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We evaluated the potential of diffusion-weighted MRI (DW-MRI) and 18F-FDG-PET for the early prediction of a triple negative breast cancer (TNBC) response to cisplatin. Cisplatin-treated TNBC tumor-bearing mice were categorized as responders or non-responders based on the tumor growth rate. DW-MRI and 18F-FDG-PET were performed before and after treatment (day 0 and days 3 and 7, respectively). The average apparent diffusion coefficient value (ADCmean), the highest standardized uptake value (SUVmax), and the metabolic tumor volume (MTV) were measured. The ratios of each parameter relative to day 0 were calculated [ΔADCmean (day 3) and (day 7), ΔSUVmax (day 3) and (day 7), and ΔMTV (day 3) and (day 7), respectively]. Overall survival rates were compared based on the thresholds determined by these parameters. Both the day 3 and day 7 ratios of ADCmean and MTV showed significant differences between the responder and non-responder groups, whereas the ratios of SUVmax did not. Mice with ΔADCmean (day 3) exceeding the threshold showed a longer overall survival rate. Mice with ΔSUVmax (day 7), ΔMTV (day 3), and ΔMTV (day 7) below the respective thresholds showed a longer overall survival rate. The ratios of ADCmean, SUVmax, and MTV have the potential to predict the therapeutic response and to screen non-responders in the ultra-early phase following cisplatin treatment in patients with TNBC.

Simple Summary Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. Here we show that the carbon ion RBE in human cancer cells correlates with the cellular uptake of 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. High RBE/64Cu-ATSM cells show greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts. These data suggest that the cellular antioxidant activity is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. Abstract Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy.

L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy, since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-[211At]-astato-α-methyl-L-phenylalanine (2-[211At]-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-[211At]-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-[211At]-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-[211At]-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-[211At-AAMP] in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-[211At]-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-[211At]-AAMP by increasing its accumulation in tumors.

Objective Radioimmunotherapy (RIT) has proven effective for patients with relapsed and refractory lymphoma. However, new types of therapy are strongly desired as B-cell lymphoma remains incurable for many patients. Photoimmunotherapy (PIT) is an emerging targeted cancer therapy that uses photosensitizer (IR700)-conjugated monoclonal antibodies (mAbs) to specifically kill cancer cells. To evaluate the usefulness and potential role of PIT for treating B-cell lymphoma in a comparison with RIT, we performed in vivo PIT and RIT studies with an IR700 or 90Y-conjugated anti-CD20 mAb, NuB2. Methods IR700 or 90Y were conjugated to NuB2. Since cell aggressiveness greatly affects the therapeutic effect, we selected both an indolent (RPMI 1788) and an aggressive (Ramos) type of B-cell lymphoma cell line. The in vitro therapeutic effect of PIT and the biodistribution profiles of IR700–NuB2 were evaluated. In vivo PIT and RIT studies were performed with 100 or 500 μg of IR700–NuB2 and 150 μCi/20 μg of 90Y-NuB2, respectively, in two types of B-cell lymphoma-bearing mice. Results The in vitro studies revealed that Ramos was more sensitive than RPMI 1788 to PIT. The therapeutic effect of PIT with 500 µg IR700–NuB2 was superior to any other therapies against aggressive Ramos tumors, whereas RIT showed the highest therapeutic effect in indolent RPMI 1788 tumors. Since the uptake levels and intratumoral distribution of IR700–NuB2 were comparable in both tumors, a possible cause of this difference is the tumor growth rate. The PIT with 500 µg (IR700–NuB2) group showed a significantly greater therapeutic effect than the PIT with 100 µg group due to the higher and more homogeneous tumor distribution of IR700–NuB2. Conclusions PIT was effective for both indolent and aggressive B-cell lymphoma, and the higher dose provided a better therapeutic effect. In aggressive tumors, PIT was more effective than RIT. Thus, PIT would be a promising strategy for the locoregional treatment or control of B-cell lymphoma. Since PIT and RIT have distinctive advantages over each other, they could play complementary rather than competitive roles in B-cell lymphoma treatment.

Positron emission tomography (PET) imaging with

Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer.A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSHAlWe successfully prepared Al

Macrophage mannose receptor (MMR/CD206) is a promising target for the detection and identification of sentinel lymph node (SLN). MMR-targeting probes have been developed using mannosylated dextran, however, impairment of efficient targeting of SLN was often caused because of retention of injection site in which macrophages and dendritic cells exist. In this study, we prepared new MMR-targeting probes from yeast mannan (85 kDa), and its bioditribution was investigated. In-vivo evaluation showed that 11.9% of injected dose of

Metallic radionuclides provide target-specific radiolabeled probes for molecular imaging in radiochemical yields sufficient for administration to subjects without purification. However, unlabeled ligands in the injectate can compete for targeted molecules with radiolabeled probes, which eventually necessitates postlabeling purification. Herein we describe a "1 to 3" design to circumvent the issue by taking advantage of inherent coordination properties of technetium-99m ((99m)Tc). A monovalent RGD ligand possessing an isonitrile as a coordinating moiety (CN-RGD) was reacted with [(99m)Tc(CO)3(OH2)3](+) to prepare [(99m)Tc(CO)3(CN-RGD)3](+) in over 95% radiochemical yields. This complex exhibited higher integrin αvβ3 binding affinity than its unlabeled monovalent ligand, primarily due to its multivalency. This compound visualized a murine tumor without removing unlabeled ligands, while a (99m)Tc-labeled monovalent probe derived from a monovalent ligand could not. The metal coordination-mediated synthesis of radiolabeled multivalent probes thereby can be a useful approach for preparing ready-to-use target-specific probes labeled with (99m)Tc and other metallic radionuclides of interest.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two NIR-light sources; light emitting diodes (LEDs) and Lasers, for their effectiveness in NIR-PIT. A photosensitizer, IRDye-700DX, conjugated to panitumumab (pan-IR700), was incubated with EGFR-expressing A431 and MDA-MB-468-luc cells. NIR-light was provided by LEDs or Lasers at the same light dose. Laser-light produced more cytotoxicity and greater reductions in IR700-fluorescence intensity than LED-light. Laser-light also produced more cytotoxicity in vivo in both cell lines. Assessment of super-enhanced permeability and retention (SUPR) effects were stronger with Laser than LED. These results suggest that Laser-light produced significantly more cytotoxic effects compared to LEDs. Although LED is less expensive, Laser-light produces superior results in NIR-PIT.

