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Hyaluronic acid (HA) with one reactive moiety grafted to the backbone is a commonly used matrix in tissue engineering. The addition of a second orthogonal moiety to the backbone allows for greater control in bioactive signal tethering and gelation. In this study, thiol and azide functional groups were grafted to the HA backbone at separate modification sites. NMR, FT-IR, colorimetric assay, and radio-TLC activity were used to confirm and quantify thiol and azide grafting to the HA backbone. Various ratios of di-functional HA (dif HA) and methacrylate HA (mHA) were used to encapsulate mouse embryonic stem cells in order to examine the neural differentiation of the cells. Greater neural maturation was observed in hydrogels containing a higher percentage of dif HA compared to mHA over a six day neural differentiation time course. This formulation was then tested in a contusion spinal cord injury model for biological effect and was found to reduce the ED1+ area in the spinal cord compared to control and allow for host axon extension into the matrix filled lesion area. These results indicate that dif HA is supportive of neural differentiation and can reduce inflammation without additional bioactive signal tethering. dif HA is a promising matrix base for the central nervous system, which should be further developed.

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1–4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing β-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.

A water-soluble octanuclear cluster, [Fe 8 ], was studied with regard to its properties as a potential contrast enhancing agent in magnetic resonance imaging (MRI) in magnetic fields of 1.3, 7.2 and 11.9 T and was shown to have transverse relaxivities r 2 = 4.01, 10.09 and 15.83 mM s −1 , respectively. A related hydrophobic [Fe 8 ] cluster conjugated with 5 kDa hyaluronic acid (HA) was characterized by 57 Fe-Mossbauer and MALDI-TOF mass spectroscopy, and was evaluated in aqueous solutions in vitro with regard to its contrast enhancing properties [r 2 = 3.65 mM s −1 (1.3 T), 26.20 mM s −1 (7.2 T) and 52.18 mM s −1 (11.9 T)], its in vitro cellular cytotoxicity towards A-549 cells and COS-7 cells and its in vivo enhancement of T 2 -weighted images (4.7 T) of a human breast cancer xenografted on a nude mouse. The physiologically compatible [Fe 8 ]-HA conjugate was i.v. injected to the tumor-bearing mouse, resulting in observable, heterogeneous signal change within the tumor, evident 15 min after injection and persisting for approximately 30 min. Both molecular [Fe 8 ] and its HA-conjugate show a strong magnetic field dependence on r 2 , rendering them promising platforms for the further development of T 2 MRI contrast agents in high and ultrahigh magnetic fields.

Passive protection, the administration of antibodies to prevent infection, has garnered significant interest in recent years as a potential prophylactic countermeasure to decrease the prevalence of hospital-acquired infections. Pili, polymerized protein structures covalently anchored to the peptidoglycan wall of many Gram-positive pathogens, are ideal targets for antibody intervention, given their importance in establishing infection and their accessibility to antibody interactions. In this work, we demonstrated that a monoclonal antibody to the major component of Enterococcus faecalis pili, EbpC, labels polymerized pilus structures, diminishes biofilm formation, and significantly prevents the establishment of a rat endocarditis infection. The effectiveness of this anti-EbpC monoclonal provides strong evidence in support of its potential as a preventative. In addition, after radiolabeling, this monoclonal identified the site of enterococcal infection, providing a rare example of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications.

Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

Dual-labeled compounds containing nuclear and near-infrared fluorescence contrast have the potential to molecularly guide surgical resection of cancer by extending whole-body diagnostic imaging findings into the surgical suite. To simplify the dual labeling process for antibody-based agents, we designed a multimodality chelation (MMC) scaffold which combined a radiometal chelating agent and fluorescent dye into a single moiety. Three dye-derivatized MMC compounds were synthesized and radiolabeled. The IRDye 800CW conjugate, 4, had favorable optical properties and showed rapid clearance in vivo. Using 4, an epithelial cell adhesion molecule (EpCAM) targeting MMC-immunoconjugate was prepared and dual-labeled with (64)Cu. In vitro binding activity was confirmed after MMC conjugation. Multimodal imaging studies showed higher tumor accumulation of (64)Cu-7 compared to nontargeted (64)Cu-4 in a prostate cancer model. Further evaluation in different EpCAM-expressing cell lines is warranted as well as application of the MMC dual labeling approach with other monoclonal antibodies.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein–coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody–drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580–90. ©2016 AACR.

