PEG Conjugated Zein Nanoparticles for In Vivo Use.

Zein can be utilized to form nanoscale particles for drug delivery applications. Despite the ease of synthesis, these particles often aggregate when exposed to physiologically relevant conditions (e.g., pH and salt concentrations). This instability has prevented their further development in applications requiring intravenous administration. To mitigate this colloidal instability, this research explored Zein nanoparticles (NP)s that were modified with polyethylene glycol (PEG) either through functionalized PEG pre- or post-NP formation. The results suggest that the pre-functionalization of the Zein using N-hydroxysuccinimide ester terminated PEG is the method of choice for synthesizing Zein NPs with conjugated PEG (Zein:PEG-Zein NPs). Zein:PEG-Zein NPs formed using this method displayed excellent stability in physiologically relevant conditions over 72 h and were stable at 4 °C for at least 3 months. When the NPs were cultured with cells for 72 h, no cytotoxicity or early signs of apoptosis were identified. Cellular uptake of the Zein:PEG-Zein NPs did not seem to be impacted by the amount of PEG incorporated in the NP but were concentration-, time-, and temperature-dependent. The lowest percent, stable Zein:PEG-Zein NP formulation (80% unmodified Zein and 20% PEG-modified Zein) induced no observable toxicity over 14 days in CD-1 mice dosed at 70 mg/kg via the tail vein. However, repeat dose pharmacokinetic (PK) studies demonstrated that following the first dose, the second dose caused health issues that required euthanasia shortly after administration. For those animals that survived, there was faster plasma elimination of the Zein:PEG-Zein NPs. Despite this, the Zein:PEG-Zein NPs represent a significantly improved formulation approach, one that displays a long circulation half-life and is suitable for single-use administration. Repeat dose applications will require additional methods to silence the immune response that is generated when using these NPs intravenously.

Gold-Protein Composite Nanoparticles for Enhanced X-ray Interactions: A Potential Formulation for Triggered Release.

Drug-delivery vehicles have been used extensively to modulate the biodistribution of drugs for the purpose of maximizing their therapeutic effects while minimizing systemic toxicity. The release characteristics of the vehicle must be balanced with its encapsulation properties to achieve optimal delivery of the drug. An alternative approach is to design a delivery vehicle that preferentially releases its contents under specific endogenous (e.g., tissue pH) or exogenous (e.g., applied temperature) stimuli. In the present manuscript, we report on a novel delivery system with potential for triggered release using external beam radiation. Our group evaluated Zein protein as the basis for the delivery vehicle and used radiation as the exogenous stimulus. Proteins are known to react with free radicals, produced during irradiation in aqueous suspensions, leading to aggregation, fragmentation, amino acid modification, and proteolytic susceptibility. Additionally, we incorporated gold particles into the Zein protein matrix to create hybrid Zein-gold nanoparticles (ZAuNPs). Zein-only nanoparticles (ZNPs) and ZAuNPs were subsequently exposed to kVp radiation (single dose ranging from 2 to 80 Gy; fractionated doses of 2 Gy delivered 10 times) and characterized before and after irradiation. Our data indicated that the presence of gold particles within Zein particles was correlated with significantly higher levels of alterations to the protein, and was associated with higher rates of release of the encapsulated drug compound, Irinotecan. The aggregate results demonstrated a proof-of-principle that radiation can be used with gold nanoparticles to modulate the release rates of protein-based drug-delivery vehicles, such as ZNPs.

Tuning the Surface Chemistry of Second-Harmonic-Active Lithium Niobate Nanoprobes Using a Silanol-Alcohol Condensation Reaction.