Somatostatin receptors are highly expressed in neuroendocrine tumors, and many radiolabeled somatostatin analogs for diagnosis and treatment have been developed. To simultaneously detect not only primary cancer but also bone metastases, this study aimed to develop a positron emission tomography probe using generator-produced nuclide Gallium-68 (T1/2 = 68 min), in which a carrier for primary cancer, a carrier for bone metastases lesions, and a stable gallium complex are introduced into the one molecule. Based on this strategy, the somatostatin receptor-targeted peptide, [Tyr3]-octreotate (TATE), aspartic acid peptide (Dn) with high binding affinity for hydroxyapatite, and Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a stable gallium complex were selected. The novel complexes, Ga-DOTA-Dn-TATE (n = 0, 2, 5, 8, or 11), were designed, synthesized, and evaluated. The radiogallium complexes were prepared using the easy-to-handle radioisotope 67Ga due to relatively long half-life. The radiogallium complex precursor DOTA-Dn-TATE was synthesized by the Fmoc-based solid-phase method and by the air oxidation method to form the disulfide bond. [67Ga]Ga-DOTA-Dn-TATE was synthesized by reacting DOTA-Dn-TATE and 67Ga. Hydroxyapatite binding assays, in vitro cellular uptake experiments in AR42J tumor cells, in biodistribution experiments in AR42J tumor-bearing mice, were performed using [67Ga]Ga-DOTA-Dn-TATE. The radiochemical purities of [67Ga]Ga-DOTA-Dn-TATE were > 96.0%. In in vitro and in vivo experiments, [67Ga]Ga-DOTA-D11-TATE had a high affinity for hydroxyapatite and highly accumulated in bone. However, the uptake of [67Ga]Ga-DOTA-D11-TATE into somatostatin receptor-positive AR42J cells was lower than that of [67Ga]Ga-DOTA-TATE, and the accumulation of [67Ga]Ga-DOTA-D11-TATE in tumor was significantly low. Ga-DOTA-D11-TATE may not be recognized by somatostatin receptor by the introduction of D11, and the charge adjustment may be important for somatostatin receptor-positive cell uptake.

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.

During fasting, most tissues including skeletal muscle heavily rely on utilization of fatty acids (FA) and minimize glucose use. In contrast, skeletal muscle prefers carbohydrate use as exercise intensity increases. In mice deficient for CD36 (CD36−/− mice), FA uptake is markedly reduced with a compensatory increase in glucose uptake in skeletal muscle even during fasting. In this study, we questioned how exercise endurance is affected during prolonged fasting in CD36−/− mice where glucose utilization is constantly increased. With or without a 24‐h fast, a single bout of treadmill exercise was started at the speed of 10 m/min, and the speed was progressively increased up to 30 m/min until mice were exhausted. Running distance of wild type (WT) and CD36−/− mice was comparable in the fed state whereas that of CD36−/− mice was significantly reduced after a 24‐h fast. Glycogen levels in liver and skeletal muscle were depleted both in WT and CD36−/− mice after a 24‐h fast. In CD36−/− mice, FA uptake by skeletal muscle continued to be reduced during fasting. Glucose utilization also continued to be enhanced in the heart and oxidative skeletal muscle and glucose supply relative to its demand was diminished, resulting in accelerated hypoglycemia. Consequently, available energy substrates from serum and in muscle for exercise performance were very limited in CD36−/− mice during prolonged fasting, which could cause a remarkable reduction in exercise endurance. In conclusion, our study underscores the importance of CD36 for nutrient homeostasis to maintain exercise performance of skeletal muscle when nutrient supply is limited.

Shell thickness determines the acoustic response of polymer-based perfluorooctyl bromide (PFOB) nanocapsule ultrasound contrast agents. PEGylation provides stealth property and arms for targeting moieties. We investigated a modulation in the polymer formulation of carboxy-terminated poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide-co-glycolide)-block-polyethylene glycol (PLGA-b-PEG) to produce thin-shelled PFOB nanocapsules while keeping its echogenicity, stealth property, and active targeting potential. Polymer formulation contains 40% PLGA-PEG that yields the PEGylated PFOB nanocapsules of approximately 150 nm size with average thickness-to-radius ratio down to 0.15, which adequately hindered phagocytosis. Functionalization with antibody enables in vitro tumor-specific targeting. Despite the acoustic response improvement, the in vivo tumor accumulation was inadequate to generate an observable acoustic response to the ultrasound power at the clinical level. The use of PLGA and PLGA-PEG polymer blend allows the production of thin-shelled PFOB nanocapsules with echogenicity improvement while maintaining its potential for specific targeting.

The overexpression of vascular endothelial growth factor (VEGF) in varying types of solid tumor renders radioimmunotherapy (RIT) with the anti-VEGF antibody bevacizumab (BV) a promising treatment. However, the slow blood clearance of BV, which may increase the occurrence risk of hematotoxicity, hinders the application of BV-RIT. Using the avidin chase is a long-known blood clearance enhancement strategy for biotinylated-mAb. To enhance RIT efficacy by increasing the radioactivity dose, we evaluated the ability of avidin to accelerate the blood clearance of yttrium-90 (90Y)-labeled biotinylated BV (90Y-Bt-BV) in a xenograft mouse model of triple-negative breast cancer (TNBC). The biodistribution study in the TNBC xenograft mice confirmed the high and specific tumor accumulation of the indium-111 (111In)-BV. The blood clearance enhancement effect of the avidin chase was demonstrated in the normal mouse studies with 111In-Bt-BV. In the subsequent biodistribution studies with the tumor-bearing mice, an optimi...

In the amyloid cascade hypothesis, β-amyloid (Aβ) plaques is one of the major pathological biomarkers in the Alzheimer’s disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [18F]4-dimethylamino-4′-fluoro-chalcone ([18F]DMFC) and [18F]4′-fluoro-4-methylamino-chalcone ([18F]FMC), as Aβ imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by 18F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [18F]DMFC and [18F]FMC. In a biodistribution study using normal mice, [18F]DMFC and [18F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2 min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30 min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aβ imaging agent ([18F]Florbetapir), meaning the improvement of the probability of detecting Aβ plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aβ peptides, [18F]DMFC and [18F]FMC showed high binding affinity to recombinant Aβ aggregates at the Kd values of 4.47 and 6.50 nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [18F]DMFC and [18F]FMC markedly accumulated only in a region with abundant Aβ plaques, indicating that they clearly recognized human Aβ plaques in vitro. These encouraging results suggest that [18F]DMFC and [18F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.

