Synthesis, Radiolabeling, and Preliminary in vivo Evaluation of [68Ga] IPCAT-NOTA as an Imaging Agent for Dopamine Transporter.

INTRODUCTION: Novel radiotracer development for imaging dopamine transporters is a subject of interest because although [99mTc]TRODAT-1, [123I]ß-CIT, and [123I]FP-CIT are commercially available; 99Mo/99mTc generator is in short supply and 123I production is highly dependent on compact cyclotron. Therefore, we designed a novel positron emission tomography (PET) tracer based on a tropane derivative through C-2 modification to conjugate NOTA for chelating 68Ga, a radioisotope derived from a 68Ge/68Ga generator. METHODS: IPCAT-NOTA 22 was synthesized and labeled with [68Ga]GaCl4 - at room temperature. Biological studies on serum stability, LogP, and in vitro autoradiography (binding assay and competitive assay) were performed. Furthermore, ex vivo autoradiography, biodistribution, and dynamic PET imaging studies were performed in Sprague Dawley rats. RESULTS: [68Ga]IPCAT-NOTA 24 obtained had a radiochemical yield of ≥90% and a specific activity of 4.25 MBq/nmol. [68Ga]IPCAT-NOTA 24 of 85% radiochemical purity (RCP%) was stable at 37°C for up to 60 minutes in serum with a lipophilicity of 0.88. The specific binding ratio (SBR%) reached 15.8 ± 6.7 at 60 minutes, and the 85% specific uptake could be blocked through co-injection at 100- and 1000-fold of the cold precursor in in vitro binding studies. Tissue regional distribution studies in rats with [68Ga]IPCAT-NOTA 24 showed striatal uptake (0.02% at 5 minutes and 0.007% at 60 minutes) with SBR% of 6%, 25%, and 62% at 5-15, 30-40, and 60-70 minutes, respectively, in NanoPET studies. The RCP% of [68Ga]IPCAT-NOTA 24 at 30 minutes in vivo remained 67.65%. CONCLUSION: Data described here provide new information on the design of PET probe of conjugate/pendent approach for DAT imaging. Another chelator or another direct method of intracranial injection must be used to prove the relation between [68Ga]IPCAT-NOTA 24 uptake and transporter localization.

Evaluation of 5-[18F]fluoro-2'-deoxycytidine as a tumor imaging agent: A comparison of [18F]FdUrd, [18F]FLT and [18F]FDG.

One of the hallmarks of cancer is increased cell proliferation. Measurements of cell proliferation by estimation of DNA synthesis with several radiolabeled nucleosides have been tested to assess tumor growth. Deoxycytidine can be phosphorylated by deoxycytidine kinase (dCK) and is incorporated into DNA. This study evaluated a radiofluorinated deoxycytidine analog, 5-[18F]fluoro-2'-deoxycytidine ([18F]FdCyd), as a proliferation probe and compared it with 5-[18F]fluoro-2'-deoxyuridine ([18F]FdUrd), 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), and [18F]fluorodeoxyglucose ([18F]FDG) in a tumor-bearing mouse model. [18F]FdCyd was synthesized from two precursors by direct electrophilic substitution. The serum stability and partition coefficient of [18F]FdCyd were evaluated in vitro. Positron emission topography (PET) imaging of Lewis lung carcinoma (LLC)-bearing mice with [18F]FdCyd, [18F]FdUrd, [18F]FLT, and [18F]FDG were evaluated. [18F]FdCyd was stable in mouse serum and normal saline for up to 4 h. With all radiotracers except [18F]FLT, PET can clearly delineate the tumor lesion. [18F]FdCyd and [18F]FdUrd showed high accumulation in the liver and kidney. The SUV and tumor-to-muscle (T/M) ratios derived from PET imaging of the radiotracers were [18F]FDG > [18F]FdCyd > [18F]FdUrd > [18F]FLT. Selective retention in tumors with a favorable tumor/muscle ratio makes [18F]FdCyd a protential candidate for further investigation as a proliferation imaging agent.

Evaluation of the Biological Behavior of a Gold Nanocore-Encapsulated Human Serum Albumin Nanoparticle (Au@HSANP) in a CT-26 Tumor/Ascites Mouse Model after Intravenous/Intraperitoneal Administration.