The surface functionalization of nanoparticles (NPs) is of great interest for improving the use of NPs in, for example, therapeutic and diagnostic applications. The conjugation of specific molecules with NPs through the formation of covalent linkages is often sought to provide a high degree of colloidal stability and biocompatibility, as well as to provide functional groups for further surface modification. NPs of lithium niobate (LiNbO3) have been explored for use in second-harmonic-generation (SHG)-based bioimaging, expanding the applications of SHG-based microscopy techniques. The efficient use of SHG-active LiNbO3 NPs as probes will, however, require the functionalization of their surfaces with molecular reagents such as polyethylene glycol and fluorescent molecules to enhance their colloidal and chemical stability and to enable a correlative imaging platform. Herein, we demonstrate the surface functionalization of LiNbO3 NPs through the covalent attachment of alcohol-based reagents through a silanol-alcohol condensation reaction. Alcohol-based reagents are widely available and can have a range of terminal functional groups such as carboxylic acids, amines, and aldehydes. Attaching these molecules to NPs through the silanol-alcohol condensation reaction could diversify the reagents available to modify NPs, but this reaction pathway must first be established as a viable route to modifying NPs. This study focuses on the attachment of a linear alcohol functionalized with carboxylic acid and its use as a reactive group to further tune the surface chemistry of LiNbO3 NPs. These carboxylic acid groups were reacted to covalently attach other molecules to the NPs using copper-free click chemistry. This derivatization of the NPs provided a means to covalently attach polyethylene glycols and fluorescent probes to the NPs, reducing NP aggregation and enabling multimodal tracking of SHG nanoprobes, respectively. This extension of the silanol-alcohol condensation reaction to functionalize the surfaces of LiNbO3 NPs can be extended to other types of nanoprobes for use in bioimaging, biosensing, and photodynamic therapies.

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

BACKGROUND & AIMS: Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. METHODS: We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. RESULTS: Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. CONCLUSIONS: In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

Spatially Specific Liposomal Cancer Therapy Triggered by Clinical External Sources of Energy.

This review explores the use of energy sources, including ultrasound, magnetic fields, and external beam radiation, to trigger the delivery of drugs from liposomes in a tumor in a spatially-specific manner. Each section explores the mechanism(s) of drug release that can be achieved using liposomes in conjunction with the external trigger. Subsequently, the treatment's formulation factors are discussed, highlighting the parameters of both the therapy and the medical device. Additionally, the pre-clinical and clinical trials of each triggered release method are explored. Lastly, the advantages and disadvantages, as well as the feasibility and future outlook of each triggered release method, are discussed.

Inactivation of the Kinase Domain of CDK10 Prevents Tumor Growth in a Preclinical Model of Colorectal Cancer, and Is Accompanied by Downregulation of Bcl-2.

Cyclin-dependent kinase 10 (CDK10), a CDC2-related kinase, is highly expressed in colorectal cancer. Its role in the pathogenesis of colorectal cancer is unknown. This study examines the function of CDK10 in colorectal cancer, and demonstrates its role in suppressing apoptosis and in promoting tumor growth in vitro and in vivo Modulation of CDK10 expression in colorectal cancer cell lines demonstrates that CDK10 promotes cell growth, reduces chemosensitivity and inhibits apoptosis by upregulating the expression of Bcl-2. This effect appears to depend on its kinase activity, as kinase-defective mutant colorectal cancer cell lines have an exaggerated apoptotic response and reduced proliferative capacity. In vivo, inhibiting CDK10 in colorectal cancer following intratumoral injections of lentivirus-mediated CDK10 siRNA in a patient-derived xenograft mouse model demonstrated its efficacy in suppressing tumor growth. Furthermore, using a tissue microarray of human colorectal cancer tissues, the potential for CDK10 to be a prognostic biomarker in colorectal cancer was explored. In tumors of individuals with colorectal cancer, high expression of CDK10 correlates with earlier relapse and shorter overall survival. The findings of this study indicate that CDK10 plays a role in the pathogenesis in colorectal cancer and may be a potential therapeutic target for treatment. Mol Cancer Ther; 16(10); 2292-303. ©2017 AACR.

The differential effects of metronomic gemcitabine and antiangiogenic treatment in patient-derived xenografts of pancreatic cancer: treatment effects on metabolism, vascular function, cell proliferation, and tumor growth.

