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Background In pancreatic cancer surgery, accurate identification and resection of intrapancreatic residual tumors are quite difficult. We have developed a novel open-typed PET system (called 'OpenPET'), which enables high-resolution PET-guided surgery in real time, and demonstrated that OpenPET-guided surgery with intraperitoneally administered 64Cu-labeled anti-epidermal growth factor receptor antibody cetuximab is useful to detect and resect primary pancreatic cancer. Here, we investigated applicability of OpenPET-guided surgery for unexpected residual intrapancreatic tumors and examined its survival benefit over conventional surgery. Methods A mouse model with large (>1 cm) resectable pancreatic cancer of xPA-1-DC cells expressing red fluorescent protein was used. OpenPET-guided surgery was conducted 24 h after intraperitoneal administration of 64Cu-labeled cetuximab (7.4 MBq/mouse). For comparison, similar surgical procedures were conducted, and conventional tumor resection was attempted using only the naked eye (control). Survival rate after OpenPET-guided surgery was compared to that after control operations. Results Intraoperative OpenPET guidance enabled detection and resection of small residual tumors. Ten residual tumor specimens (3-10 mm in diameter) were intraoperatively isolated with OpenPET guidance (n = 7 mice). All isolated specimens showed tumor RFP signals. No resection of tumor tissue was performed in control group because the tumor could not be clearly detected with the naked eye alone. Mice after OpenPET-guided surgery showed significantly longer survival rates than those in control group. Conclusions OpenPET-guided surgery with 64Cu-labeled-cetuximab enabled intraoperative identification and resection of intrapancreatic small residual tumors. This technology could be useful to prevent tumor residuals during surgery and improve pancreatic cancer survival.

Pancreatic cancer (PC) has a poor prognosis owing to difficulties in the diagnosis of resectable PC at early stages. Several clinical studies have indicated that the detection and surgery of small resectable PC (64Cu-labeled anti-epidermal growth factor receptor antibody cetuximab was intraperitoneally administered into mice. It clearly identified PC tumors ≥3 mm. In contrast, neither OpenPET with intravenous-administered 64Cu-cetuximab nor intraperitoneal/intravenous-administered 18F-FDG (a traditional PET probe) could detect PC in this model. Immuno-OpenPET-guided surgery accurately resected small PC in mice and achieved significantly prolonged survival. This technology could provide a novel diagnostic and therapeutic strategy for small resectable PC to improve patient survival.

Peritoneal dissemination is a common cause of death from gastrointestinal cancers and is difficult to treat using current therapeutic options, particularly late-phase disease. Here, we investigated the feasibility of integrated therapy using 64Cu-intraperitoneal radioimmunotherapy (ipRIT), alone or in combination with positron emission tomography (PET)-guided surgery using a theranostic agent (64Cu-labeled anti-epidermal growth factor receptor antibody cetuximab) to treat early- and late-phase peritoneal dissemination in mouse models. In this study, we utilized the OpenPET system, which has open space for conducting surgery while monitoring objects at high resolution in real time, as a novel approach to make PET-guided surgery feasible. 64Cu-ipRIT with cetuximab inhibited tumor growth and prolonged survival with little toxicity in mice with early-phase peritoneal dissemination of small lesions. For late-phase peritoneal dissemination, a combination of 64Cu-ipRIT for down-staging and subsequent OpenPET-guided surgery for resecting large tumor masses effectively prolonged survival. OpenPET clearly detected tumors (≥3 mm in size) behind other organs in the peritoneal cavity and was useful for confirming the presence or absence of residual tumors during an operation. These findings suggest that integrated 64Cu therapy can serve as a novel treatment strategy for peritoneal dissemination.

Glioblastoma is the most aggressive malignant brain tumor in humans and is difficult to cure using current treatment options. Hypoxic regions are frequently found in glioblastoma, and increased levels of hypoxia are associated with poor clinical outcomes of glioblastoma patients. Hypoxia plays important roles in the progression and recurrence of glioblastoma because of drug delivery deficiencies and induction of hypoxia-inducible factor-1α in tumor cells, which lead to poor prognosis. We focused on a promising hypoxia-targeted internal radiotherapy agent, 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), to address the need for additional treatment for glioblastoma. This compound can target the overreduced state under hypoxic conditions within tumors. Clinical positron emission tomography studies using radiolabeled Cu-ATSM have shown that Cu-ATSM accumulates in glioblastoma and its uptake is associated with high hypoxia-inducible factor-1α expression. To evaluate the therapeutic potential of this agent for glioblastoma, we examined the efficacy of 64Cu-ATSM in mice bearing U87MG glioblastoma tumors. Administration of single dosage (18.5, 37, 74, 111, and 148 MBq) and multiple dosages (37 MBq × 4) of 64Cu-ATSM was investigated. Single administration of 64Cu-ATSM in high-dose groups dose-dependently inhibited tumor growth and prolonged survival, with slight and reverse signs of adverse events. Multiple dosages of 64Cu-ATSM remarkably inhibited tumor growth and prolonged survival. By splitting the dose of 64Cu-ATSM, no adverse effects were observed. Our findings indicate that multiple administrations of 64Cu-ATSM have effective antitumor effects in glioblastoma without side effects, indicating its potential for treating this fatal disease.