Colorectal cancer is one of the major causes of cancer-related death in Taiwan and worldwide. Patients with peritoneal metastasis from colorectal cancer have reduced overall survival and poor prognosis. Hybrid protein-inorganic nanoparticle systems have displayed multifunctional applications in solid cancer theranostics. In this study, a gold nanocore-encapsulated human serum albumin nanoparticle (Au@HSANP), which is a hybrid protein-inorganic nanoparticle, and its radioactive surrogate 111In-labeled Au@HSANP (111In-Au@HSANP), were developed and their biological behaviors were investigated in a tumor/ascites mouse model. 111In-Au@HSANP was injected either intravenously (iv) or intraperitoneally (ip) in CT-26 tumor/ascites-bearing mice. After ip injection, a remarkable and sustained radioactivity retention in the abdomen was noticed, based on microSPECT images. After iv injection, however, most of the radioactivity was accumulated in the mononuclear phagocyte system. The results of biodistribution indicated that ip administration was significantly more effective in increasing intraperitoneal concentration and tumor accumulation than iv administration. The ratios of area under the curve (AUC) of the ascites and tumors in the ip-injected group to those in the iv-injected group was 93 and 20, respectively. This study demonstrated that the ip injection route would be a better approach than iv injections for applying gold-albumin nanoparticle in peritoneal metastasis treatment.

68Ga-labelled NOTA-RGD-GE11 peptide for dual integrin and EGFR-targeted tumour imaging.

INTRODUCTION: Multiple peptide receptors are co-expressed in many types of cancers. Arg-Gly-Asp (RGD) and GE11 peptides specifically target integrin αVß3 and EGFR, respectively. Recently, we designed and synthesized a heterodimer peptide NOTA-c(RGDyK)-GE11 (NOTA-RGD-GE11). The aim of this study was to investigate the characteristics of NOTA-RGD-GE11 for dual receptor imaging. METHODS: NOTA-RGD-GE11 heterodimer was labelled with 68Ga. The dual receptor binding affinity was investigated by antibody competition binding assay. The in vitro and in vivo characteristics of [68Ga]Ga-NOTA-RGD-GE11 were investigated and compared with that of monomeric peptides [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11. RESULTS: NOTA-RGD-GE11 had binding affinities with both integrin αVß3 and EGFR. The dual receptor targeting property of [68Ga]Ga-NOTA-RGD-GE11 was validated by blocking studies in a NCI-H292 tumour model. [68Ga]Ga-NOTA-RGD-GE11 showed higher tumour uptake than [68Ga]Ga-NOTA-RGD and [68Ga]Ga-NOTA-GE11 in biodistribution and PET/CT imaging studies. CONCLUSION: The dual receptor targeting and enhanced tumour uptake of [68Ga]Ga-NOTA-RGD-GE11 warrant its further investigation for dual integrin αVß3 and EGFR-targeted tumour imaging.

Use of 111In-Hexavalent Lactoside for Liver Reserve Estimation in Rodents with Thioacetamide-Induced Hepatic Fibrosis.