It is important to detect mediastinal lymph node metastases in patients with lung cancer to improve outcomes, and it is possible that activatable fluorescence imaging with indocyanine green (ICG) can help visualize metastatic lymph nodes. Therefore, we investigated the feasibility of applying this method to mediastinal lymph node metastases in an epidermal growth factor receptor (EGFR)-positive squamous cell carcinoma of the lung. Tumors were formed by injecting H226 (EGFR-positive) and H520 (EGFR-negative) cell lines directly in the lung parenchyma of five mice each. When computed tomography revealed tumors exceeding 8 mm at their longest or atelectasis that occupied more than half of lateral lung fields, a panitumumab (Pan)–ICG conjugate was injected in the tail vein (50 μg/100 μL). The mice were then sacrificed 48 hours after injection and their chests were opened for fluorescent imaging acquisition. Lymph node metastases with the five highest fluorescent signal intensities per mouse were chosen for statistical analysis of the average signal ratios against the liver. Regarding the quenching capacity, the Pan–ICG conjugate had almost no fluorescence in phosphate-buffered saline, but there was an approximate 61.8-fold increase in vitro after treatment with 1% sodium dodecyl sulfate. Both the fluorescent microscopy and the flow cytometry showed specific binding between the conjugate and H226, but almost no specific binding with H520. The EGFR-positive mediastinal lymph node metastases showed significantly higher average fluorescence signal ratios than the EGFR-negative ones (n = 25 per group) 48 hours after conjugate administration (70.1% ± 4.5% vs. 13.3% ± 1.8%; p < 0.05). Thus, activatable fluorescence imaging using the Pan–ICG conjugate detected EGFR-positive mediastinal lymph node metastases with high specificity.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

UNLABELLED Radiolabeled amino acids are superior PET tracers for the imaging of malignant tumors, and amino acids labeled with (76)Br, an attractive positron emitter because of its relatively long half-life (16.2 h), could potentially be a widely usable tumor imaging tracer. In this study, in consideration of its stability and tumor specificity, we designed two (76)Br-labeled amino acid derivatives, 2-(76)Br-bromo-α-methyl-l-phenylalanine (2-(76)Br-BAMP) and 4-(76)Br-bromo-α-methyl-l-phenylalanine (4-(76)Br-BAMP), and investigated their potential as tumor imaging agents. METHODS Both (76)Br- and (77)Br-labeled amino acid derivatives were prepared. We performed in vitro and in vivo stability studies and cellular uptake studies using the LS180 colon adenocarcinoma cell line. Biodistribution studies in normal mice and in LS180 tumor-bearing mice were performed, and the tumors were imaged with a small-animal PET scanner. RESULTS Both (77)Br-BAMPs were stable in the plasma and in the murine body. Although both (77)Br-BAMPs were taken up by LS180 cells and the uptake was inhibited by L-type amino acid transporter 1 inhibitors, 2-(77)Br-BAMP exhibited higher uptake than 4-(77)Br-BAMP. In the biodistribution studies, 2-(77)Br-BAMP showed more rapid blood clearance and lower renal accumulation than 4-(77)Br-BAMP. More than 90% of the injected radioactivity was excreted in the urine by 6 h after the injection of 2-(77)Br-BAMP. High tumor accumulation of 2-(77)Br-BAMP was observed in tumor-bearing mice, and PET imaging with 2-(76)Br-BAMP enabled clear visualization of the tumors. CONCLUSION 2-(77)Br-BAMP exhibited preferred pharmacokinetics and high LS180 tumor accumulation, and 2-(76)Br-BAMP enabled clear visualization of the tumors by PET imaging. These findings suggest that 2-(76)Br-BAMP could constitute a potential new PET tracer for tumor imaging and may eventually enable the wider use of amino acid tracers.

The high and persistent radioactivity at the injection site hinders the accuracy and expansion of sentinel lymph node (SLN) mapping. We investigated the mechanism underlying the undesirable radioactivity after subcutaneous injection of (99m)Tc-labeled mannosylated dextran ((99m)Tc(CO)3-DCM20), a SLN mapping agent targeting mannose receptors on macrophages and dendritic cells, in a mouse model. Biodistribution studies were performed 1 h after subcutaneous injection of (99m)Tc(CO)3-DCM20 from the rear footpad of mice in the presence of varying molar amounts of DCM20 or DC15, a modified dextran without mannose. Biodistribution studies were also conducted after subcutaneous injection of [(125)I]radioiodinated mannosyl-neoglycoalbumin ((125)I-NMA) from the rear footpad. The distribution of fluorescence-labeled DCM20 and DC15 at the injection site was also compared 1 h after subcutaneous injection by immunofluorescent histochemistry. The radioactivity levels of (99m)Tc(CO)3-DCM20 at the injection site and popliteal lymph node, a SLN in this model, decreased with an increase in the molar amounts of DCM20, whereas no significant changes in biodistribution were observed after injection of (99m)Tc(CO)3-DCM20 with varying molar amounts of DC15. (125)I-NMA exhibited rapid elimination of radioactivity from both the popliteal lymph node and the injection site. The fluorescence-labeled DCM20 colocalized well with CD68-positive cells such as macrophages and dendritic cells at the injection site. While partial colocalization was observed between DC15 and CD68-positive cells, the signal intensity was very weak. These findings suggest that specific binding of (99m)Tc(CO)3-DCM20 to the mannose receptor on macrophages and dendritic cells would be responsible for the sustained radioactivity levels at the injection site. These results also imply that discriminated blockage of (99m)Tc(CO)3-DCM20 binding to mannose receptors at the injection sites would reduce the radioactivity at the injection site.

Radionuclide therapy with low-energy auger electron emitters may provide high antitumor efficacy while keeping the toxicity to normal organs low. Here we evaluated the usefulness of an auger electron emitter and compared it with that of a beta emitter for tumor treatment in in vitro models and conducted a dosimetry simulation using radioiodine-labeled metaiodobenzylguanidine (MIBG) as a model compound. We evaluated the cellular uptake of 125I-MIBG and the therapeutic effects of 125I- and 131I-MIBG in 2D and 3D PC-12 cell culture models. We used a Monte Carlo simulation code (PHITS) to calculate the absorbed radiation dose of 125I or 131I in computer simulation models for 2D and 3D cell cultures. In the dosimetry calculation for the 3D model, several distribution patterns of radionuclide were applied. A higher cumulative dose was observed in the 3D model due to the prolonged retention of MIBG compared to the 2D model. However, 125I-MIBG showed a greater therapeutic effect in the 2D model compared to the 3D model (respective EC50 values in the 2D and 3D models: 86.9 and 303.9 MBq/cell), whereas 131I-MIBG showed the opposite result (respective EC50 values in the 2D and 3D models: 49.4 and 30.2 MBq/cell). The therapeutic effect of 125I-MIBG was lower than that of 131I-MIBG in both models, but the radionuclide-derived difference was smaller in the 2D model. The dosimetry simulation with PHITS revealed the influence of the radiation quality, the crossfire effect, radionuclide distribution, and tumor shape on the absorbed dose. Application of the heterogeneous distribution series dramatically changed the radiation dose distribution of 125I-MIBG, and mitigated the difference between the estimated and measured therapeutic effects of 125I-MIBG. The therapeutic effect of 125I-MIBG was comparable to that of 131I-MIBG in the 2D model, but the efficacy was inferior to that of 131I-MIBG in the 3D model, since the crossfire effect is negligible and the homogeneous distribution of radionuclides was insufficient. Thus, auger electrons would be suitable for treating small-sized tumors. The design of radiopharmaceuticals with auger electron emitters requires particularly careful consideration of achieving a homogeneous distribution of the compound in the tumor.