// Yukie Yoshii 1 , Mitsuyoshi Yoshimoto 2 , Hiroki Matsumoto 3 , Takako Furukawa 1, 4 , Ming-Rong Zhang 1 , Masayuki Inubushi 5 , Atsushi B. Tsuji 1 , Yasuhisa Fujibayashi 1 , Tatsuya Higashi 1 and Tsuneo Saga 1, 6 1 National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan 2 Division of Functional Imaging, National Cancer Center Hospital East, Kashiwa, Japan 3 Research Centre, Nihon Medi-Physics Co., Ltd., Sodegaura, Japan 4 Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan 5 Department of Nuclear Medicine, Kawasaki Medical School, Kurashiki, Japan 6 Department of Diagnostic Radiology, Kyoto University Hospital, Kyoto, Japan Correspondence to: Yukie Yoshii, email: yoshii.yukie@qst.go.jp Keywords: 64 Cu-ATSM, angiogenesis, bevacizumab, hypoxia, vascular decrease Received: July 19, 2017 Accepted: August 17, 2017 Published: September 28, 2017 ABSTRACT Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, is an antiangiogenic agent clinically used for various cancers. However, repeated use of this agent leads to tumor-decreased vascularity and hypoxia with activation of an HIF-1 signaling pathway, which results in drug delivery deficiency and induction of malignant behaviors in tumors. Here, we developed a novel strategy to treat tumors with bevacizumab-induced vascular decrease and hypoxia using 64 Cu-diacetyl-bis ( N 4 -methylthiosemicarbazone) ( 64 Cu-ATSM), a potential theranostic agent, which possesses high tissue permeability and can target over-reduced conditions under hypoxia in tumors, with a human colon carcinoma HT-29 tumor-bearing mouse model. The long-term treatment with bevacizumab caused decreased blood vessel density and activation of an HIF-1 signaling pathway; increased uptake of 64 Cu-ATSM was also observed despite limited blood vessel density in HT-29 tumors. In vivo high-resolution SPECT/PET/CT imaging confirmed reduced vascularity and increased proportion of 64 Cu-ATSM uptake areas within the bevacizumab-treated tumors. 64 Cu-ATSM therapy was effective to inhibit tumor growth and prolong survival of the bevacizumab-treated tumor-bearing mice without major adverse effects. In conclusion, 64 Cu-ATSM therapy effectively enhanced anti-tumor effects in tumors with bevacizumab-induced vascular decrease and hypoxia. 64 Cu-ATSM therapy could represent a novel approach as an add-on to antiangiogenic therapy.

Hypoxia in tumors is known to contribute to the malignant characteristics such as therapy resistance and metastatic potential. Cu-diacetyl-bis (N4-methylthiosemicarbazone) (Cu-ATSM), labeled with several Cu radioisotopes (60Cu, 62Cu and 64Cu) is a potential PET imaging agent that targets tumor hypoxia with over-reduced conditions. Clinical studies of Cu-ATSM PET has been conducted worldwide in recent years and the usefulness of this agent is being recognized. Multicenter clinical study of 62Cu-ATSM PET has been conducted in Japan. Among Cu-ATSM tracers, 64Cu-ATSM can be used not only as a PET imaging agent but also as a therapeutic agent, because 64Cu emits β− particle and Auger electron, which can damage the cells. Here, we will discuss the present status and future prospects of Cu-ATSM.

Pancreatic cancer (PC) has a very poor prognosis. Surgery is the primary treatment for patients with resectable PC; however, local recurrence, hepatic metastasis, and peritoneal dissemination often occur even after extensive surgery. Adjuvant chemotherapy, typically with gemcitabine, has been used clinically but with only a modest survival benefit. To achieve a better outcome, we investigated the efficacy of (64)Cu-intraperitoneal radioimmunotherapy (ipRIT) with (64)Cu-labeled antiepidermal growth factor receptor antibody cetuximab as an adjuvant treatment after PC surgery using an orthotopic xenografted mouse model. Methods: The efficacy of adjuvant (64)Cu-ipRIT was investigated in a human PC mouse model harboring orthotopic xenografts of xPA-1-DC cells. To reproduce the clinical situation, PC xenografts were surgically resected when pancreatic tumors were readily visible but not metastatic tumors. Increasing doses of (64)Cu-cetuximab were intraperitoneally injected, and the mice were monitored for toxicity to determine the safe therapeutic dose. For adjuvant (64)Cu-ipRIT, the day after tumor resection, the mice were intraperitoneally administered 22.2 MBq of (64)Cu-PCTA-cetuximab and the survival was compared with that in surgery-only controls. For comparison, adjuvant chemotherapy with gemcitabine was also examined using the same model. Results: The mouse model not only developed primary tumors in the pancreas but also subsequently reproduced local recurrence, hepatic metastasis, and peritoneal dissemination after surgery, which is similar to the manifestations that occur with human PC. Adjuvant (64)Cu-ipRIT with (64)Cu-labeled cetuximab after surgery effectively suppressed local recurrence, hepatic metastasis, and peritoneal dissemination in this model. Significant improvement of the survival with minimal toxicity was achieved by adjuvant (64)Cu-ipRIT compared with that in control mice that underwent surgery only. Adjuvant chemotherapy with gemcitabine nominally prolonged the survival, but the effect was not statistically significant. Conclusion: (64)Cu-ipRIT with cetuximab can be an effective adjuvant therapy after PC surgery.