BACKGROUND: Metronomic chemotherapy has shown promising activity against solid tumors and is believed to act in an antiangiogenic manner. The current study describes and quantifies the therapeutic efficacy, and mode of activity, of metronomic gemcitabine and a dedicated antiangiogenic agent (DC101) in patient-derived xenografts of pancreatic cancer. METHODS: Two primary human pancreatic cancer xenograft lines were dosed metronomically with gemcitabine or DC101 weekly. Changes in tumor growth, vascular function, and metabolism over time were measured with magnetic resonance imaging, positron emission tomography, and immunofluorescence microscopy to determine the anti-tumor effects of the respective treatments. RESULTS: Tumors treated with metronomic gemcitabine were 10-fold smaller than those in the control and DC101 groups. Metronomic gemcitabine, but not DC101, reduced the tumors' avidity for glucose, proliferation, and apoptosis. Metronomic gemcitabine-treated tumors had higher perfusion rates and uniformly distributed blood flow within the tumor, whereas perfusion rates in DC101-treated tumors were lower and confined to the periphery. DC101 treatment reduced the tumor's vascular density, but did not change their function. In contrast, metronomic gemcitabine increased vessel density, improved tumor perfusion transiently, and decreased hypoxia. CONCLUSION: The aggregate data suggest that metronomic gemcitabine treatment affects both tumor vasculature and tumor cells continuously, and the overall effect is to significantly slow tumor growth. The observed increase in tumor perfusion induced by metronomic gemcitabine may be used as a therapeutic window for the administration of a second drug or radiation therapy. Non-invasive imaging could be used to detect early changes in tumor physiology before reductions in tumor volume were evident.

Overexpression of HER-2 in MDA-MB-435/LCC6 Tumours is Associated with Higher Metabolic Activity and Lower Energy Stress.

Overexpresssion of HER-2 in the MDA-MB-435/LCC6 (LCC6(HER-2)) tumour model is associated with significantly increased hypoxia and reduced necrosis compared to isogenic control tumours (LCC6(Vector)); this difference was not related to tumour size or changes in vascular architecture. To further evaluate factors responsible for HER-2-associated changes in the tumour microenvironment, small animal magnetic resonance imaging (MRI) and positron emission tomography (PET) were used to measure tumour tissue perfusion and metabolism, respectively. The imaging data was further corroborated by analysis of molecular markers pertaining to energy homeostasis, and measurements of hypoxia and glucose consumption. The results showed a strong trend towards higher perfusion rates (~58% greater, p = 0.14), and significantly higher glucose uptake in LCC6(HER-2) (~2-fold greater; p = 0.025), relative to control tumours. The expression of proteins related to energy stress (P-AMPK, P-ACC) and glucose transporters (GLUT1) were lower in LCC6(HER-2) tumours (~2- and ~4-fold, respectively). The in vitro analysis showed that LCC6(HER-2) cells become more hypoxic in 1% oxygen and utilise significantly more glucose in normoxia compared to LCC6(Vector)cells (p < 0.005). Amalgamation of all the data points suggests a novel metabolic adaptation driven by HER-2 overexpression where higher oxygen and glucose metabolic rates produce rich energy supply but also a more hypoxic tumour mass.

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization.

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

Nitroimidazole-Containing H2dedpa and H2CHXdedpa Derivatives as Potential PET Imaging Agents of Hypoxia with (68)Ga.

(68)Ga is an attractive radiometal for use in positron emission tomography (PET) imaging. The success of (68)Ga-based agents is dependent on a chelator that exhibits rapid radiometal incorporation, and strong kinetic inertness to prevent transchelation of (68)Ga in vivo. The linear chelating agents H2dedpa (1,2-[[6-carboxypyridin-2-yl]methylamino]ethane) and H2CHXdedpa (CHX = cyclohexyl/cyclohexane) (N4O2) have recently been developed that bind Ga(3+) quickly and under mild conditions, ideal properties to be incorporated into a (68)Ga PET imaging agent. Herein, nitroimidazole (NI) derivatives of H2dedpa and H2CHXdedpa to investigate specific targeting of hypoxic tumor cells are investigated, given that NI can be reduced and retained exclusively in hypoxic cells. Nine N,N'-bis-alkylated derivatives of H2dedpa and H2CHXdedpa have been synthesized; they have been screened for their ability to bind gallium, and cyclic voltammetry of nonradioactive complexes was performed to probe the redox cycling mechanism of NI. The compounds were radiolabeled with (67)Ga and (68)Ga and show promising radiolabeling efficiencies (>99%) when labeled at 10(-5) M for 10 min at room temperature. Moreover, stability studies (via apo-transferrin challenge, 37 °C) show that the (67)Ga complexes exhibit exceptional stability (86-99% intact) after 2 h. In vitro uptake studies under hypoxic (0.5% O2) and normoxic (21% O2) conditions in three cancerous cell lines [HT-29 (colon), LCC6(HER-2) (breast), and CHO (Chinese hamster ovarian)] were performed. Of the four H2dedpa or H2CHXdedpa NI derivatives tested, all showed preferential uptake in hypoxic cells compared to normoxic cells with hypoxic/normoxic ratios as high as 7.9 ± 2.7 after 120 min. The results suggest that these novel bis-alkylated NI-containing H2dedpa and H2CHXdedpa ligands would be ideal candidates for further testing in vivo for PET imaging of hypoxia with (68)Ga.

