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Introduction Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared 64 Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. Methods DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with 64 Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant ( K d ) and maximum number of binding sites ( B max ) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. Results 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2–3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. 64 Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while 64 Cu-DOTA-mAb7 had higher K d and B max values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for 64 Cu-NODAGA-mAb7. 64 Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo . This was further confirmed by the higher blood activity of 64 Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for 64 Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with 64 Cu-NODAGA-mAb7 in our prostate cancer model. Conclusions To the best of our knowledge, this study represents the first comparison of 64 Cu-labeled DOTA and NODAGA immunoconjugates in vivo . Our results show favorable in vivo performance for 64 Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.

The presence of brown adipose tissue responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Owing to limitations of current methods for brown adipose tissue detection, analysing the abundance and localization of brown adipose tissue in the body has remained challenging. Here we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of brown adipose tissue, but not of intraperitoneal white adipose tissue. We show that in addition to brown adipose tissue, this peptide probe also recognizes the vasculature of brown adipose tissue-like depots of subcutaneous white adipose tissue. Our results indicate that the CPATAERPC peptide localizes to brown adipose tissue even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify brown adipose tissue depots in mice by whole-body near-infrared fluorescence imaging.

Nuclear and near-infrared fluorescence (NIRF) imaging provide non-invasive monitoring of disease. Merging the respective contrast agents through dual labeling could facilitate NIRF validation using proven nuclear imaging methodology while expanding the utility of diagnostic radiotracers.

Summary Chemotherapeutic regimens incorporating taxanes significantly improve outcomes for patients with squamous cell carcinomas of the head and neck (SCCHN). However, treatment with taxanes is limited by toxicities, including bone marrow suppression and peripheral neuropathies. We proposed that conjugating taxanes to targeting carrier molecules would increase antitumor efficacy and decrease toxicity. The cell surface proteoglycan, CD44, is expressed on most SCCHNs, and we hypothesized that it is an attractive candidate for targeted therapy via its natural ligand, hyaluronic acid (HA). We determined whether HA–paclitaxel conjugates were able to decrease tumor growth and improve survival in orthotopic nude mouse human SCCHN xenograft models. HA–paclitaxel concentration-dependent growth inhibition of human SCCHN cell lines OSC-19 and HN5 in vitro, very similarly to free paclitaxel treatment. Tumor cell uptake of FITC-labeled HA–paclitaxel was significantly blocked with free HA, indicating the dependence of uptake on CD44. HA–paclitaxel administered intravenously once per week for three weeks at 120 mg/kg paclitaxel equivalents, far above the paclitaxel maximum tolerated dose, exerted superior tumor growth control to that of paclitaxel in both orthotopic OSC-19-luciferase and HN5 xenograft models in vivo. Mouse survival following HA–paclitaxel administration was prolonged compared with that of controls in mice implanted with either of these xenografts. Mice treated with HA–paclitaxel displayed increased TUNEL(+) cells in tumor tissue, as well as markedly reduced microvessel density compared to those treated with free paclitaxel. No acute histopathological changes were observed in mice treated with HA–paclitaxel. Thus, we conclude that HA–paclitaxel effectively inhibits tumor growth in human SCCHN xenografts via an HA-mediated mechanism and this conjugate should be considered for further preclinical development for this disease.

BACKGROUND Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance. METHODS We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II. RESULTS In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment. CONCLUSIONS Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC. Prostate 67: 1699–1717, 2007. © 2007 Wiley-Liss, Inc.