Background and purpose The aim of this study was to compare the accumulation of 4-borono-2- 18 F-fluoro-phenylalanine ( 18 F-BPA) with that of 18 F-fluorodeoxyglucose ( 18 F-FDG) in head and neck cancers, and to assess the usefulness of 18 F-FDG PET for screening candidates for boron neutron capture therapy (BNCT). Material and methods Twenty patients with pathologically proven malignant tumors of the head and neck were recruited from March 2012 to January 2014. All patients underwent both whole-body 18 F-BPA PET/CT and 18 F-FDG PET/CT within 2weeks of each other. The uptakes of 18 F-BPA and 18 F-FDG at 1h after injection were evaluated using the maximum standardized uptake value (SUVmax). Results The accumulation of 18 F-FDG was significantly correlated with that of 18 F-BPA. The SUVmax of 18 F-FDG ⩾5.0 is considered to be suggestive of high 18 F-BPA accumulation. Conclusions 18 F-FDG PET might be an effective screening method performed prior to 18 F-BPA for selecting patients with head and neck cancer for treatment with BNCT.

Lung cancer is one of the leading causes of cancer-related deaths worldwide, including Japan. Although computed tomography (CT) can detect small lung lesions such as those appearing as ground glass opacity, it cannot differentiate between malignant and non-malignant lesions. Previously, we have shown that single photon emission computed tomography (SPECT) imaging using (111)In-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-cyclo-(Arg-Gly-Asp-D-Phe-Lys) (DOTA-c(RGDfK)), an imaging probe of αvβ3 integrin, is useful for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. In this study, we aimed to assess the usefulness of SPECT/CT with (111)In-DOTA-c(RGDfK) for the evaluation of the malignancy of lung cancer.Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice. Twenty-six weeks after the urethane treatment, SPECT was performed an hour after injection of (111)In-DOTA-c(RGDfK). Following this, the radioactivity ratios of tumor to normal lung tissue were measured by autoradiography (ARG) in the excised lung samples. We also examined the expression of αvβ3 integrin in mouse and human lung samples.Urethane treatment induced 5 hyperplasias, 41 adenomas and 12 adenocarcinomas in the lungs of 8 A/J mice. SPECT with (111)In-DOTA-c(RGDfK) could clearly visualize lung nodules, though we failed to detect small lung nodules like adenoma and hyperplasias (adenocarcinoma: 66.7%, adenoma: 33.6%, hyperplasia: 0.0%). ARG analysis revealed significant uptake of (111)In-DOTA-c(RGDfK) in all the lesions. Moreover, tumor to normal lung tissue ratios increased along with the progression of carcinogenesis. Histopathological examination using human lung tissue samples revealed clear up-regulation of αvβ3 integrin in well-differentiated adenocarcinoma (Noguchi type B and C) rather than atypical adenomatous hyperplasia.Although there are some limitations in evaluating the malignancy of small lung tumors using (111)In-DOTA-c(RGDfK), SPECT with (111)In-DOTA-c(RGDfK) might be a useful non-invasive imaging approach for evaluating the characteristics of lung tumors in mice, thus showing potential for use in humans.