Many biochemical tests detecting the presence of liver disease are not liver-specific and may be abnormal in nonhepatic conditions. The asialoglycoprotein receptor (ASGPR) is a hepatocyte-specific receptor for Gal/GalNAc-terminated glycopeptide or glycoprotein. The number of these receptors decreases in patients with chronic liver diseases. Here, we aimed to evaluate the use of 111In-hexavalent lactoside, a known ASGPR imaging biomarker, as a more sensitive probe to detect small changes in liver reserve in animal models of chronic liver injury. Thioacetamide (TAA) treatment via intraperitoneal injection every 2 days in BALB/c mice continued for 1, 2, 3, or 4 months. The liver fibrosis stages were determined by Sirius Red staining and were based on the METAVIR classification method. Serum transaminase enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)), alkaline phosphatase, albumin, and bilirubin were measured using a FUJI FDC3500 i/s analyzer. The ASGPR staining was performed by immunohistocytochemical stain. The percentages of fibrosis and ASGPR were calculated using ImageJ software after collagen staining and anti-ASGPR staining, respectively. A nanoSPECT/CT was used for molecular imaging and liver uptake measurement. We observed fibrosis grades of F0-F1 in mice treated with TAA for 1 month, F2 in mice treated for 2 months, F3-F4 in mice treated for 3 months, and F4 in mice treated for 4 months. The levels of ALT and albumin were not significantly different in the TAA groups from those in the controls. Although the average levels of AST, alkaline phosphatase, and bilirubin in the TAA groups were different from those in the control group, there was little difference between TAA groups. More sensitive distinctions among TAA groups were detected in 111In-hexavalent lactoside uptake of ASGPR, ASGPR staining, and fibrosis % than when using the conventional AST, ALT, albumin, alkaline phosphatase, and bilirubin tests. The absorption and distribution of 111In-hexavalent lactoside were lower in the chronic hepatitis models than the normal controls. The liver reserves measured by 111In-hexavalent lactoside uptake were 71.7 ± 7.5% and 50.9 ± 5.6% after 1 and 2 months, respectively, of TAA treatment. As an ASGPR biomarker, 111In-hexavalent lactoside has higher sensitivity than traditional liver function tests and collagen stain to provide more objective data for evaluating compensated cirrhosis or changes in liver damage. ASGPR staining can reflect the regenerated hepatocytes, but the need for a biopsy limits its use. 111In-hexavalent lactoside measurement is comparable with ASGPR staining, which suggests that 111In-hexavalent lactoside measurement will be more useful as a practical, noninvasive test of chronic liver injury.

Development of single vial kits for preparation of 68 Ga-labelled hexavalent lactoside for PET imaging of asialoglycoprotein receptor.

The aim of this study was to formulate and evaluate the freeze-dried kit of NOTA-hexavalent lactoside (NOTA-HL) for the preparation of 68 Ga-labeled glycoligand for PET imaging of the asialoglycoprotein receptor (ASGPR). 68 GaCl3 was obtained from a commercial 68 Ge/68 Ga generator. Single-vial kits of HL were formulated. Optimization of radiolabeling with 68 Ga, various evaluations of NOTA-HL kits, and in vitro stability study of 68 Ga-HL were carried out. PET/CT imaging of normal mice injected with 68 Ga-NOTA-HL was performed. NOTA-HL kit was successfully formulated. High radiochemical yields (>95%) were obtained by 68 Ga radiolabeling. The NOTA-HL kits were stable for at least 12 months, and 68 Ga-NOTA-HL exhibited good in vitro stability. PET studies in normal mice revealed high specific accumulation of activity in the liver. The NOTA-HL kit was developed for fast 68 Ga labeling. 68 Ga-NOTA-HL showed high specific uptake in liver. The availability of ready-to-use NOTA-HL kits combined with 68 Ge/68 Ga generators would provide an efficient approach for PET imaging of ASGPR.

Development and validation of a reversed-phase HPLC method for analysis of radiochemical purity in [123I]IBZM.

[123I]IBZM is used widely for in vivo imaging of D2 receptors in human brain and shows relatively fast kinetics and a greater susceptibility to synaptic dopamine release than other single-photon emission computed tomography (SPECT) radioligands. A reliable and reversed-phase HPLC method using UV/VIS and radiometric detectors has been developed for qualitative analysis of BZM and IBZM and radiochemical purity in [123I]IBZM preparations. The method uses gradient elution on a Zorbax XDB C-18 column with a mobile phase that consists of 10mM 3,3-dimethylglutaric acid (DMGA), pH 7.0 and acetonitrile (ACN). The flow rate was 1.0mL/min with detection at λ=254nm. The method was validated for system suitability, precision, accuracy, specificity, linearity, robustness, limit of detection (LOD) and limit of quantification (LOQ), as described in ICH guidelines. The results are described as follows: (1) The system suitability includes the tailing factor, theoretical plate number and resolution, which are 0.962, 10656.11 and 9.367, respectively. (2) For specificity, the BZM and [123I]NH4I did not interfere with the retention time of the [123I]IBZM. (3) The percentage coefficient of variation for analysis of precision, including repeatability and intermediate precision, is less than 2.0%. (4) Accuracy of the method is within the range of 85-100%. (5) The range of linearity is from 100% to 70% radiochemical purity (%RCP) of [123I]IBZM, with the correlation coefficient (R) always being above 0.995. (6) The data of method robustness are within acceptance criteria. (7) The LOD and LOQ for impurity (BZM) are 0.145 and 0.50µg/mL, respectively. All of the analysis results demonstrate that this method is sensitive, specific and suitable for routine analysis of the radiochemical purity in [123I]IBZM preparations.

