receptor at baseline but expresses abundant receptor after treatment with doxycycline. We injected [64Cu]Pep-1L into mice bearing IL13RA2-inducible melanoma tumors and performed in vivo PET/CT and post-necropsy biodistribution studies and found that tumors that were induced to express IL13RA2 receptor by doxycycline pretreatment bound radiolabeled Pep-1L 3-4 fold greater than uninduced tumors, demonstrating receptor specificity. This work demonstrates that [64Cu]Pep-1L selectively binds hIL13RA2-expressing tumors and validates Pep-1L as an effective platform to deliver diagnostics and therapeutics to IL13RA2-expressing cancers.

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [18F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [18F]KBM-1 was carried out through KHF2 assisted substitution of [18F]- from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [18F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5min to 60min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30min to 60min post injection. Tumor uptake in subset of 30min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [18F]KBM-1 as a RAR-α imaging agent.

We automated radiochemical synthesis of 1-[ 11 C]acetoacetate in a commercially available radiochemistry module, TRASIS AllInOne by [ 11 C]carboxylation of the corresponding enolate anion generated in situ from isopropenylacetate and MeLi, and purified by ion-exchange column resins.1-[ 11 C]acetoacetate was synthesized with high radiochemical purity (95%) and specific activity (~ 66.6 GBq/µmol, n = 30) with 35% radiochemical yield, decay corrected to end of synthesis. The total synthesis required ~ 16 min. PET imaging studies were conducted with 1-[ 11 C]acetoacetate in vervet monkeys to validate the radiochemical synthesis. Tissue uptake distribution was similar to that reported in humans.

Chronic neuropathic pain is a frequent comorbidity following spinal cord injury (SCI) and often fails to respond to conventional pain management strategies. Preventive administration of docosahexaenoic acid (DHA) or the consumption of a diet rich in omega-3 polyunsaturated fatty acids (O3PUFAs) confers potent prophylaxis against SCI and improves functional recovery. The present study examines whether this novel dietary strategy provides significant antinociceptive benefits in rats experiencing SCI-induced pain. Rats were fed control chow or chow enriched with O3PUFAs for 8weeks before being subjected to sham or cord contusion surgeries, continuing the same diets after surgery for another 8 more weeks. The paw sensitivity to noxious heat was quantified for at least 8weeks post-SCI using the Hargreaves test. We found that SCI rats consuming the preventive O3PUFA-enriched diet exhibited a significant reduction in thermal hyperalgesia compared to those consuming the normal diet. Functional neurometabolomic profiling revealed a distinctive deregulation in the metabolism of endocannabinoids (eCB) and related N-acyl ethanolamines (NAEs) at 8weeks post-SCI. We found that O3PUFAs consumption led to a robust accumulation of novel NAE precursors, including the glycerophospho-containing docosahexaenoyl ethanolamine (DHEA), docosapentaenoyl ethanolamine (DPEA), and eicosapentaenoyl ethanolamine (EPEA). The tissue levels of these metabolites were significantly correlated with the antihyperalgesic phenotype. In addition, rats consuming the O3PUFA-rich diet showed reduced sprouting of nociceptive fibers containing CGRP and dorsal horn neuron p38 mitogen-activated protein kinase (MAPK) expression, well-established biomarkers of pain. The spinal cord levels of inositols were positively correlated with thermal hyperalgesia, supporting their role as biomarkers of chronic neuropathic pain. Notably, the O3PUFA-rich dietary intervention reduced the levels of these metabolites. Collectively, these results demonstrate the prophylactic value of dietary O3PUFA against SCI-mediated chronic pain.

The cerebral vasculature incorporates several fail-safes that must be breached before an irreversible ischemic event takes place. In particular, when autoregulatory vasodilatation fails secondary to falling cerebral perfusion pressure (CPP; stage I hemodynamic failure), increases in the oxygen extraction fraction work to maintain the cerebral metabolic rate of oxygen. Previously, failure of this mechanism, stage II hemodynamic failure, or misery perfusion, has been imaged via positron emission tomography/computed tomography (PET/CT). Current susceptibility-weighted sequences (SWI) allow for more efficient imaging of this physiology. In this case, we identify an incident of reversible ischemia caused by spontaneous carotid artery dissection using a combination of diffusion weighted imaging (DWI) and SWI. The level of hemodynamic failure identified by the imaging sequences elevated the urgency of neurointervention, expediting the patient’s arrival to the neurointerventional table and thus avoiding impending irreversible ischemia.