Early detection of pancreatic cancer is key to overcoming its poor prognosis. α(v)β(3)-integrin is often overexpressed in pancreatic tumor cells, whereas it is scarcely expressed in normal pancreatic cells. In this study, we investigated the usefulness of SPECT imaging with (111)In-1,4,7,10-tetraazacylododecane-N,N',N″,N'''-tetraacetic acid-cyclo-(Arg-Gly-Asp-d-Phe-Lys) [(111)In-DOTA-c(RGDfK)], an imaging probe of α(v)β(3)-integrin, for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model.Hamsters were subcutaneously injected with the pancreatic duct carcinogen N-nitrosobis(2-oxopropyl)amine to induce pancreatic cancer. N-nitrosobis(2-oxopropyl)amine-treated hamsters underwent in vivo SPECT with (111)In-DOTA-c(RGDfK). After imaging, the tumor-to-normal pancreatic tissue radioactivity ratios in excised pancreatic samples were measured with autoradiography (ARG) and compared with the immunopathologic findings for α(v)β(3)-integrin. In a mouse model in which inflammation was induced with turpentine, the uptake of (111)In-DOTA-c(RGDfK) in inflammatory regions was evaluated with ARG and compared with that of (18)F-FDG.(111)In-DOTA-c(RGDfK) was clearly visualized in pancreatic cancer lesions as small as 3 mm in diameter. ARG analysis revealed high tumor-to-normal pancreatic tissue radioactivity ratios (4.6 ± 1.0 [mean ± SD] in adenocarcinoma and 3.3 ± 1.4 in atypical hyperplasia). The uptake of (111)In-DOTA-c(RGDfK) strongly correlated with α(v)β(3)-integrin expression. In the inflammatory model, inflammation-to-muscle ratios for (18)F-FDG and (111)In-DOTA-c(RGDfK) were 8.37 ± 4.37 and 1.98 ± 0.60, respectively. These results imply that (111)In-DOTA-c(RGDfK) has a lower rate of false-positive tumor detection than (18)F-FDG.Our findings suggest that SPECT with (111)In-DOTA-c(RGDfK) has great potential for the early and accurate detection of pancreatic cancer.

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, the radioiodinated 4-(3-iodoanilino)-6,7-diethoxyquinazoline ([(125)I]m-IPQ) has been reported to possess good characteristics as a tumor imaging agent; however, it was also found to have low in vivo stability. To improve the in vivo stability, m-IPQ derivatives, 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) and 4-(3-iodobenzylamino)-6,7-diethoxyquinazoline (BAY) were designed and synthesized, and the biological studies of [(125)I]PHY and [(125)I]BAY were performed to evaluate these new ligands as in vivo tumor diagnosis agents.PHY and BAY were synthesized according to previous reports. The EGFR-TK inhibitory potency of these new compounds was measured and compared to other EGFR-TK inhibitors. Radiolabeled [(125)I]PHY and [(125)I]BAY were synthesized by an iododestannylation reaction. Biodistribution studies of these radioligands were conducted in normal mice and tumor-bearing mice. Furthermore, selectivity and binding characteristics of [(125)I]PHY were analyzed by in vitro blocking studies and a binding assay.The new derivatives were found to have high inhibitory potency against EGFR-TK (PHY: IC50 = 12.7 ± 7.2 nM, BAY: IC50 = 51.0 ± 8.9 nM). [(125)I]PHY and [(125)I]BAY were conveniently synthesized from tributylstannyl precursors. In in vivo biodistribution studies, [(125)I]PHY and [(125)I]BAY were observed to have lower uptake in the stomach, an indication of deiodination, than [(125)I]m-IPQ. Moreover, [(125)I]PHY showed high uptake and prolonged retention in tumors and low accumulation in blood and muscle tissue resulting in a good tumor-to-blood ratio (0.94-1.50) and tumor-to-muscle ratio (1.02-1.95). The EGFR-TK selectivity of [(125)I]PHY was confirmed by pretreatment experiments with specific EGFR-TK inhibitors.New radioiodinated quinazoline derivatives were synthesized, which were found to have improved in vivo stability. In particular, [(125)I]PHY showed higher tumor accumulation than the other ligands which was indicative of selective binding to EGFR-TK. These desirable characteristics for [(125)I]PHY suggest that the (123)I-labeled counterpart, [(123)I]PHY, could be a possible candidate for cancer diagnosis radiopharmaceutical.

A radioiodinated analog of PD153035 (m-IPQ) was evaluated as a potential epidermal growth factor receptor tyrosine kinase (EGFR-TK) activity imaging ligand for SPECT. The 50% inhibition concentration (IC50) value of m-IPQ for EGFR-TK phosphorylation inhibition was evaluated and compared to various EGFR-TK inhibitors. [125I]m-IPQ was synthesized by iododestannylation reaction. Biodistribution study of [125I]m-IPQ was conducted in normal mice and tumor-bearing mice. The selectivity and binding characteristics (B max and K d) were analyzed. The quinazoline derivative m-IPQ was found to have high inhibitory potency (IC50: 50.5 ± 3.5 nM) and selectivity toward EGFR-TK. In vivo biodistribution studies of [125I]m-IPQ demonstrated its rapid clearance and low retention in normal tissue. On the other hand, high tumor uptake was observed. However, the increase in [125I]m-IPQ uptake in the stomach as a deiodination parameter was found. Thus, [125I]m-IPQ showed low in vivo stability. The selectivity toward EGFR-TK of m-IPQ was confirmed by the pretreatment experiment with EGFR-TK specific inhibitors, PD153035, Genistein. [125I]m-IPQ bound to single population of binding sites with high affinity and kinetic parameter. In addition, [125I]m-IPQ was bound to EGFR-TK according to the amount of EGFR-TK expression in the tumor. [125I]m-IPQ showed a relatively high tumor accumulation with selective EGFR-TK binding. Moreover, the tumor uptake of [125I]m-IPQ might be reflected in the amount of EGFR-TK expression in the tumor. These good characteristics of [125I]m-IPQ suggested that a 123I-labeled counterpart, [123I]m-IPQ, would have great potential for EGFR-TK imaging with SPECT. However, the in vivo stability of this compound needs to improve.