18F- or 131I-labeled (4-fluoro-3-iodobenzyl)guanidine (FIBG) has been a promising yet unattainable derivative of radioiodine-labeled meta-iodobenzylguanidine (MIBG), because of the complex radiofluorination method. In this study, we proposed a 2-step radiosynthetic method to obtain 18F-FIBG and evaluated the diagnostic and therapeutic potential of 18F-FIBG and 131I-FIBG in a pheochromocytoma model (PC-12). Methods:18F-FIBG was prepared from a (mesityl)(aryl)iodonium salt precursor in the presence of a copper catalyst. Biodistribution studies, PET imaging, and a therapeutic study were performed on the PC-12 xenograft mice with either 18F- or 131I-FIBG. The association between the therapeutic effect and the tumor uptake of pretherapy 18F-FIBG PET was also evaluated. Results: The copper-mediated radiofluorination method readily yielded 18F-FIBG, as well as its regioisomer, 18F-IFBG. The isolated 18F-FIBG showed a higher accumulation in the PC-12 xenograft tumor than in any other tissue. The high tumor uptake of 18F-FIBG allowed clear tumor visualization in the PET images as early as 1 h after injection, with an excellent tumor-to-background ratio. A biodistribution study with 131I-FIBG revealed its higher and prolonged retention in the tumor in comparison with 125I-MIBG. As a result, a therapeutic dose of 131I-FIBG delayed tumor growth significantly more than did 131I-MIBG. The tumor uptake of 18F-FIBG was proportional to the therapeutic effect of 131I-FIBG. Conclusion: These results suggest the potential usefulness of FIBG as a diagnostic and therapeutic agent for the management of norepinephrine transporter (NET)-expressing tumors.

We evaluated the potential of diffusion-weighted MRI (DW-MRI) andCisplatin-treated TNBC tumor-bearing mice were categorized as responders or non-responders based on the tumor growth rate. DW-MRI andBoth the day 3 and day 7 ratios of ADCThe ratios of ADC

Background Myocardial contractile dysfunction in sepsis has been attributed mainly to increased inflammatory cytokines, insulin resistance, and impaired oxidative phosphorylation of fatty acids (FAs). However, precise molecular mechanisms underlying the cardiac dysfunction in sepsis remain to be determined. We previously reported major shift in myocardial energy substrates from FAs to glucose, and increased hepatic ketogenesis in mice lacking fatty acid-binding protein 4 (FABP4) and FABP5 (DKO). Purpose We sought to determine whether a shift of energy substrates from FAs to glucose and increased availability of ketone bodies are beneficial or detrimental to cardiac function under the septic condition. Methods Lipopolysaccharide (LPS, 10 mg/kg) was intraperitoneally injected into wild-type (WT) and DKO mice. Twelve hours after injection, cardiac function was assessed by echocardiography and serum and hearts were collected for further analyses. Results Cardiac contractile function was more deteriorated by LPS injection in DKO mice than WT mice despite comparable changes in pro-inflammatory cytokine production. LPS injection reduced myocardial uptake of FA tracer by 30% in both types of mice, while uptake of the glucose tracer did not significantly change in either group of mice in sepsis. Storage of glycogen and triacylglycerol in hearts was remarkably increased by LPS injection in both mice. Metabolome analysis revealed that LPS-induced suppression of pool size in the TCA cycle was more enhanced in DKO hearts. A tracing study with 13C6-glucose further revealed that LPS injection substantially reduced glucose-derived metabolites in the TCA cycle and related amino acids in DKO hearts. Consistent with these findings, glucose oxidation in vitro was similarly and markedly reduced in both mice. Serum concentration of β-hydroxybutyrate and cardiac expression of genes associated with ketolysis were reduced in septic mice. Conclusions Our study demonstrated that LPS-induced cardiac contractile dysfunction is associated with the robust suppression of catabolism of energy substrates including FAs, glucose and ketone bodies and accumulation of glycogen and triacylglycerol in the heart. Thus, a fuel shift from FAs to glucose and/or ketone bodies may be detrimental rather than protective under septic conditions.

Aims The metabolism of the failing heart is characterized by an increase in glucose uptake with reduced fatty acid (FA) oxidation. We previously found that the genetic deletion of FA-binding protein-4 and -5 [double knockout (DKO)] induces an increased myocardial reliance on glucose with decreased FA uptake in mice. However, whether this fuel switch confers functional benefit during the hypertrophic response remains open to debate. To address this question, we investigated the contractile function and metabolic profile of DKO hearts subjected to pressure overload. Methods and results Transverse aortic constriction (TAC) significantly reduced cardiac contraction in DKO mice (DKO-TAC), although an increase in cardiac mass and interstitial fibrosis was comparable with wild-type TAC (WT-TAC). DKO-TAC hearts exhibited enhanced glucose uptake by 8-fold compared with WT-TAC. Metabolic profiling and isotopomer analysis revealed that the pool size in the TCA cycle and the level of phosphocreatine were significantly reduced in DKO-TAC hearts, despite a marked increase in glycolytic flux. The ingestion of a diet enriched in medium-chain FAs restored cardiac contractile dysfunction in DKO-TAC hearts. The de novo synthesis of amino acids as well as FA from glycolytic flux was unlikely to be suppressed, despite a reduction in each precursor. The pentose phosphate pathway was also facilitated, which led to the increased production of a coenzyme for lipogenesis and a precursor for nucleotide synthesis. These findings suggest that reduced FA utilization is not sufficiently compensated by a robust increase in glucose uptake when the energy demand is elevated. Glucose utilization for sustained biomass synthesis further enhances diminishment of the pool size in the TCA cycle. Conclusions Our data suggest that glucose is preferentially utilized for biomass synthesis rather than ATP production during pressure-overload-induced cardiac hypertrophy and that the efficient supplementation of energy substrates may restore cardiac dysfunction caused by energy insufficiency.

The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of (67)Ga-labeled Fabs for low renal radioactivity levels.