The involvement of insulin-like growth factor 2 binding protein 3 (IMP3) in pancreatic cancer cell migration, invasion, and adhesion.

BACKGROUND: Over-expression of insulin-like growth factor 2 mRNA binding protein 3 (IMP3) is correlated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Previous studies examining other cancer types have implicated IMP3 in the regulation of several cellular functions that are characteristic of tumour cells. However, the role of this oncofetal protein in PDAC progression remained unclear. METHODS: Using siRNA, we examined the effect of IMP3 inhibition on the motility, invasive ability, and matrix adhesion of PDAC cells. In addition, we also evaluated the expression of cytoskeleton-associated genes following IMP depletion. RESULTS: Knockdown of IMP3 significantly decreased the motility, invasion, and extracellular matrix adhesion of select PDAC cells in vitro. In addition, IMP3-depleted cells exhibited lower levels of CD44 protein and KIF11 mRNA. Moreover, we also observed a reduction in downstream RhoA signaling following IMP3 knockdown, indicating that IMP3 modulates the levels of proteins involved in cytoskeletal organization. CONCLUSIONS: These results suggest that IMP3 facilitates PDAC progression by enhancing the pro-metastatic behaviour of tumour cells.

Irinophore C™, a lipid nanoparticulate formulation of irinotecan, improves vascular function, increases the delivery of sequentially administered 5-FU in HT-29 tumors, and controls tumor growth in patient derived xenografts of colon cancer.

PURPOSE: A liposomal formulation of irinotecan, Irinophore C™ (IrC™) is efficacious in a panel of tumor models, normalizes tumor vasculature, and increases the accumulation of a second drug in the same tumor. We now show that Irinophore C™ is also effective against patient derived xenografts (PDX) of colon cancer, and examine the kinetics of vascular normalization in the HT-29 tumor model and assess how these changes might be used with 5-FU sequentially. MATERIALS AND METHODS: Rag2M mice bearing HT-29 tumors were treated with IrC™ (25mg/kg; Q7D×3) for up to three weeks. Groups of tumors were harvested for analysis at 7, 14 and 21days after the start of treatment. Drug and lipid levels in the tumor were evaluated using HPLC and scintillation counts, respectively. Changes in tumor morphology (H&E), vasculature (CD31), perfusion (Hoechst 33342) and apoptosis (TUNEL) were quantified using microscopy. The accumulation of a second drug ([(14)C]-5-FU, 40mg/kg) given 3h before sacrifice was determined using liquid scintillation. The efficacy of IrC™ (Q7D×3) followed by 5-FU treatment (Q7D×3) was assessed in mice bearing established HT-29 tumors. The efficacy of IrC™ was also evaluated in primary human colorectal tumors grown orthotopically in NOD-SCID mice. RESULTS: Following a single dose of IrC™ the active lactone forms of irinotecan and its metabolite SN-38 were measurable in HT-29 tumors after 7days. The treatment reduced tumor cell density and increased apoptosis. Hoechst 33342 perfusion and accumulation of [(14)C]-5-FU in the treated tumors increased significantly on days 7 and 14. This was accompanied by an increase in the number of endothelial cells relative to total nuclei in the tumor sections. Pre-treatment with IrC™ (Q7D×3) followed by 5-FU (Q7D×3) delayed the time taken for tumors to reach 1cm(3) by 9days (p<0.05). IrC™ was just as effective as free irinotecan when used on patient derived xenografts of colorectal cancer. CONCLUSIONS: Treatment with IrC™ reduces tumor cell viability and appears to normalize the vascular function of the tumor after a single treatment cycle. A concomitant increase in the accumulation of a second drug (5-FU) in the tumor was observed in tumors from IrC™ treated animals and this was correlated with changes in vascular structure consistent with normalization. The treatment effects of sequential 5-FU dosing following IrC™ are additive with no additional toxicity in contrast to previous studies where concurrent 5-FU and IrC™ treatment exacerbated 5-FU toxicity. The studies with PDX tumors also indicate that IrC™ is just as effective as free irinotecan on PDX tumors even though the delivered dose is halved.