The alpha(v)beta(3) integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress alpha(v)beta(3) integrin, as in tumor neovascularization, and alpha(v)beta(3) integrin expression in other microvascular beds and organs is limited. Therefore, alpha(v)beta(3) integrin is a suitable receptor for tumor-targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to alpha(v)beta(3) integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor-targeting properties and the therapeutic potential of 111In- and 90Y-labeled monomeric RGD peptides in BALB/c nude mice with SKOV-3 human ovarian carcinoma tumors. DOTA-c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to alpha(v)beta(3) integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In-DOTA-c(RGDfK) and 90Y-DOTA-c(RGDfK) showed high uptake in tumors (7.3 +/- 0.6% ID/g and 4.6 +/- 0.8% ID/g, respectively) and gradually decreased over time (2.3 +/- 0.4% ID/g and 1.5 +/- 0.5% ID/g at 24 hr, respectively). High tumor-to-blood and -muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple-dose administration of 90Y-DOTA-c(RGDfK) (3 x 11.1 MBq) suppressed tumor growth in comparison to the control group and a single-dose administration (11.1 MBq). Monomeric RGD peptides, 111In-DOTA-c(RGDfK) and (90)Y-DOTA-c(RGDfK), could be promising tracers for alpha(v)beta(3) integrin-targeting imaging and radiotherapy.

64 Cu-labeled diacetyl-bis(N 4 -methylthiosemicarbazone) ( 64 Cu-ATSM) is a promising agent for internal radiation therapy and imaging of hypoxic tissues. In the present study, the intra-tumoral distribution of 64 Cu-ATSM was investigated by comparing it to that of [ 18 F]FDG and histological findings. VX2 tumors were implanted into Japanese white rabbits subcutaneously. 64 Cu-ATSM and [ 18 F]FDG were co-injected intravenously and the tumor was dissected and cut into 1 mm thick slices 1 h after the injection. The uptake of 64 Cu-ATSM and [ 18 F]FDG was measured using a dual-tracer autoradiographic technique. Histological cell biology was estimated from the optical microscopy of tumor sections. The major accumulation of 64 Cu-ATSM was observed around the outer rim of the tumor masses which consisted mainly of active cells and expected to be hypoxic. [ 18 F]FDG was distributed more widely with highest levels in the inner regions where pre-necrotic cells were mainly observed. 64 Cu-ATSM appears to be useful for the detection of hypoxic but active tumor cell regions in vivo.

During the past decade, the efficacy of new molecular targeted drugs such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies has been proven worldwide, and molecular targeted therapies have become the mainstream in cancer therapy. However, clinical use of these new drugs presents unexpected adverse effects or poor therapeutic effects. Therefore, we require diagnostic tools to estimate the target molecule status in cancer tissues and predict therapeutic efficacy and adverse effects. Although immunohistochemical, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) analyses of biopsy samples are conventional and popular for this diagnostic purpose, molecular imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) are also useful for noninvasive estimation of gene and protein expression and drug pharmacokinetics. In this review, we introduce new radiolabeled TKIs, antibodies, and their clinical application in molecular targeted therapy and discuss the issues of these imaging probes.

Epidermal growth factor receptor (EGFR) is attractive target for tumor diagnosis and therapy, as it is specifically and abundantly expressed in tumor cells. EGFR-tyrosine kinase (TK) inhibitors such as gefitinib and erlotinib are widely used in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated whether radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), which is a candidate EGFR-TK imaging agent for single photon emission computed tomography (SPECT) is able to predict gefitinib sensitivity. We used four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid carcinoma cell line, A431 (wild-type EGFR). Cell proliferation assay and Western blotting revealed that A431 and H3255 with high EGFR expression showed high sensitivity to gefitinib. On the other hand, A549, H1650 and H1975 showed much lower sensitivity to gefitinib. The blocking study revealed that gefitinib decreased tumor uptake in (125)I-PHY in A431-bearing mice. Moreover, in vivo tumor uptake of (125)I-PHY was correlated with the IC50 of gefitinib for cell proliferation. In the present study, tumor uptake of (125)I-PHY was correlated with the gefitinib sensitivity and this uptake was based on expression levels of EGFR, but not on mutation status. Although the mutation status is the most important factor for predicting gefitinib sensitivity, the abundant expression of EGFR is essential for therapy with EGFR-TK inhibitors. Therefore, radioiodinated PHY is a potential imaging agent to predict gefitinib sensitivity based on EGFR expression levels though further modifications of the imaging agent is needed to accurately estimate the mutation status.