EGFR-targeted micelles containing near-infrared dye for enhanced photothermal therapy in colorectal cancer.

The purpose of this research was to investigate the effectiveness of epidermal growth factor receptor (EGFR) targeted micelles loaded with IR-780 (Cetuximab/IR-780/micelles) for generating tumor targeting, multimodal images, and photothermal therapy (PTT). We initially studied the cellular uptake of these micelles using the HCT-116 and SW-620 cell lines. HCT-116 (high expression of EGFR) and SW-620 (low expression of EGFR) cell lines were used to examine biodistribution and antitumor effects of Cetuximab/IR-780/micelles. Time-lapse near-IR fluorescence (NIRF) images also indicated the highest IR-780 accumulation from Cetuximab/IR-780/micelles in HCT-116 tumors (p<0.05). HCT-116 tumors in tumor-bearing mice exhibited significantly higher accumulations of Cetuximab/IR-780/111In-micelles than SW-620 tumors in Micro-SPECT/CT imaging and biodistribution studies (p<0.05). Dual-radioisotope Nano-SPECT/CT imaging of Cetuximab/131I-IR-780/111In-micelles demonstrated simultaneous high accumulation of both IR-780 and micelles in HCT-116 tumors, but not in SW-620 tumors. Regarding antitumor effects, following the Cetuximab/IR-780/micelles with PPT on day 6, all HCT-116 tumor-bearing mice were cured. In contrast, SW-620 tumors relapsed at 13days after treatment. In summary, we expect that the Cetuximab/IR-780/micelles could enhance the antitumor effects by PTT in EGFR overexpression colorectal cancers through effective drug delivery nanoparticles.

111In-cetuximab as a diagnostic agent by accessible epidermal growth factor (EGF) receptor targeting in human metastatic colorectal carcinoma.

Colorectal adenocarcinoma is a common cause death cancer in the whole world. The aim of this study is to define the 111In-cetuximab as a diagnosis tracer of human colorectal adenocarcinoma. In this research, cell uptake, nano-SPECT/CT scintigraphy, autoradiography, biodistribution and immunohitochemical staining of EGF receptor were included. HCT-116 and HT-29 cell expressed a relatively high and moderate level of EGF receptor, respectively. The nano-SPECT/CT image of 111In-cetuximab showed tumor radiation uptake of subcutaneous HCT-116 xenograft tumor was higher than SW-620. Autoradiography image also showed that tumor of HCT-116 had high 111In-cetuximab uptake. Mice that bearing CT-26 in situ xenograft colorectal tumors showed similar high uptake in vivo and ex vivo through nano-SPECT/CT imaging at 72 hours. Metastatic HCT-116/Luc tumors demonstrated the highest uptake at 72 hours after the injection of 111In-cetuximab. Relatively, results of 111In-DTPA showed that metabolism through urinary system, especially in the kidney. The quantitative analysis of biodistribution showed count value of metastatic HCT-116/Luc tumors that treated with 111In-cetuximab had a significant difference (P < 0.05) compared with that treated with 111In-DTPA after injection 72 hours. Result of immunohistologic staining of EGF receptor also showed high EGF receptor expression and uptake in metastatic colorectal tumors. In summary, we suggested that 111In-cetuximab will be a potential tool for detecting EGF receptor expression in human metastatic colorectal carcinoma.

Synthesis of (68)Ga-labeled NOTA-RGD-GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor-targeted tumor imaging.