A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the x(C)⁻ transporter.

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).

Dual mode fluorescent (18)F-PET tracers: efficient modular synthesis of rhodamine-[cRGD]2-[(18)F]-organotrifluoroborate, rapid, and high yielding one-step (18)F-labeling at high specific activity, and correlated in vivo PET imaging and ex vivo fluorescence.

The design of dual mode fluorescent-PET peptidic tracers that can be labeled with [(18)F]fluoride at high specific activity and high yield has been challenged by the short half-life of (18)F and its aqueous indolence toward nucleophilic displacement, that often necessitates multistep reactions that start with punctiliously dry conditions. Here we present a modular approach to constructing a fluorescent dimeric peptide with a pendant radioprosthesis that is labeled in water with [(18)F]fluoride ion in a single, user-friendly step. The modular approach starts with grafting a new zwitterionic organotrifluoroborate radioprosthesis onto a pentaerythritol core with three pendent alkynes that enable successive grafting of a bright fluorophore (rhodamine) followed by two peptides (cylcoRGD). The construct is labeled with [(18)F]fluoride via isotope exchange within 20 min in a single step at high specific activity (>3 Ci/µmol) and in good yield to provide 275 mCi and high radiochemical purity. Neither drying of the [(18)F]fluoride ion solution nor HPLC purification of the labeled tracer is required. Facile chemical synthesis of this dual mode tracer along with a user-friendly one-step radiolabeling method affords very high specific activity. In vivo PET images of the dual mode tracer are acquired at both high and low specific activities. At very high specific activity, i.e., 3.5 Ci/µmol, tumor uptake is relatively high (5.5%ID/g), yet the associated mass is below the limits of fluorescent detection. At low specific activity, i.e., 0.01 Ci/µmol, tumor uptake in the PET image is reduced by approximately 50% (2.9%ID/g), but the greater associated mass enables fluorescence detection in the tumor. These data highlight a facile production of a dual mode fluorescent-PET tracer which is validated with in vivo and ex vivo images. These data also define critical limitations for the use of dual mode tracers in small animals.

Autophagy inhibition augments the anticancer effects of epirubicin treatment in anthracycline-sensitive and -resistant triple-negative breast cancer.

PURPOSE: Triple-negative breast cancers (TNBC) are defined by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2). Although initially responsive to chemotherapy, most recurrent TNBCs develop resistance, resulting in disease progression. Autophagy is a lysosome-mediated degradation and recycling process that can function as an adaptive survival response during chemotherapy and contribute to chemoresistance. Our goal was to determine whether autophagy inhibition improves treatment efficacy in TNBC cells in tumors either sensitive or refractory to anthracyclines. EXPERIMENTAL DESIGN: We used in vitro and in vivo models of TNBC using cell lines sensitive to epirubicin and other anthracyclines, as well as derivative lines, resistant to the same drugs. We assessed basal autophagy levels and the effects of chemotherapy on autophagy in parental and resistant cells. Applying various approaches to inhibit autophagy alone and in combination with chemotherapy, we assessed the effects on cell viability in vitro and tumor growth rates in vivo. RESULTS: We demonstrated that epirubicin induced autophagic flux in TNBC cells. Epirubicin-resistant lines exhibited at least 1.5-fold increased basal autophagy levels and, when treated with autophagy inhibitors, showed a significant loss in viability, indicating dependence of resistant cells on autophagy for survival. Combination of epirubicin with the autophagy inhibitor hydroxychloroquine resulted in a significant reduction in tumor growth compared with monotherapy with epirubicin. CONCLUSION: Autophagy inhibition enhances therapeutic response in both anthracycline-sensitive and -resistant TNBC and may be an effective new treatment strategy for this disease.