No-carrier-added 177Lu produced via the 176Yb(n, γ)177Yb → 177Lu process was separated from the macroscopic amounts of the Yb target using reversed-phase ion-pair liquid chromatography. To produce a highly purified 177Lu solution capable of labeling antibodies, the metallic impurities were removed using cation, chelating ion, and anion exchange columns. After the elimination of metallic impurities, the concentrations of Ca, Fe, and Zn were reduced from 87, 340, and 77 ppb to 13, 18, and 9 ppb, respectively. Consequently, the labeling yield of the 177Lu-labeled antibody increased from

Near infrared (NIR) fluorescent probes are ideal for in vivo imaging because they offer deeper tissue penetration and lower background autofluorescence. Although most fluorophores in this range are cyanine-based dyes, several new classes of fluorescent NIR probes have been developed. In this study, we developed organic bacteriochlorin derivatives, NMP4 and NMP5, which are excited with a single green light and emit different narrow, well-resolved bands in the NIR (peak of 739 and 770 nm for NMP4 and NMP5, respectively). When conjugated to galactosyl-human serum albumin (hGSA) or glucosyl-human serum albumin (glu-HSA), both targeting H-type lectins, including the β-d-galactose receptor expressing on ovarian cancer, these agents become targeted, activatable, single excitation, multicolor NIR fluorescence probes. After conjugation to either glu-HSA or hGSA, substantial quenching of fluorescence occurs that is reversed after cell binding and internalization. In vitro studies showed higher cancer cell uptake with NMP4 or NMP5 conjugated to hGSA compared to the same conjugates with glu-HSA. In vivo single excitation two-color imaging was performed after intraperitoneal injection of these agents into mice with disseminated ovarian cancer. Excited with a single green light, distinct NIR emission spectra from each fluorophore were detected and could be distinguished with spectral unmixing. In vivo results using a red fluorescence protein (RFP) labeled tumor model of disseminated ovarian cancer demonstrated high sensitivity and specificity for all probes. The success of single excitation, 2-color NIR fluorescence imaging with a new class of bacteriochlorin-based activatable fluorophores, NMP4 and NMP5, paves the way for further exploration of noncyanine dye-based NIR fluorophores.

Background The aim of this study is to investigate the underlying biologic mechanisms of 2-[18F]-fluoro-2-deoxy- d -glucose (18F-FDG) uptake on positron emission tomography (PET) in non-small cell lung cancer (NSCLC). Methods One-hundred forty patients with NSCLC who underwent 18F-FDG PET were included in the study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), GLUT3, hypoxia-inducible factor-1 alpha (HIF-1α), hexokinase I, vascular endothelial growth factor (VEGF), microvessels (CD34), epidermal growth factor receptor (EGFR), and molecules relevant to PI3K/Akt/mTOR signaling pathway (PTEN, p-Akt, p-mTOR and p-S6). We also conducted in vitro studies of 18F-FDG uptake and mTOR inhibition in NSCLC cells. Results High 18F-FDG uptake was significantly associated with poor prognosis in NSCLC patients. 18F-FDG uptake was significantly correlated with GLUT1, hexokinase I, HIF-1α, VEGF, CD34, p-Akt, p-mTOR and EGFR. PTEN expression showed inverse correlation with 18F-FDG uptake. In in vitro study, 18F-FDG uptake was markedly decreased by the inhibition of GLUT1 and GLUT1 upregulation by the induction of HIF-1α increased the 18F-FDG uptake. Inhibition of both mTOR complex1 (mTORC1) and mTORC2 suppressed cell growth, but activity of mTORC1 regulated the 18F-FDG uptake. NCI-H1650 cells with PTEN loss showed the highest 18F-FDG uptake and the least sensitivity to mTOR inhibitors. Conclusion The amount of 18F-FDG accumulation is associated with molecules relevant to glucose metabolism, hypoxia, angiogenesis and mTOR signaling pathway. Especially, PTEN status may affect not only 18F-FDG uptake but also effect of mTOR inhibitors on the growth of NSCLC.

Blood contamination, such as bloody ascites or hemorrhages during surgery, is a potential hazard for clinical application of fluorescence imaging. In order to overcome this problem, we investigate if fluorescence-lifetime imaging helps to overcome this problem. Samples were prepared at concentrations ranging 0.3-2.4 μm and mixed with 0-10% of blood. Fluorescence intensities and lifetimes of samples were measured using a time-domain fluorescence imager. Ovarian cancer SHIN3 cells overexpressing the D-galactose receptor were injected into the peritoneal cavity 2.5 weeks before the experiments. Galactosyl serum albumin-rhodamine green (GSA-RhodG), which bound to the D-galactose receptor and was internalized thereafter, was administered intraperitoneally to peritoneal ovarian cancer-bearing mice with various degrees of bloody ascites. In vitro study showed a linear correlation between fluorescence intensity and probe concentration (r(2) > 0.99), whereas the fluorescence lifetime was consistent (range, 3.33 ± 0.15-3.75 ± 0.04 ns). By adding 10% of blood to samples, fluorescence intensities decreased to

Photoimmunotherapy (PIT) is a new cancer treatment that combines the specificity of an- tibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two commonly available anti-EGFR mono- clonal antibodies, cetuximab and panitumumab, for their effectiveness as PIT agents in EGFR positive tumor models. A photosensitizer, IR-700, conjugated to either cetuximab (cet-IR700) or panitumumab (pan-IR700), was evaluated using EGFR-expressing A431 and MDAMB468-luc cells in 2D- and 3D-culture. PIT was conducted with irradiation of NIR light after exposure of the sample or animal to each conjugate. In vivo PIT was performed with fractionated exposure of NIR light after injection of each agent into A431 xenografts or a MDAMB468-luc orthotopic tumor bearing model. Cet-IR700 and pan-IR700 bound with equal affinity to the cells in 2D-culture and penetrated equally into the 3D-spheroid, resulting in identical PIT cytotoxic effects in vitro. In contrast, in vivo anti-tumor effects of PIT with cet-IR700 were inferior to that of pan-IR700. Assess- ment of the biodistribution showed lower accumulation into the tumors and more rapid hepatic catabolism of cet-IR700 compared to pan-IR700. Although cet-IR700 and pan- IR700 showed identical in vitro characteristics, pan-IR700 showed better therapeutic tumor responses than cet-IR700 in in vivo mice models due to the prolonged retention of the con- jugate in the circulation, suggesting that retention in the circulation is advantageous for tu- mor responses to PIT. These results suggest that the choice of monoclonal antibody in photosensitizer conjugates may influence the effectiveness of PIT. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies.