Purpose (64)Cu-diacetyl-bis (N (4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with (64)Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during (64)Cu-ATSM IRT. Methods Biodistribution was evaluated in HT-29 tumor-bearing mice injected with (64)Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between (64)Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after (64)Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic (64)Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses. Results Penicillamine reduced (64)Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after (64)Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic (64)Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine. Conclusions We developed a novel strategy to reduce radiation exposure in critical organs during (64)Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other (64)Cu-labeled compounds.

Objective Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, radioiodinated 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) was reported to possess good characteristics as a tumor imaging agent. We have explored the feasibility of developing tumor diagnosis ligands superior to radioiodinated PHY. Methods New phenoxyquinazoline derivatives were designed with various side chains introduced to the 6th position of PHY. The IC50 values of the new derivatives to interrupt EGFR-TK phosphorylation were evaluated and compared to well-known EGFR-TK inhibitors. Tumor uptake studies of the new 125I-labeled derivatives were conducted with A431 tumor-bearing mice. Selectivity and binding characteristics were analyzed by in vitro blocking studies and a binding assay. Furthermore, SPECT/CT scans were performed using A431 tumor-bearing mice. Results Six quinazoline derivatives were designed and synthesized, and among these, 6a–d were found to have relatively high EGFR-TK inhibitory potency. In tumor uptake studies, [125I]6a ([125I]PYK) was found to have the highest tumor uptake and longest retention in tumors. In contrast, [125I]PYK was rapidly cleared from peripheral tissues, resulting in a high tumor-to-tissue ratio 24 h after injection. Moreover, the EGFR-TK selectivity of [125I]PYK was confirmed by pretreatment experiments with specific EGFR-TK inhibitors. Furthermore, [125I]PYK provided clear SPECT images of tumors. Conclusions Radioiodinated PYK, one of the newly synthesized quinazoline derivatives, was found to be a desirable ligand for EGFR-TK SPECT imaging. [125I]PYK showed high tumor accumulation and selective EGFR-TK binding and also succeeded in delivering high contrast imaging of tumors. These favorable characteristics of [125I]PYK suggest that the 123I-labeled counterpart, [123I]PYK, would have great potential for diagnostic SPECT tumor imaging.

Tumor interiors are never homogeneous and in vivo visualization of intratumoral heterogeneity would be an innovation that contributes to improved cancer therapy. But, conventional nuclear medicine tests have failed to visualize heterogeneity in vivo because of limited spatial resolution. Recently developed single photon emission computed tomographic (SPECT) scanners dedicated for small animal imaging are of interest due to their excellent spatial resolution of1 mm, but few studies have focused on the evaluation of intratumoral heterogeneity. We investigated the optimal conditions related to high resolution imaging of heterogeneous tumor interiors using a small animal SPECT scanner.The conditions related to SPECT/CT visualization of heterogeneous tumor interiors were investigated using phantoms with (111)In and simulations of actual small animal imaging. The optimal conditions obtained were validated by in vivo imaging of sarcoma 180-bearing mice.Larger number of counts must be obtained within limited acquisition time to visualize tumor heterogeneity in vivo in animal imaging, compared to cases that simply detect tumors. At an acquisition time of 30 min, better image quality was obtained with pinhole apertures diameter of 1.4 mm than of 1.0 mm. The obtained best spatial resolution was 1.3 mm, it was acceptable for our purpose, though a little worse than the best possible performance of the scanner (1.0 mm). Additionally, the reconstruction parameters, such as noise suppression, voxel size, and iteration/subset number, needed to be optimized under the limited conditions and were different from those found under the ideal condition. The minimal radioactivity concentration for visualization of heterogeneous tumor interiors was estimated to be as high as 0.2-0.5 MBq/mL. Liposomes containing (111)In met this requirement and were administered to tumor-bearing mice. SPECT imaging successfully showed heterogeneous (111)In distribution within the tumors in vivo with good spatial resolution. A threshold of 0.2 MBq/g for clear visualization of tumor heterogeneity was validated. Autoradiograms obtained ex vivo of excised tumors confirmed that the in vivo SPECT images accurately depicted the heterogeneous intratumoral accumulation of liposomes.Intratumoral heterogeneity was successfully visualized under the optimized conditions using a SPECT/CT scanner.