CPI-613 is a lipoate analogue that has been shown to inhibit the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes selectively in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). These two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle and play a central role in mitochondrial metabolism. PDH converts pyruvate into acetyl-CoA, which in turn can enter the TCA cycle for cellular respiration. Since pyruvate is the final product of glycolysis, PDH serves to link glycolysis to the TCA cycle. Early clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. Moreover, apparently homeostatic responses of tumor cells to CPI-613 inhibition of mitochondrial metabolism include compensatory upregulation of glucose uptake. Thus, we hypothesized that we can use 18F-Fludeoxyglucose (FDG)-PET/CT, a translational imaging approach that measures cellular glucose uptake, as a biomarker for CPI-613 cellular delivery and its targeting of mitochondrial metabolism. Therefore, in this work we evaluated the in vitro and in vivo glucose uptake in cancer cells and tumor xenografts after treatment with CPI-613. To measure glucose uptake in vitro we pulsed BxPc3 pancreatic cancer cells with 3H 2-deoxyglucose for 15 or 60 minutes following 2 hours of treatment with CPI-613. We observed a significant upregulation (~100% increase) of cellular 2-deoxyglucose uptake, consistent with a compensatory increase in glucose uptake as a result of successful targeting of mitochondrial metabolism. We therefore examined whether this upregulation occurs in vivo using FDG-PET/CT. Mice bearing BxPc3 flank tumors were treated with 50mg/kg of CPI-613 and underwent FDG-PET/CT scans 4 hours and 24 hours post-CPI-613 treatment. Similar to the in vitro response, tumors treated with CPI-613 exhibited a 75% increase in 18F-FDG uptake compared to untreated controls at 4 hours post therapy. In the 24 hour post-therapy scans, tumor 18F-FDG was significantly decreased, indicating tumor cell killing, which corresponds to previously published data that demonstrated efficacy of CPI-613 against BxPc3 tumor xenografts. These results indicate the potential for using the initial FDG flare seen on PET imaging as a biomarker to detect mitochondrial targeting by CPI-613 immediately after CPI-613 treatment, a strategy that may be used, after further clinical validation, to stratify responders to this novel mitochondrial inhibitor. Citation Format: Kiran Solingapuram Sai, Zuzana Zachar, Frankis Almaguel, Shawn D. Stuart, Michael S. Dahan, Moises Guardado, Stephanie Rideout, Minghui Wang, Anirudh Sattiraju, Paul M. Bingham, Boris Pasche, Akiva Mintz. FDG-PET imaging as a potential biomarker of mitochondrial targeting by CPI-613, a novel inhibitor of mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2017-2867

Background Hormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity. Conclusion These results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.

The tumour microenvironment is believed to be involved in development, growth, metastasis, and therapy resistance of many cancers. Here we show survivin, a member of the inhibitor of apoptosis protein (IAP) family, implicated in apoptosis inhibition and the regulation of mitosis in cancer cells, exists in a novel extracellular pool in tumour cells. Furthermore, we have constructed stable cell lines that provide the extracellular pool with either wild-type survivin (Surv-WT) or the previously described dominant-negative mutant survivin (Surv-T34A), which has proven pro-apoptotic effects in cancer cells but not in normal proliferating cells. Cancer cells grown in conditioned medium (CM) taken from Surv-WT cells absorbed survivin and experienced enhanced protection against genotoxic stresses. These cells also exhibited an increased replicative and metastatic potential, suggesting that survivin in the tumour microenvironment may be directly associated with malignant progression, further supporting survivin's function in tumourigenesis. Alternatively, cancer cells grown in CM taken from the Surv-T34A cells began to apoptose through a caspase-2- and caspase-9-dependent pathway that was further enhanced by the addition of other chemo- and radiotherapeutic modalities. Together our findings suggest a novel microenvironmental function for survivin in the control of cancer aggressiveness and spread, and should result in the genesis of additional cancer treatment modalities.