Objective: To investigate the role of transporter proteins in gadolinium (Gd) distribution and retention in the brain after one high-dose injection of Gd-based contrast agent (GBCA). Methods and materials: 30 ddY mice were randomly divided into three treatment groups to be intravenously injected with either Gadodiamide (linear GBCA), Gadobutrol (macrocyclic GBCA), or Gadoterate (macrocyclic GBCA) at a dose of 5 mmol/kg, while five mice in the control group received 250 µL saline. Five minutes (5 min) and ten days (10d) post-injection, the cerebrospinal fluid (CSF), choroid plexus (CP), and meninges and associated vasculature (MAV) were collected. The brain was then dissected to obtain the olfactory bulb, cerebral cortex, hippocampus, cerebellum, and brainstem. Proteins were extracted and separated by a size-exclusion high-performance liquid chromatography (SEC) system, and Gd concentrations were quantified by inductively coupled plasma mass spectrometry (ICP-MS). Results: 5 m post-injection, the Gadodiamide group had the highest Gd concentration, while Gadoterate had the lowest Gd concentration in all parts of the brain (p < .05). Gd concentration was highest in the cerebrospinal fluid (CSF) of the Gadodiamide group (578.4 ± 135.3 nmol), while Gd concentration was highest in MAV in the Gadobutrol group (379.7 ± 75.4 nmol) at 5 min post-injection. At 10d, in spite of the significant decrease of Gd from all GBCAs ( p < 0.01), retained Gd from Gadodiamide was detected all over the brain in several molecules that varied in size. Gd from Gadobutrol detected in the olfactory bulb (8.7 ± 4.5 nmol) was significantly higher than in other parts of the brain. Although most Gd from Gadobutrol was found in molecules similar in size to Gadobutrol, it was also found in several protein molecules of molecular size larger than the contrast agents. Only a small amount of Gd from Gadoterate was found in the brain. Conclusion: GBCAs may be able to pass through intact brain barriers, and the chemical structures of GBCAs may affect the penetration capability of Gd into the brain. Retained Gd in the brain tissue from Gadodiamide and Gadobutrol may be bound to some organic molecules, including proteins. Advances in knowledge: Intact GBCA are able to penetrate a series of brain barrier immediately after administration regardless the type of the chelate. Gd may be bound with macromolecules that may cause Gd retention in the brain.

Objective— Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium. Approach and Results— Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-( p -iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-β and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis. Conclusions— Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Objective: Almost all small animal SPECT is performed with pinhole collimators (PH), including single-PH (SPH) and multi-PH (MPH). In the clinical study, not only PH but also parallel-hole collimator (PAH) is often used in planar and SPECT imaging. However, there have been no comparative studies on image quality with various collimators on the small animal imaging. This study compared the basic characteristics of PH and PAH in small animal imaging. Methods: Performance of planar and SPECT images was evaluated using 99mTcO4 - and SPH, MPH and PAH with low energy and high resolution on the SPECT/CT scanner FX3200. We measured sensitivity, resolution, concentration linearity and uniformity. Planar imaging of mice with 99mTc-labeled mercaptoacetyltriglycine (99mTc-MAG3) was performed using SPH and PAH. SPECT imaging with 99mTc-methylene diphosphonate (99mTc-MDP) was performed using all collimators. Results: With SPH, MPH and PAH, sensitivity was 43.5, 211.2 and 926.5 cps/MBq, respectively, and spatial resolution was 0.60/0.56, non/0.96, 5.20/5.34 mm full-width half maximum (planar/SPECT), respectively. There were marked correlations between the radioactivity counts on images and radioactivity with all collimators. Values of % standard deviation on planar imaging showed small differences between the SPH and PAH, while the values were the smallest on SPECT imaging with MPH. On imaging of mice, SPH yielded high-quality 99mTc-MAG3-planar images when compared with PAH. MPH yielded sharper 99mTc-MDP-SPECT images than SPH and PAH. Conclusions: The characteristics of PH and PAH differed on small animal imaging. Although sensitivity was higher with PAH, PH showed higher resolution. Among the PH collimators, SPH was more appropriate for planar imaging, and MPH was more suitable for SPECT imaging in a small animal imaging scanner with CZT detector. © 2013 The Japanese Society of Nuclear Medicine., Thesis of Yusuke Higaki / 檜垣 佑輔 博士論文 金沢大学医薬保健学総合研究科（保健学専攻）

We examined the possible efficacy of the yttrium-90 ((90)Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin.MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111((111)In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of (90)Y-labeled MAb-6 ((90)Y-MAb-6) to EBC, H1373, and SW948-inoculated mice.Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of (111)In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of (90)Y-MAb-6 (100 μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of (90)Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity.Our findings demonstrate that CDH3/P-cadherin-targeting RIT with (90)Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 ((111)In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with (111)In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH(3) and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In (111)In radiolabeling reactions, C-ODTA provided (111)In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The (111)In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the (111)In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare (111)In-labeled peptides of high specific activities in high radiochemical yields.

Metallic radionuclides provide target-specific radiolabeled probes for molecular imaging in radiochemical yields sufficient for administration to subjects without purification. However, unlabeled ligands in the injectate can compete for targeted molecules with radiolabeled probes, which eventually necessitates postlabeling purification. Herein we describe a “1 to 3” design to circumvent the issue by taking advantage of inherent coordination properties of technetium-99m (99mTc). A monovalent RGD ligand possessing an isonitrile as a coordinating moiety (CN-RGD) was reacted with [99mTc(CO)3(OH2)3]+ to prepare [99mTc(CO)3(CN-RGD)3]+ in over 95% radiochemical yields. This complex exhibited higher integrin αvβ3 binding affinity than its unlabeled monovalent ligand, primarily due to its multivalency. This compound visualized a murine tumor without removing unlabeled ligands, while a 99mTc-labeled monovalent probe derived from a monovalent ligand could not. The metal coordination-mediated synthesis of radiolabele...

Introduction 3-[ 18 F]fluoro-α-methyl-l-tyrosine ([ 18 F]FAMT) is a useful amino acid tracer for positron emission tomography (PET) imaging of malignant tumors. FAMT analogs labeled with 76 Br, a positron emitter with a long half-life ( t 1/2 =16.1 h), could potentially be widely used as amino acid tracers for tumor imaging. In this study, 3-[ 76 Br]bromo-α-methyl-l-tyrosine ([ 76 Br]BAMT) was designed, and its usefulness was evaluated as a novel PET tracer for imaging malignant tumors. Methods In this study, both [ 76 Br]BAMT and [ 77 Br]BAMT were prepared. The in vitro and in vivo stability of [ 77 Br]BAMT was evaluated by HPLC analysis. Cellular uptake and retention of [ 77 Br]BAMT and [ 18 F]FAMT were evaluated using LS180 colon adenocarcinoma cells. Biodistribution studies were performed in normal mice and in LS180 tumor-bearing mice, and the tumors were imaged with a small-animal PET scanner. Results [ 77 Br]BAMT was stable in vitro but was catabolized after administration in mice. Cellular accumulation and retention of [ 77 Br]BAMT were significantly higher than those of [ 18 F]FAMT. In biodistribution studies, the tumor accumulation of [ 77 Br]BAMT was higher than that of [ 18 F]FAMT. However, some level of debromination was seen, which caused more retention of radioactivity in the blood and organs than was seen with [ 18 F]FAMT. PET imaging with [ 76 Br]BAMT enabled clear visualization of the tumor, and the whole-body image using [ 76 Br]BAMT was similar to that using [ 18 F]FAMT. Conclusions [ 77 Br]BAMT showed high levels of tumor accumulation, and [ 76 Br]BAMT enabled clear visualization of the tumor by PET imaging. Although an improvement in stability is still needed, 76 Br-labeled FAMT analogs could potentially serve as PET tracers for the imaging of malignant tumors.