N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic ductal carcinomas and early ductal lesions in Syrian hamsters have been reported to show histopathological resemblance to those in humans. Specific protein expression profiles have been found in human carcinomas, but a detailed molecular approach regarding the dissection of BOP-induced pancreatic carcinogenesis has yet to be determined. The present immunohistochemical study of early and advanced hamster lesions focused on five proteins reported to be overexpressed in human patients, to clarify interspecies phenotype similarity. Integrin α(V)β(3) was found to be overexpressed in the epithelial cells of 13 of 14 atypical hyperplasias and 6 of 6 adenocarcinomas. This overexpression was more frequent than in the remaining four proteins. However, immunoreactivity for α-enolase in epithelial cells and for kallikrein 7 and galectin-1/3 in both epithelial and stromal cells was also evident at various frequencies. Thus, similarities of tumor-associated protein expression between human and hamster pancreatic ductal lesions were confirmed, and integrin α(V)β(3) was identified as a potentially useful immunohistochemical marker for early lesions in hamsters.

X-ray computed tomography (CT) has been used for diagnoses of human pancreatic cancer. Although micro-CT is a useful approach to evaluate macromorphology of organs/tissue also in animal models, reports on pancreatic tumors are limited. In this study, the utility of micro-CT was assessed in characterizing chemically induced pancreatic tumors in Syrian hamsters. Hamsters treated with or without N-nitrosobis(2-oxopropyl)amine (BOP) were injected with the antispasmodic agent, scopolamine butylbromide, and contrast agents, 5 or 10 mL/kg body weight of iopamidol or Fenestra VC at 18-38 weeks, then examined by micro-CT scanning with a respiratory gating system. Both peristaltic and respiratory movements were substantially suppressed by the combination of scopolamine butylbromide treatment and the respiratory gating system, resulting in improvements of image qualities. Iopamidol clearly visualized the pancreatic parenchyma and contrasted the margins among the pancreas and other abdominal organs/tissue. Meanwhile Fenestra VC predominantly contrasted abdominal vascular systems, but the margins among pancreas and other organs/tissue remained obscure. Six pancreatic tumors of 4-13 mm in diameter were detected in four of 15 animals, but not the five tumors of 1-4 mm in diameter. The inner tumor images were heterogeneously or uniformly visualized by iopamidol and Fenestra VC. Overall, iopamidol could clearly contrast between pancreatic parenchyma and the tumors as compared with Fenestra VC. All tumors confirmed were histopathologically diagnosed as pancreatic ductal adenocarcinomas. Thus, micro-CT could be useful to evaluate the carcinogenic processes and preventive methods of pancreatic cancer in hamsters and to assess the novel contrast agents for detection of small pancreatic cancer in humans.

Background We have studied the possibility that low-dose treatment utilizing the inhibition that may occur between two drugs at the same site of human serum albumin (HSA) improves the pharmacological effects. The purpose is to elucidate the differences in the binding capacities of sites I and II of HSA between pre-haemodialysis (HD) and post-HD in patients with end-stage renal disease. Methods We evaluated free fractions of site probes, (14)C-warfarin (site I) and (14)C-diazepam (site II), by ultrafiltration in serum between pre-HD and post-HD. To investigate effects on the binding capacities of HSA sites, free fractions of site probes were calculated from the radioactivities measured with a liquid scintillation counter. Endogenous uraemic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulphate (IS) and hippurate (HA), were determined by HPLC. Free fatty acid (FFA) as an endogenous substance was determined with an automatic multi-item simultaneous analyser. Results The concentrations of HSA and FFA increased significantly (post-HD/pre-HD ratio: 1.18 +/- 0.10, 5.46 +/- 4.91), the concentrations of IS and HA decreased significantly (post-HD/pre-HD ratio: 0.69 +/- 0.10, 0.33 +/- 0.15) and CMPF concentrations did not alter significantly (post-HD/pre-HD ratio: 0.97 +/- 0.12, P = 0.471). The free fractions of (14)C-warfarin decreased in all 14 patients at site I at post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59 +/- 0.13). The free fractions of (14)C-diazepam at site II remarkably decreased in 10 of 14 patients (post-HD/pre-HD ratio: 0.61 +/- 0.17) and unexpectedly increased in 4 (post-HD/pre-HD ratio: 1.08 +/- 0.06) post-HD compared to pre-HD. In these four patients, when we investigated the influences of these variation factors on the reduction of the binding capacities of site II, [FFA]/[HSA] increased significantly post-HD, compared to pre-HD (post-HD/pre-HD ratio: 6.91 +/- 6.58). ([FFA]/[HSA] ratios of the 4 patients were from 1.22 to 3.55, the highest for the 14 patients post-HD, but the ratios of the other 10 were below 1.2 post-HD.) Conclusion The binding capacity of site II was unexpectedly decremented by the effects of the remarkable elevation of FFA. Therefore, monitoring the binding capacity of site II in HD is important for patients with end-stage renal disease in the efficacious administration plan using the binding inhibition of HSA.