Functional imaging of oxidative stress with a novel PET imaging agent, 18F-5-fluoro-L-aminosuberic acid.

UNLABELLED: Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc(-)) represents a potentially attractive PET biomarker to enable in vivo quantification of xc(-) activity in response to oxidative stress associated with disease. We have developed a system xc(-) substrate that incorporates characteristics of both natural substrates, L-cystine and L-glutamate (L-Glu). L-aminosuberic acid (L-ASu) has been identified as a more efficient system xc(-) substrate than L-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, (18)F-5-fluoro-aminosuberic acid ((18)F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. METHODS: (18)F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to L-cystine and L-Glu. Affinity toward system xc(-) was determined via in vitro uptake and inhibition studies using oxidative stress-induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). RESULTS: In vitro assay results determined that L-ASu inhibited system xc(-) as well as or better than L-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc(-) uptake of L-ASu than L-Glu. Radiosynthesis of (18)F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. (18)F-FASu uptake was approximately 5 times greater than (18)F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. CONCLUSION: (18)F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. (18)F-FASu may provide more sensitive detection than (18)F-FDG in certain tumors.

Increasing the bioavailability of Ru(III) anticancer complexes through hydrophobic albumin interactions.

A series of pyridine-based derivatives of the clinically successful Ru(III)-based complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (KP1339) have been synthesized to probe the effect of hydrophobic interactions with human serum albumin (hsA) on anticancer activity. The solution behavior and protein interactions of the new compounds were characterized by using electron paramagnetic resonance (EPR) and UV/Vis spectroscopy. These studies have revealed that incorporation of hydrophobic substituents at the 4'-position of the axial pyridine ligand stabilizes non-coordinate interactions with hsA. As a consequence, direct coordination to the protein is inhibited, which is expected to increase the bioavailability of the complexes, thus potentially leading to improved anticancer activity. By using this approach, the lifetimes of hydrophobic protein interactions were extended from 2 h for the unsubstituted pyridine complex, to more than 24 h for several derivatives. Free complexes were tested for their anticancer activity against the SW480 human colon carcinoma cell line, exhibiting low cytotoxicity. Pre-treatment with hsA improved the solubility of every compound and led to some changes in activity. Particularly notable was the difference in activity between the methyl- and dibenzyl-functionalized complexes. The former shows reduced activity after incubation with hsA, indicating reduced bioavailability due to protein coordination. The latter exhibits little activity on its own but, following treatment with hsA, exhibited significant cytotoxicity, which is consistent with its ability to form non-coordinate interactions with the protein. Overall, our studies demonstrate that non-coordinate interactions with hsA are a viable target for enhancing the activity of Ru(III)-based complexes in vivo.

Synthesis, characterization, and biological studies of emissive rhenium-glutamine conjugates.

Two new rhenium complexes containing pyridine-triazole (pyta) and quinoline-triazole (quinta) ligands with attached glutamine-targeting agents have been characterized and tested for uptake in the HT-29 human colon adenocarcinoma cell line. The glutamine moiety in Re(CO)3Br(pyta) (1) and Re(CO)3Br(quinta) (2) remains pendant in solution. Both complexes exhibit absorptions in the 300-400-nm range with metal-to-ligand charge transfer (MLCT) character, as predicted by time-dependent density functional theory calculations. Geometrical analysis by theoretical calculations provides information on the cationic complexes 1 (+) and 2 (+) resulting from aquo for halide ligand exchange under aqueous conditions. The emissive properties of both complexes were studied under aqueous conditions, and the measured quantum yields were 0.46 % for 1 (+) and 0.18 % for 2 (+). The large Stokes shifts and oxygen sensitivity of the emission suggest a (3)MLCT process for both complexes. Cell studies in the HT-29 cell line demonstrate that both complexes are nontoxic over a large concentration range (0-1.4 mM). Preliminary uptake studies show that 2 (+), but not 1 (+), displays significant concentration-dependent uptake at 3 and 24 h.