Radiolabeled Arg-Gly-Asp (RGD) peptide analogs have been extensively studied for αvß3 integrin-targeted angiogenesis imaging. According to recently presented evidence, the dodecapeptide GE11 has high affinity to the epidermal growth factor receptor (EGFR), which is overexpressed in many types of cancer. Dual-receptor molecular imaging probes with two different heterodimeric peptides exhibit improved cancer targeting efficacy. In the present study, the design and synthesis of a new RGD-GE11 peptide heterodimer for dual αvß3 integrin/EGFR-targeted cancer imaging are described. The RGD-GE11 heterodimer was linked with 6-aminohexanoic acid (6-Ahx) and cysteine and conjugated with 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) to form NOTA-RGD-cys-6-Ahx-GE11. The monomeric peptides, NOTA-cys-6-Ahx-GE11 and c(RGDyK), were formed by a peptide synthesizer. The peptide heterodimer NOTA-RGD-GE11 was obtained by NOTA-cys-6-Ahx-GE11 and maleimidopropyl-c(RGDyK) conjugation with a thioether linkage. The NOTA peptide conjugate was labeled with freshly eluted (68)Ga and purified using reversed-phase high-performance liquid chromatography. The (68)Ga-NOTA-RGD-cys-6-Ahx-GE11 was successfully prepared, in this study, with a radiochemical yield of 85% and a radiochemical purity of >98%. These results warrant further investigation of this heterodimeric peptide's binding affinity to the receptors.

The specificity and accuracy of (111)In-hexavalent lactoside in estimating liver reserve and its threshold value for mortality in mice.

BACKGROUND & AIMS: The asialoglycoprotein receptor on hepatocyte membranes recognizes the galactose residues of glycoproteins. We investigated the specificity, accuracy and threshold value of asialoglycoprotein receptor imaging for estimating liver reserve via scintigraphy using (111)In-hexavalent lactoside in mouse models. METHODS: (111)In-hexavalent lactoside scintigraphy for asialoglycoprotein receptor imaging was performed on groups of normal mice, orthotopic SK-HEP-1-bearing mice, subcutaneous HepG2-bearing mice, mice with 20-80% partial hepatectomy and mice with acute hepatitis induced by acetaminophen. Liver reserve was measured by relative liver uptake and compared with normal mice. Asialoglycoprotein receptor blockade was performed via an in vivo asialofetuin competitive binding assay. RESULTS: A total of 73.64±7.11% of the injection dose accumulated in the normal liver tissue region, and radioactivity was barely detected in the hepatoma region. When asialoglycoprotein receptor was blocked using asialofetuin, less than 0.41±0.04% of the injection dose was detected as background in the liver. Asialoglycoprotein receptor imaging data revealed a linear correlation between (111)In-hexavalent lactoside binding and residual liver mass (R(2)=0.8548) in 20-80% of partially hepatectomized mice, demonstrating the accuracy of (111)In-hexavalent lactoside imaging for measuring the functional liver mass. Asialoglycoprotein receptor imaging data in mice with liver failure induced using 600mg/kg acetaminophen revealed 19-45% liver reserve relative to normal mice and a fatal threshold value of 25% liver reserve. CONCLUSION: The (111)In-hexavalent lactoside imaging method appears to be a good, specific, visual and quantitative predictor of functional liver reserve. The diagnostic threshold for survival was at 25% liver reserve in mice.

188Re-HYNIC-trastuzumab enhances the effect of apoptosis induced by trastuzumab in HER2-overexpressing breast cancer cells.

PURPOSE: The development of radioimmunotherapy has provided an impressive alternative approach in improving trastuzumab therapy. However, the mechanisms of trastuzumab and radiation treatment combined to increase therapeutic efficacy are poorly understood. Here, we try to examine the efficacy of cytotoxicity and apoptosis induction for (188)Re-HYNIC-trastuzumab in cancer cell lines with various levels of Her2. MATERIALS AND METHODS: Fluorescence flow cytometry was used to detect the alterations of apoptosis induction after (188)Re-HYNIC-trastuzumab treatment in two breast cancer cell lines with different levels of HER2 (BT-474 and MCF-7) and a colorectal carcinoma cell line (HT-29) for control. RESULTS: Our results indicated that (188)Re-HYNIC-trastuzumab led to cell death of breast cancer cells specifically in HER2 level-dependent and radioactivity dose-dependent fashions. In BT-474 cells, 370 kBq/ml of (188)Re-HYNIC-trastuzumab enhanced the cytotoxicity to a level nearly 100-fold that of trastuzumab-alone treatment. The results also revealed that the mitochondria-dependent pathway attenuated irradiation-induced apoptosis in HER2-expressing breast cancer cells after (188)Re-HYNIC-trastuzumab treatment. In contrast, only after 48 h of (188)Re-HYNIC-trastuzumab treatment, BT-474 cells exhibited typical apoptotic changes, including exposure of phospholipid phosphatidylserine on the cell surface, or fragmented DNA formation, in a radioactivity dose-dependent manner. CONCLUSION: Briefly, our study demonstrates that (188)Re-labeled HYNIC-trastuzumab not only enhances cell death in a radioactivity dose-dependent fashion, but may also prolong the effects of apoptosis involved with the mitochondria-dependent pathway in HER2-overexpressing breast cancer cells. It is possible that the (188)Re-HYNIC-trastuzumab treatment induced a second round of apoptosis to prolong the effects of cell kill in these cancer cells. These data revealed that (188)Re-HYNIC-trastuzumab has the potential for use as a therapeutic radiopharmaceutical agent in HER2-overexpressing breast cancer cell treatment.