Paclitaxel (Taxol) has demonstrated clinical activity in non–small-cell lung cancer (NSCLC), but its use has not led to marked improvements in survival. This ineffectiveness can in part be attributed to inadequate delivery of effective drug levels to the lung via systemic administration and to drug resistance mechanisms. Locoregional drug administration and the use of drug copolymers are possible approaches to address these issues. In this study, we evaluated the activity of a poly(l-glutamic acid)-paclitaxel (PGA-TXL) formulation administered by intratracheal injection to mice bearing orthotopic human NSCLC tumors (H460, H358). H460 cells were found to be sensitive to paclitaxel and PGA-TXL in vitro, in a time- and concentration-dependent manner. In preliminary acute toxicity studies, PGA-TXL administered by intratracheal injection was found to be much less toxic than paclitaxel, as anticipated. Mice into which H460 cells had been implanted by intratracheal injection were given single-dose intratracheal treatments with paclitaxel (1.2 or 2.4 mg/kg) or with PGA-TXL (15 mg/kg, paclitaxel equivalents) 1 week later. When the mice were sacrificed at up to 65 days after tumor implantation, they were evaluated grossly for tumor at bronchial, neck, and lung sites. Control mice had tumors in 60% of all three sites, and all of the control mice had tumors in at least one site. The low- and high-dose Taxol groups had fewer incidences at these three sites (27–33%) and 60–80% of these mice had tumors in at least one site. The PGA-TXL mice displayed a low (13%) incidence at these sites, and only 40% had detectable tumors. In a subsequent survival study with the intratracheal H358 model, control mice had a mean life span of 95 days, whereas both the intratracheal Taxol (2.5 mg/kg, every 7th day for three doses) and the intratracheal PGA-TXL (20 mg/kg, paclitaxel equivalents, every 7th day for three doses) groups had improved survival (mean life spans: 133.5 and 136.5 days, respectively). In pilot studies intended to compare the feasibility of the development of paclitaxel aerosols suitable for clinical application, based either on Cremophor solutions or on PGA backbones, only the latter gave acceptable particle size distributions and flow rates. These results encourage the development and application of Cremophor-free copolymer formulations of paclitaxel for locoregional treatment (e.g., as aerosol) of endobronchial malignant diseases.

Specifically designed series of molecules (landers), comprising a central rigid polyaromatic core and several spacers that decouple the board from the metallic surface, have been synthesized. UHV-STM studies, on one hand, have shown important distortions of the molecule by interaction with the substrate. On the other hand, surface restructuring results from the presence of the molecules.

Objectives: Positron Emission Tomography (PET) plays a pivotal role in the surgical care of cancer patients pre- and post-operatively, however, no functional imaging approaches are currently available for real-time surgical guidance. Recently, antibodies dual-labeled with radioactive and near-infrared fluorescent (NIRF) labels have emerged as potential agents to extend whole-body nuclear imaging capabilities into the operating room, but structural limitations in conventional labeling reagents necessitate a random, two-step labeling process which can reduce bioactivity and batch reproducibility. Here, we evaluate the utility of a site-specific multimodal chelator (MMC) that combines PET/NIRF labeling into a single moiety for enhanced production of immunoconjugates for hybrid imaging. Methods: An anti-epithelial cell adhesion molecule (EpCAM)-specific antibody, mAb7, was treated with varying amounts of the reducing agent dithiothreitol (DTT) to partially reduce the interchain disulfides and permit conjugation with MMC-maleimide. The number of MMC moieties per mAb were quantified by isotopic dilution using non-radioactive Cu and immunoreactivity was assessed by ELISA. 64Cu labeling was performed and samples were purified by size exclusion prior to HPLC analysis. Stability of the immunoconjugates was evaluated by HPLC. Multimodal PET/NIRF imaging was performed in an orthotopic prostate tumor model to assess in vivo targeting. Results: mAbs treated with increasing amounts of DTT had higher conjugation ratios, with values ranging from 1-8 MMC moieties per mAb. ELISAs showed no differences in binding potency for the MMC-immunoconjugates. Radiochemical yields were high for all samples (>80%) and increased as a function of conjugation ratio. Interestingly, initial 64Cu labeling of the immunoconjugate with a MMC/mAb ratio of 4 had a corresponding HPLC trace showing >95% radiochemical purity, however, labeling 8 weeks later revealed formation of a second, prominent radioactive peak. Conversely, the HPLC profile of the immunoconjugate with a MMC/mAb ratio of 2 was consistent throughout the study, suggesting a possible correlation between the extent of partial reduction and protein stability. This observation was supported by in vivo imaging findings as lesions were better visualized in mice receiving 64Cu-MMC-mAb7 with a MMC/mAb ratio of 2. Conclusions: The results indicate that site-specific conjugation of MMC is effective for dual labeling antibodies. However, partial reduction conditions must be optimized in order to obtain a multimodal immunoconjugate with suitable imaging properties and stability. Citation Format: Sukhen C. Ghosh, Barrett R. Harvey, Holly Robinson, Kenneth L. Pinkston, Nathaniel Wilganowski, Banghe Zhu, Eva M. Sevick-Muraca, Ali Azhdarinia. Comparison of dual labeling strategies for NIRF/PET hybrid imaging. [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 4298. doi:10.1158/1538-7445.AM2014-4298