Imaging tumor vasculature noninvasively with positron emission tomography and RGD peptides labeled with copper 64 using the bifunctonal chelates DOTA, oxo-DO3a. and PCTA.

Two novel bifunctional chelates, 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (Oxo-DO3A), were found to radiolabel antibodies with copper 64 (64Cu) well for positron emission tomography (PET). In this study, the same chelators were used to radiolabel peptides with 64Cu for PET imaging of angiogenesis. PCTA, Oxo-DO3A, and 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) were conjugated to cyclic-(RGDyK), and their binding affinities were confirmed. Conditions for 64Cu radiolabeling were optimized for maximum yield and specific activity. The in vitro stability of the radiolabeled compounds was challenged with serum incubation. PET studies were carried out in a non-αvß3-expressing tumor model to evaluate the compounds' specificity for proliferating tumor vasculature and their in vivo pharmacokinetics. The PCTA and Oxo-DO3A bioconjugates were labeled with 64Cu at higher effective specific activity and radiochemical yield than the DOTA bioconjugate. In the imaging studies, all the 64Cu bioconjugates could be used to visualize the tumor and the radiotracer uptake was blocked with cyclic-(RGDyK). Target uptake of each bioconjugate was similar, but differences in other tissues were observed. 64Cu-PCTA-RGD showed the best clearance from nontarget tissue and the highest tumor to nontarget ratios. PCTA was the most promising bifunctional chelate for 64Cu peptide imaging and warrants further investigation.

(68)Ga small peptide imaging: comparison of NOTA and PCTA.

In this study, a bifunctional version of the chelate PCTA was compared to the analogous NOTA derivative for peptide conjugation, (68)Ga radiolabeling, and small peptide imaging. Both p-SCN-Bn-PCTA and p-SCN-Bn-NOTA were conjugated to cyclo-RGDyK. The resulting conjugates, PCTA-RGD and NOTA-RGD, retained their affinity for the peptide target, the α(v)ß(3) receptor. Both PCTA-RGD and NOTA-RGD could be radiolabeled with (68)Ga in >95% radiochemical yield (RCY) at room temperature within 5 min. For PCTA-RGD, higher effective specific activities, up to 55 MBq/nmol, could be achieved in 95% RCY with gentle heating at 40 °C. The (68)Ga-radiolabeled conjugates were >90% stable in serum and in the presence of excess apo-transferrin over 4 h; (68)Ga-PCTA-RGD did have slightly lower stability than (68)Ga-NOTA-RGD, 93 ± 2% compared to 98 ± 1%, at the 4 h time point. Finally, the tumor and nontarget organ uptake and clearance of (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD was compared in mice bearing HT-29 colorectal tumor xenografts. Activity cleared quickly from the blood and muscle tissue with >90% and >70% of the initial activity cleared within the first 40 min, respectively. The majority of activity was observed in the kidney, liver, and tumor tissue. The observed tumor uptake was specific with up to 75% of the tumor uptake blocked when the mice were preinjected with 160 nmol (100 µg) of unlabeled peptide. Uptake observed in the blocked tumors was not significantly different than the background activity observed in muscle tissue. The only significant difference between the two (68)Ga-radiolabeled bioconjugates in vivo was the kidney uptake. (68)Ga-radiolabeled PCTA-RGD had significantly lower (p < 0.05) kidney uptake (1.1 ± 0.5%) at 2 h postinjection compared to (68)Ga-radiolabeled NOTA-RGD (2.7 ± 1.3%). Overall, (68)Ga-radiolabeled PCTA-RGD and NOTA-RGD performed similarly, but the lower kidney uptake for (68)Ga-radiolabeled PCTA-RGD may be advantageous in some imaging applications.