Meta-iodobenzylguanidine (MIBG) labeled with (123)I or (131)I has been widely used for the diagnosis and radiotherapy of norepinephrine transporter (NET)-expressing tumors. However, (123)I/(131)I-MIBG has limitations for detecting small lesions because of its lower spatial resolution than PET tracers. In this study, meta-bromobenzylguanidine (MBBG) labeled with (76)Br (half-life, 16.1 h), an attractive positron emitter, was prepared and evaluated as a potential PET tracer for imaging NET-expressing tumors.(76)Br-MBBG was prepared by a halogen-exchange reaction between the (76)Br and iodine of nonradioactive MIBG. The stability of MBBG was evaluated in vitro and in vivo by high-performance liquid chromatography analysis. Cellular uptake studies with or without NET inhibitors were performed in NET-positive PC-12 cell lines. Biodistribution studies were performed in PC-12 tumor-bearing nude mice by administration of a mixed solution of MBBG, MIBG, and (18)F-FDG. The tumor was imaged using (76)Br-MBBG and (18)F-FDG with a small-animal PET scanner.MBBG was stable in vitro, but some time-dependent dehalogenation was observed after administration in mice. MBBG showed high uptake in PC-12 tumor cells that was significantly decreased by the addition of NET inhibitors. In biodistribution studies, MBBG showed high tumor accumulation (32.0 +/- 18.6 percentage injected dose per gram at 3 h after administration), and the tumor-to-blood ratio reached as high as 54.4 +/- 31.9 at 3 h after administration. The tumor uptake of MBBG correlated well with that of MIBG (r = 0.997) but not with that of (18)F-FDG. (76)Br-MBBG PET showed a clear image of the transplanted tumor, with high sensitivity, which was different from the lesion shown by (18)F-FDG PET.(76)Br-MBBG showed high tumor accumulation, which correlated well with that of MIBG, and provided a clear PET image. These results indicated that (76)Br-MBBG would be a potential PET tracer for imaging NET-expressing neuroendocrine tumors and could provide useful information for determining the indications for (131)I-MIBG therapy.

Objective l-3-[18F]-fluoro-α-methyl tyrosine (FAMT) is transported into cancer cells by l-type amino acid transporter 1 (LAT1). The purpose of the present study is to correlate the uptake of FAMT and FDG with the cellular proliferative activity measured by the Ki-67 labeling index (Ki-67 LI) in oral squamous cell carcinoma (OSCC).

Matrix metalloproteinases (MMPs) are endopeptidases responsible for degrading the extracellular matrix (ECM) and remodeling tissue in both physiological and pathological processes. MMP2 and membrane-type 1 MMP (MT1-MMP) have been associated with tumor invasion, metastasis and angiogenesis; therefore, a molecular imaging strategy assessing their activity may help to predict the malignancy of tumors. Here, we established a novel method of specifically tagging the surface of MMP2- and MT1-MMP-positive cells, and applied it to the development of an optical imaging probe. We constructed a protein-based probe composed of a glutathione-S-transferase (GST)-tag (Inhibitory [I]-domain), a polypeptide as a specific substrate for both MMP2 and MT1-MMP (Cleaved [C]-domain), a transmembrane domain of the epidermal growth factor receptor (Transmembrane [TM]-domain), and DsRed2 (Fluorescent [F]-domain). In vitro experiments clearly demonstrated that, after the probe was cleaved at the C-domain by the MMPs, the resultant TM–F-domain was inserted into the cellular membrane. Optical imaging experiments in vivo demonstrated that the probe was cleaved and specifically remained in tumor xenografts in a MMP-dependent manner. These results indicate that the release of the I–C-domain through the proteolytic cleavage of the C-domain by MMP2 and MT1-MMP triggers the tagging of cellular membranes with the TM–F-domain. The present feasibility study opens the door to the development of a novel imaging probe for tumor malignancy using positron emission tomography as well as an optical imaging device.

Clinical application of FDG-PET in head and neck cancer includes identification of metastases, unknown primary head and neck malignancy, or second primary carcinoma, and also recurrent tumor after treatment. In this study, the additional value of PET/CT fusion images over PET images alone was evaluated in patients with initial staging and follow up of head and neck malignancy.Forty patients with suspected primary head and neck malignancy and 129 patients with suspected relapse after treatment of head and neck malignancy were included. FDG-PET/CT study was performed after the intravenous administration of FDG (5 MBq/kg). Target of evaluation was set at primary tumor, cervical lymph node, and whole body. PET images and PET with CT fusion images were compared. Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Results of PET and PET/CT were compared with postoperative histopathological examination, and case by case comparison of PET and PET/CT results for each region was performed. The additional value of CT images over PET only images was assessed. Statistical differences in sensitivity and specificity were evaluated.In the comparative evaluation of 507 targets by PET alone and PET/CT, 401 targets showed agreement of the results. Of the 106 discordant targets, 103 showed a positive result on PET alone and negative result on PET/CT. These results showed a significant difference (p0.01). Sensitivity of PET/CT was slightly higher than that of PET without statistical significance, while specificity of PET/CT was significantly higher than that of PET alone (Initial staging: 90.5% vs. 62.2%, p0.01; Follow up: 97.2% vs. 74.4%, p0.01). In Fisher's direct probability test, a significant difference was noted in the sensitivity (Initial staging: 91.3% vs. 87.0%, p0.01; Follow up: 93.9% vs. 91.4%, p0.01).Combined PET/CT showed improved diagnostic performance than PET alone by decreasing the number of false positive findings in patients with initial staging and follow up of head and neck malignancy.