Monitoring Tumor Response After Histone Deacetylase Inhibitor Treatment Using 3'-Deoxy-3'-[18F]-fluorothymidine PET.

PURPOSE: This study employed 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]FLT) microPET scanning to assess the treatment response of histone deacetylase inhibitors (HDACi), e.g., N1-hydroxy-N8-phenyloctanediamide (SAHA) and its iodinated derivative ISAHA, in a hepatoma mouse model. PROCEDURES: The in vitro cytotoxicity of HDACi in various hepatoma cell lines was determined by MTT assay and flow cytometry. ISAHA and SAHA were used to treat HepG2 hepatoma xenograft-bearing mice. The treatment responses were characterized in terms of tumor burden, microPET imaging, and immunohistochemical staining of tumor sections. RESULTS: ISAHA effectively inhibited HepG2 hepatoma cell survival and tumor growth. A significantly reduced tumor uptake during HDACi treatment was noticed in [(18)F]FLT microPET imaging, which was consistent with the findings in immunohistochemical staining. CONCLUSIONS: ISAHA can suppress tumor cell proliferation both in vitro and in vivo. [(18)F]FLT PET is a promising modality for evaluating the in vivo therapeutic efficacy of HDACi at the early stage of treatment.

Therapeutic and scintigraphic applications of polymeric micelles: combination of chemotherapy and radiotherapy in hepatocellular carcinoma.

This study evaluated a multifunctional micelle simultaneously loaded with doxorubicin (Dox) and labeled with radionuclide rhenium-188 ((188)Re) as a combined radiotherapy and chemotherapy treatment for hepatocellular carcinoma. We investigated the single photon emission computed tomography, biodistribution, antitumor efficacy, and pathology of (188)Re-Dox micelles in a murine orthotopic luciferase-transfected BNL tumor cells hepatocellular carcinoma model. The single photon emission computed tomography and computed tomography images showed high radioactivity in the liver and tumor, which was in agreement with the biodistribution measured by γ-counting. In vivo bioluminescence images showed the smallest size tumor (P<0.05) in mice treated with the combined micelles throughout the experimental period. In addition, the combined (188)Re-Dox micelles group had significantly longer survival compared with the control, (188)ReO4 alone (P<0.005), and Dox micelles alone (P<0.01) groups. Pathohistological analysis revealed that tumors treated with (188)Re-Dox micelles had more necrotic features and decreased cell proliferation. Therefore, (188)Re-Dox micelles may enable combined radiotherapy and chemotherapy to maximize the effectiveness of treatment for hepatocellular carcinoma.

Synthesis and preclinical characterization of [18F]FPBZA: a novel PET probe for melanoma.

INTRODUCTION: Benzamide can specifically bind to melanoma cells. A 18F-labeled benzamide derivative, [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy) ethoxy)ethoxy]benzamide ([18F]FPBZA), was developed as a promising PET probe for primary and metastatic melanoma. METHODS: [18F]FPBZA was synthesized via a one-step radiofluorination in this study. The specific uptake of [18F]FPBZA was studied in B16F0 melanoma cells, A375 amelanotic melanoma cells, and NB-DNJ-pretreated B16F0 melanoma cells. The biological characterization of [18F]FPBZA was performed on mice bearing B16F0 melanoma, A375 amelanotic melanoma, or inflammation lesion. RESULTS: [18F]FPBZA can be prepared efficiently with a yield of 40-50%. The uptake of [18F]FPBZA by B16F0 melanoma cells was significantly higher than those by A375 tumor cells and NB-DNJ-pretreated B16F0 melanoma cells. B16F0 melanoma displayed prominent uptake of [18F]FPBZA at 2 h (7.81±0.82%ID/g), compared with A375 tumor and inflammation lesion (3.00±0.71 and 1.67±0.56%ID/g, resp.). [18F]FPBZA microPET scan clearly delineated B16F0 melanoma but not A375 tumor and inflammation lesion. In mice bearing pulmonary metastases, the lung radioactivity reached 4.77±0.36%ID/g at 2 h (versus 1.16±0.23%ID/g in normal mice). CONCLUSIONS: Our results suggested that [18F]FPBZA PET would provide a promising and specific approach for the detection of primary and metastatic melanoma lesions.