Introduction Based on the concept of bifunctional radiopharmaceuticals, we have previously developed 186 Re-complex-conjugated bisphosphonate analogs for palliation of painful bone metastases and have demonstrated the utility of these compounds. By applying a similar concept, we hypothesized that a bone-specific directed 90 Y-labeled radiopharmaceutical could be developed. Methods In this study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as the chelating site, and DOTA was conjugated with 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. [ 90 Y]DOTA-complex-conjugated bisphosphonate ([ 90 Y]DOTA-HBP) was prepared by coordination with 90 Y, and its biodistribution was studied in comparison to [ 90 Y]citrate. Results In biodistribution experiments, [ 90 Y]DOTA-HBP and [ 90 Y]citrate rapidly accumulated and resided in the bone. Although [ 90 Y]citrate showed a higher level of accumulation in the bone than [ 90 Y]DOTA-HBP, the clearances of [ 90 Y]DOTA-HBP from the blood and from almost all soft tissues were much faster than those of [ 90 Y]citrate. As a result, the estimated absorbed dose ratios of soft tissues to osteogenic cells (target organ) of [ 90 Y]DOTA-HBP were lower than those of [ 90 Y]citrate. Conclusions [ 90 Y]DOTA-HBP showed superior biodistribution characteristics as a bone-seeking agent and led to a decrease in the level of unnecessary radiation compared to [ 90 Y]citrate. Since the DOTA ligand forms a stable complex not only with 90 Y but also with lutetium ( 177 Lu), indium ( 111 In), gallium ( 67/68 Ga), gadolinium (Gd) and so on, complexes of DOTA-conjugated bisphosphonate with various metals could be useful as agents for palliation of metastatic bone pain, bone scintigraphy and magnetic resonance imaging.

Fanconi syndrome is a renal dysfunction characterized by various combinations of renal tubular transport dysfunction involving amino acids, glucose, protein and other substances. Most reabsorption of amino acids occurs in proximal renal tubule segment 1 (S1). The present study evaluated the possibility of early detection of drug-induced Fanconi syndrome, based on decreased renal accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine (125I-IMT), an amino acid transport marker, in the S1 region of renal cortex. The present experimental model used maleate (MAL)-induced Fanconi syndrome in mice. Results were compared between 125I-IMT and 3 other clinical renal radiopharmaceuticals: 99mTc-2,3-dimercaptosuccinic acid (99mTc-DMSA); 99mTc-mercaptoacetylglycylglycylglycine (99mTc-MAG3); and 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA).Male ddY mice (age, 6 weeks; body weight, 25 g) were used to create a Fanconi model of renal dysfunction. A single dose of maleate disodium salt was administered by intraperitoneal injection (6 mmol/kg). Hematoxylin and eosin (HE) staining of the renal cortex, renal autoradiography and measurement of renal radioactivity of labeled compounds were performed at 30, 60, 90 and 120 min after MAL injection. At 5 min after injection of labeled compounds (18.5 kBq for accumulation experiment, 670 kBq for autoradiography), animals were sacrificed by ether overdose and kidneys were removed. For the accumulation experiment, radioactivity was measured using a well-type scintillation counter. For autoradiography, 20-microm sections of frozen kidney were used with Bio-Imaging Analyzer.At 30 min after MAL injection, HE staining showed pyknosis in some proximal tubule cells. At that time, accumulations of 125I-IMT and 99mTc-DMSA in the S1 region were approximately 67% and 55% of control levels (p0.005). MAL increased accumulation of 99mTc-DTPA in the S1 region, but had no effect on accumulation of 99mTc-MAG3 in the S 1 region.Decreased accumulation of 123I-IMT in the S1 region appears to represent a useful marker for detection of MAL-induced Fanconi syndrome. In future, we plan to assess the efficacy of using 125I-IMT to monitor renal dysfunction induced by nephrotoxic clinical drugs.

Dynamic changes in the cerebral glucose metabolic rate (CMRglc) before and after thiamine replenishment were investigated in living brain slices obtained from pyrithiamine-treated (PT) and pair-fed control rats by use of a positron autoradiography technique. Fresh rat brain slices (300 microm thick) were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, during which serial two-dimensional images of [18F]FDG uptake in the slices were constructed on the imaging plates. The net influx constant (=K) of [18F]FDG was determined by a Patlak graphical method of the image data. Prior to thiamine pyrophosphate (TPP)-loading, the K value in the neurologically symptomatic PT was higher in all brain regions except the thalamus and mammillary body than the control, suggesting compensatory enhanced glycolysis. The rapid decrease in this heightened net influx constant immediately after TPP-loading was surmised to be due to activation of pyruvate oxidation with lactate as the substrate, with this inhibiting the glycolysis. From > or = 150 min after TPP-loading, the K value continued to show low values in the thalamus and mammillary body, which are regarded as the responsible sites for Korsakoff syndrome, whereas in all other sites recovery to control values was observed. These findings suggest that using this technique the quantitative evaluation of serial local changes in CMRglc from thiamine deficiency to after its replenishment may be useful in elucidating the pathophysiology and prognosis of Wernicke's encephalopathy.