Introduction We have developed a method for producing no-carrier-added 64 Cu by using chelating resin bearing iminodiacetic acid groups. Methods We optimized the conditions for the selective separation of radioactive Cu from Ni and Co using the chelating resin and produced no-carrier-added 64 Cu under the optimized conditions. We analyzed the amounts of the metal ions present in 64 Cu by inductively coupled-plasma mass spectroscopy (ICP-MS) and optical emission spectroscopy (ICP-OES), and performed radiolabeling of monoclonal antibodies in order to investigate the quality of the 64 Cu produced in this study. Results Radioactive Cu was separated from Ni and Co with 0.1 and 2 M HCl solutions. The yield of 64 Cu isolated from the 64 NiO target was almost 87%. The radiochemical purity of 64 Cu obtained from different amounts of 64 NiO targets was >99% in all cases. We found that the 64 Cu solution presented extremely low amounts of the metal ions and showed high specific activity (average: 595 GBq/μmol). Moreover, the antibodies were labeled with 64 Cu with a high average efficiency (average: 88%). Conclusions We could efficiently separate 64 Cu by using short ion-exchange columns. The chelating ion-exchange method provides a high quality of 64 Cu that is sufficient for the synthesis of 64 Cu-labeled antibodies and medical applications.

Frizzled homolog 10 (FZD10) is expressed at high levels on the cell surface of almost all synovial sarcoma tissues, but is absent in most normal organs. In a previous study, yttrium-90 ((90)Y)-labeled anti-FZD10 antibody (MAb 92-13) showed considerable therapeutic efficacy in synovial sarcoma cell-bearing mice. The purpose of the present study was to elucidate the factors associated with this therapeutic efficacy of (90)Y-MAb 92-13.FZD10 expression levels of SYO-1 (FZD10-overexpressing synovial sarcoma cell line) and DLD-1/FZD10 (FZD10-transfected DLD-1 cell) were determined by the cell binding assay, and their radiosensitivity was evaluated by incubation with (90)Y-MAb 92-13 in vitro. Biodistribution study of indium-111 ((111)In)-MAb 92-13 was performed in SYO-1 and DLD-1/FZD10 tumor-bearing mice. For therapeutic studies, SYO-1 and DLD-1/FZD10 tumor-bearing mice were treated with (90)Y-MAb 92-13 (100, 150, and 200 muCi), after which the change in tumor volume was measured. Immunohistochemical staining was performed on the excised tumor.Expression level of FZD10 on DLD-1/FZD10 was much greater than that on SYO-1. The accumulation of (111)In-MAb 92-13 was much higher in DLD-1/FZD10 tumor-bearing mice than in SYO-1 tumor-bearing mice (49.0 +/- 4.2 and 22.0 +/- 4.5% ID/g, respectively, at 48 h after administration). In SYO-1 tumor, substantial tumor size reduction was observed in all mice treated with (90)Y-MAb 92-13 (tumor volume decreased to less than 0.1 cm(3) at 11 days after treatment) and tumor regrowth was not observed in most of them. In contrast, only slow progression was observed in DLD-1/FZD10 tumor. When incubated with (90)Y-MAb 92-13, high radioactivity was needed to damage DLD-1/FZD10. Immunohistochemical study indicated apoptosis of SYO-1 tumor.The therapeutic efficacy of RIT seems to largely depend on the tumor radiosensitivity.

金沢大学疾患モデル総合研究センター / 金沢大学学際科学実験センターアイソトープ総合研究施設, Purpose: We have developed a 186Re-mercaptoacetylglycylglycylglycine complex-conjugated bisphosphonate (186Re-MAG3-HBP) for the treatment of painful bone metastases. We assumed competitive inhibitors of protein binding to be useful for procuring a favorable biodistribution of 186Re-MAG3-HBP for the palliation of bone pain because it has been reported that the concurrent administration of 99mTc-MAG3 and drugs with high affinity for serum protein produced competitive displacement at specific binding sites and enhanced total clearance and tissue distribution. Methods: The displacement effects of several protein-binding inhibitors on the protein binding of 186Re-MAG3-HBP were investigated. Biodistribution experiments were performed by intravenously administering 186Re-MAG3-HBP into rats with ceftriaxone as a competitive protein-binding inhibitor or saline. Results: The protein binding of 186Re-MAG3-HBP in rat serum, human serum, and a human serum albumin solution was significantly decreased by the addition of ceftriaxone, which has high affinity for binding site I on serum albumin. In the biodistribution experiments, pretreatment with ceftriaxone enhanced the clearance of the radioactivity of 186Re-MAG3-HBP in blood and nontarget tissues but had no effect on accumulation in bone. Conclusions: The findings suggested that the use of protein-binding competitive inhibitors would be effective in improving the pharmacokinetics of radiopharmaceuticals with high affinity for serum protein. © 2008 Springer-Verlag.

The radionuclide 67Cu was produced via the 68Zn(p,2p)67Cu reaction by irradiating enriched 68Zn targets with 70 MeV proton beam. Copper-67 was chemically separated from the zinc target by ion-exchange chromatography using Chelex-100 chelating ion-exchange resin. Procedure for recovery of the enriched 68Zn was developed. The target recovery yield of this method was evaluated to be more than 97%.

Since elevated levels of gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are associated with a poor prognosis in cancer patients, these enzymes are potential targets for tumor imaging. In the present study, a cyclic decapeptide, cCTTHWGFTLC (CTT), was selected as a mother compound because of its selective inhibitory activity toward gelatinases. For imaging gelatinase activity in tumors, we designed a CTT-based radiopharmaceutical taking into consideration that (1) the HWGF motif of the peptide is important for the activity, (2) hydrophilic radiolabeled peptides show low-level accumulation in the liver and (3) an increase in the negative charge of radiolabeled peptides is effective in reducing renal accumulation. Thus, a highly hydrophilic and negatively charged radiolabel, indiun-111-diethylenetriaminepentaacetic acid ( 111 In-DTPA), was attached to an N-terminal residue distant from the HWGF motif ( 111 In-DTPA-CTT). In MMP-2 inhibition assays, In-DTPA-CTT significantly inhibited the proteolytic activity in a concentration-dependent fashion. When injected into normal mice, 111 In-DTPA-CTT showed low levels of radioactivity in the liver and kidney. A comparison of the pharmacokinetic characteristics of 111 In-DTPA-CTT with those of other CTT derivatives having different physicochemical properties revealed that the increase in hydrophilicity and negative charge caused by the conjugation of 111 In-DTPA reduced levels of radioactivity in the liver and kidney. In tumor-bearing mice, a significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of 111 In-DTPA-CTT and gelatinase activity. These findings support the validity of the chemical design of 111 In-DTPA-CTT for reducing accumulation in nontarget tissues and maintaining the inhibitory activity of the mother compound. Furthermore, 111 In-DTPA-CTT derivatives would be potential radiopharmaceuticals for the imaging of gelatinase activity in metastatic tumors in vivo.