Preparation and therapeutic evaluation of (188)Re-thermogelling emulsion in rat model of hepatocellular carcinoma.

Radiolabeled Lipiodol(®) (Guerbet, Villepinte, France) is routinely used in hepatoma therapy. The temperature-sensitive hydrogel polyethylene glycol-b-poly-DL-lactic acid-co-glycolic acid-b-polyethylene glycol triblock copolymer is used as an embolic agent and sustained drug release system. This study attempted to combine the polyethylene glycol-b-poly-DL-lactic acid-co-glycolic acid-b-polyethylene glycol hydrogel and radio-labeled Lipiodol to form a new radio-thermogelling emulsion, rhenium-188-N,N'-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride-Lipiodol/hydrogel ((188)Re-ELH). The therapeutic potential of (188)Re-ELH was evaluated in a rodent hepatoma model. Rhenium-188 chelated with N,N'-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride was extracted with Lipiodol to obtain rhenium-188-N,N'-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride-Lipiodol ((188)Re-EL), which was blended with the hydrogel in equal volumes to develop (188)Re-ELH. The (188)Re-ELH phase stability was evaluated at different temperatures. Biodistribution patterns and micro-single-photon emission computed tomography/computed tomography images in Sprague Dawley rats implanted with the rat hepatoma cell line N1-S1 were observed after in situ tumoral injection of ~3.7 MBq (188)Re-ELH. The therapeutic potential of (188)Re-EL (48.58±3.86 MBq/0.1 mL, n=12) was evaluated in a 2-month survival study using the same animal model. The therapeutic effects of (188)Re-ELH (25.52±4.64 MBq/0.1 mL, n=12) were evaluated and compared with those of (188)Re-EL. The responses were assessed by changes in tumor size and survival rates. The (188)Re-ELH emulsion was stable in the gel form at 25°C-35°C for >52 hours. Biodistribution data and micro-single-photon emission computed tomography/computed tomography images of the (188)Re-ELH group indicated that most activity was selectively observed in hepatomas. Long-term (188)Re-ELH studies have demonstrated protracted reductions in tumor volumes and positive effects on the survival rates (75%) of N1-S1 hepatoma-bearing rats. Conversely, the 2-month survival rate was 13% in the control sham group. Therapeutic responses differed significantly between the two groups (P<0.005). Thus, the hydrogel enhanced the injection stability of (188)Re-EL in an animal hepatoma model. Given the synergistic results, direct (188)Re-ELH intratumoral injection is a potential therapeutic alternative for hepatoma treatment.

Biological characterization of cetuximab-conjugated gold nanoparticles in a tumor animal model.

Gold nanoparticles (AuNPs) are widely applied to the diagnosis and treatment of cancer and can be modified to contain target-specific ligands via gold-thiolate bonding. This study investigated the pharmacokinetics and microdistribution of antibody-mediated active targeting gold nanoparticles in mice with subcutaneous lung carcinoma. We conjugated AuNPs with cetuximab (C225), an antibody-targeting epidermal growth factor receptor (EGFR), and then labeled with In-111, which created EGFR-targeted AuNPs. In vitro studies showed that after a 2 h incubation, the uptake of C225-conjugated AuNPs in high EGFR-expression A549 cells was 14.9-fold higher than that of PEGylated AuNPs; furthermore, uptake was also higher at 3.8-fold when MCF7 cells with lower EGFR-expression were used. MicroSPECT/CT imaging and a biodistribution study conducted by using a A549 tumor xenograft mouse model provided evidence of elevated uptake of the C225-conjugated AuNPs into the tumor cells as a result of active targeting. Moreover, the microdistribution of PEGylated AuNPs revealed that a large portion of AuNPs remained in the tumor interstitium, whereas the C225-conjugated AuNPs displayed enhanced internalization via antibody-mediated endocytosis. Our findings suggest that the anti-EGFR antibody-conjugated AuNPs are likely to be a plausible nano-sized vehicle for drug delivery to EGFR-expressing tumors.