Purpose: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid. Metronomic dosing, the frequent administration of chemotherapeutics at substantially lower than maximum tolerated doses (MTD), has been shown to result in reduced normal tissue toxicity and to minimize “off-treatment” exposure resulting in an improved therapeutic ratio. Experimental Design: We tested the hypothesis that hyaluronic acid (HA) conjugates of paclitaxel (TXL; HA-TXL) would exert strong antitumor effects with metronomic (MET) dosing and induce antiangiogenic effects superior to those achieved with MTD administration or with free TXL. Female nude mice bearing SKOV3ip1 or HeyA8 ovarian cancer cells were treated intraperitoneally (i.p.) with MET HA-TXL regimens (or MTD administration) to determine therapeutic and biologic effects. Results: All MET HA-TXL–treated mice and the MTD group revealed significantly reduced tumor weights and nodules compared with controls (all P values < 0.05) in the chemotherapy-sensitive models. However, the MTD HA-TXL–treated mice showed significant weight loss compared with control mice, whereas body weights were not affected in the metronomic groups in HeyA8-MDR model, reflecting reduced toxicity. In the taxane-resistant HeyA8-MDR model, significant reduction in tumor weight and nodule counts was noted in the metronomic groups whereas the response of the MTD group did not achieve significance. While both MTD and metronomic regimens reduced proliferation (Ki-67) and increased apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), only metronomic treatment resulted in significant reductions in angiogenesis (CD31, microvessel density). Moreover, metronomic treatment resulted in substantial increases in thrombospondin-1 (Tsp-1), an inhibitor of angiogenesis. Conclusions: This study showed that MET HA-TXL regimens have substantial antitumor activity in ovarian carcinoma, likely via a predominant antiangiogenic mechanism. Clin Cancer Res; 18(15); 4114–21. ©2012 AACR.

BACKGROUND: Although inflammatory breast cancer (IBC) is recognized as the most lethal variant of locally advanced breast cancer, few molecular signatures of IBC have been identified that can be used as targets to develop therapeutics that effectively inhibit the aggressive phenotype displayed by IBC tumors. METHODS: Real-time polymerase chain reaction analysis, Western blot analysis, modified Boyden chamber invasion assays, vasculogenic mimicry (VM) assays, and gelatin zymography were used in the current studies. Agonists and antagonists of the prostanoid receptors EP3 and EP4 and of EP4 short-hairpin RNA (shRNA) knockdown approaches were used as tools to assess the role of prostanoid receptors EP3 and EP4 in the regulation of specific biologic activities of IBC cells. RESULTS: The current studies revealed that the IBC breast cancer cell lines SUM149 and SUM190 express high levels of cyclooxygenase-2 messenger RNA and protein, produce abundant levels of prostaglandin E2, and produce both EP3 and EP4 receptor proteins. Studies using the EP4 antagonist GW627368X and shRNA molecular knockdown approaches revealed a role for EP4 in regulating invasion of IBC cells. EP3, but not EP4, regulated the ability of SUM149 cells to undergo VM, which is the ability to form capillary-like structures, a characteristic exhibited by very aggressive tumor types. Inhibition of VM by sulprostone was associated with an inhibition of matrix metalloprotease-2 (MMP-2) enzyme activity. CONCLUSIONS: The prostanoid receptors EP3 and EP4 differentially regulate activities exhibited by IBC cells that have been associated with the aggressive phenotype of this lethal variant of breast cancer. Whereas EP4 regulates invasion, EP3 regulates VM and the associated increased MMP-2 enzyme activity. Cancer 2010;116(11 suppl):2806–14. © 2010 American Cancer Society.

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Poly- l -glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N -dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA–DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA–DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA–DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.

Numerous human tumor types, including ovarian cancer, display a significant expression of the CD44 family of cell surface proteoglycans. To develop tumortargeted drugs, we have initially evaluated whether the CD44 ligand hyaluronic acid (HA) could serve as a backbone for paclitaxel (TXL) prodrugs. HA-TXL was prepared by modification of previous techniques. The in vitro cytotoxicity of HA-TXL against the CD44(+) human ovarian carcinoma cell lines SKOV-3ip and NMP-1 could be significantly blocked by preincubation with a molar excess of free HA. Female nude mice bearing intraperitoneal implants of NMP-1 cells were treated intraperitoneally with a single sub-maximum tolerated dose dose of HA-TXL or with multiple-dose regimens of paclitaxel (Taxol; Mead Johnson, Princeton, NJ) to determine the effects of these regimens on host survival and intraperitoneal tumor burden, with the latter being assessed by magnetic resonance imaging. NMP-1 xenograffs were highly resistant to Taxol regimens, as host survival was only nominally improved compared to controls (T/C ∼ 120), whereas singledose HA-TXL treatment significantly improved survival in this model (T/C ∼ 140; P = .004). In both NMP-1 and SKOV-3ip models, MR images of abdomens of HA-TXL-treated mice obtained shortly before controls required humane sacrifice revealed markedly reduced tumor burdens compared to control mice. This study is among the first to demonstrate that HA-based prodrugs administered locoregionally have antitumor activity in vivo.

Coumarin derivatives of N,N-dimethylsphingosine (DMSP) were prepared and chemically characterized. They were apparently biologically equivalent to DMSP in terms of tumor cell cytotoxicity and were used to establish the rapid mitochondrial localization of this sphingolipid in tumor cells, followed closely by its marked reduction of mitochondrial membrane potential.

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.

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.

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