Sorafenib increases efficacy of vorinostat against human hepatocellular carcinoma through transduction inhibition of vorinostat-induced ERK/NF-κB signaling.

Sorafenib is effective for patients with advanced hepatocellular carcinoma (HCC) and particularly for those who are unsuitable to receive life-prolonging transarterial chemo-embolization. The survival benefit of sorafenib, however, is unsatisfactory. Vorinostat also known as suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor with anti-HCC efficacy in preclinical studies. SAHA induces nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity in vitro, which may lead to cancer cell progression and jeopardize cytotoxic effect of SAHA in HCC. The goal of this study was to investigate whether sorafenib enhances SAHA cytotoxicity against HCC through inhibition of SAHA-induced NF-κB activity. The human HCC cell line Huh7 transfected with dual reporter genes, luciferase (luc) and thymidine kinase (tk) with NF-κB response elements, was co-transfected with red fluorescent protein (rfp) gene for non-invasive molecular imaging to assess NF-κB activity and living cells simultaneously. Cell viability assay, DNA fragmentation, western blotting, electrophoretic mobility shift assay (EMSA) and multiple modalities of molecular imaging were used to assess the combination efficacy and mechanism of sorafenib and SAHA. The administration of high-dose SAHA (10 µM) with long treatment time (48 h) in vitro, and 25 mg/kg/day by gavage in HCC-bearing nude mice to induce NF-κB activity were performed. Sorafenib inhibited SAHA-induced NF-κB activity and the expression of NF-κB-regulated effector proteins while it increased the efficacy of SAHA against HCC both in vitro and in vivo. The mechanism of sorafenib to enhance SAHA efficacy on HCC is through the suppression of ERK/NF-κB pathway, which induces extrinsic and intrinsic apoptosis. Combination of sorafenib and SAHA may have the potential as new strategy against HCC.

Curcumin synergistically enhances the radiosensitivity of human oral squamous cell carcinoma via suppression of radiation-induced NF-κB activity.

The anticancer effect of curcumin has been widely reported. However, whether curcumin can enhance the radiosensitivity of human oral squamous cell carcinoma (OSCC) remains to be elucidated. The aim of the present study was to evaluate the efficacy of curcumin combined with radiation against OSCC. SAS cells were transfected with the luciferase gene (luc) and named SAS/luc. NF-κB/DNA binding activity, the surviving fraction and NF-κB-regulated effector protein expression were determined by electrophoretic mobility shift assay, clonogenic survival assay and western blotting, respectively. The therapeutic efficacy was evaluated in SAS/luc tumor-bearing mice by caliper measurement and bioluminescence imaging. Curcumin enhanced SAS/luc radiosensitivity through the inhibition of radiation-induced NF-κB activity and expression of effector proteins both in vitro and in vivo. With 4 Gy or greater radiation doses, synergistic effects of curcumin were observed. The combination group (curcumin plus radiation) had significantly better tumor control compared with that of curcumin or radiation alone. No significant body weight change of mice was found throughout the entire study. In conclusion, curcumin is a radiosensitizer against OSCC with negligible toxicity.

Synthesis and cellular uptake of p-[(123)I]-phenyl-amino-thiazole ((123)I-PAT) as a potential agent for targeting tubulin polymerization in tumors.

The phenyl-amino-thiazole (PAT) templates of methoxylbenzoyl-aryl-thiazole are potent agents against cancer by inhibiting tubulin polymerization in the nanomolar range. Herein, a radioiodinated PAT, [(123)I]-PAT 1, was prepared via a tributylstannyl precursor and [(123)I]iodide through electrophilic aromatic radioiodination. Radiolabelling of [(123)I]-PAT 1 was achieved in less than 15 min, with a radiochemical purity of over 99%. The accumulated radioactivity in tumor cellular uptake experiments suggested that [(123) I]-PAT could serve as a potential radioprobe for targeting tumor cells.