Izumi O. Umeda

Development of 111In-Labeled Liposomes for Vulnerable Atherosclerotic Plaque Imaging

Macrophage infiltration is a common characteristic feature of atherosclerotic-vulnerable plaques. Macrophages recognize phosphatidylserine (PS) exposed on the surface of apoptotic cells, which triggers the engulfment of the apoptotic cells by macrophages through phagocytosis. In this study, we prepared radiolabeled PS liposomes for detection of vulnerable plaques. Methods: PS liposomes were prepared by lipid film hydration. Phosphatidylcholine (PC) liposomes were prepared as controls. Liposomes (100 or 200 nm) were generated by an extruder to produce PS100, PS200, PC100, and PC200 liposomes. These were then radiolabeled by encapsulating 111In-nitrilotriacetic acid using an active-loading method. 111In liposomes were incubated with cultured macrophages for 2 h, and the uptake level was measured. For biodistribution studies, the 111In liposomes were injected intravenously into ddY mice. In addition, the 111In liposomes were injected into apolipoprotein E–deficient (apoE−/−) mice, and the aortas were harvested for autoradiography and oil red O staining. For SPECT imaging, 111In liposomes were injected intravenously into Watanabe heritable hyperlipidemic rabbits and scanned 48 h after injection. Results: The radiochemical yields were greater than 95% for all the prepared 111In liposomes. The level of in vitro uptake by macrophages was 60.5, 14.7, 32.0, and 14.4 percentage injected dose per milligram of protein for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. In biodistribution studies, high spleen uptake was seen with PC liposomes. Liver uptake was high for all liposomes but was lowest with 111In-PS200. The blood half-lives were 3.2, 22.0, 3.6, and 7.4 min for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. The distribution of 111In-labeled PS liposomes into atherosclerotic regions determined by autoradiography was well matched with the results of oil red O staining in apoE−/− mice. The target-to-nontarget ratios were 2.62, 2.23, 3.27, and 2.51 for 111In-PS100, 111In-PC100, 111In-PS200, and 111In-PC200, respectively. The aorta was successfully visualized by SPECT at 48 h after 111In-labeled PS liposome injection; however, high liver uptake was also observed. Discussion: From the in vitro uptake study, it has been demonstrated that macrophage targeting was accomplished by PS modification. Also, an atherosclerotic region was successfully detected by 111In-PS200 in apoE−/− mice and Watanabe heritable hyperlipidemic rabbits in vivo. Liposome modification to obtain slower blood clearance and lower liver uptake would be required to improve the SPECT images.

Radiolabeled liposome imaging determines an indication for liposomal anticancer agent in ovarian cancer mouse xenograft models

Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov‐3, SK‐OV‐3, KURAMOCHI, and TOV‐112D) to correlate the therapeutic effects of doxorubicin‐encapsulated liposome (Doxil®) and histological characteristics linked to the enhanced permeability and retention effect. We next generated 111In‐encapsulated liposomes to examine their capacities to determine indications for Doxil® treatment by single‐photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil® were drastically enhanced in Caov‐3, moderately in SK‐OV‐3, and minimally in KURAMOCHI and TOV‐112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov‐3 and SK‐OV‐3, indicating a close relation with the enhanced antitumor effects. Next, 111In‐encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov‐3, relatively high in SK‐OV‐3, and low in two other tumors. Importantly, as both Doxil® effects and liposomal accumulation varied in the SK‐OV‐3 group, we individually obtained SPECT/CT images of SK‐OV‐3‐bearing mouse (n = 11) before Doxil® treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil® was confirmed (R2 = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents.

In Vivo SPECT Imaging with 111In-DOTA-c(RGDfK) to Detect Early Pancreatic Cancer in a Hamster Pancreatic Carcinogenesis Model

Early detection of pancreatic cancer is key to overcoming its poor prognosis. αvβ3-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 111In-1,4,7,10-tetraazacylododecane-N,N′,N″,N′′′-tetraacetic acid-cyclo-(Arg-Gly-Asp-d-Phe-Lys) [111In-DOTA-c(RGDfK)], an imaging probe of αvβ3-integrin, for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. Methods: 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 111In-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 αvβ3-integrin. In a mouse model in which inflammation was induced with turpentine, the uptake of 111In-DOTA-c(RGDfK) in inflammatory regions was evaluated with ARG and compared with that of 18F-FDG. Results: 111In-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 111In-DOTA-c(RGDfK) strongly correlated with αvβ3-integrin expression. In the inflammatory model, inflammation-to-muscle ratios for 18F-FDG and 111In-DOTA-c(RGDfK) were 8.37 ± 4.37 and 1.98 ± 0.60, respectively. These results imply that 111In-DOTA-c(RGDfK) has a lower rate of false-positive tumor detection than 18F-FDG. Conclusion: Our findings suggest that SPECT with 111In-DOTA-c(RGDfK) has great potential for the early and accurate detection of pancreatic cancer.

Evaluation of [125I]IPOS as a molecular imaging probe for hypoxia-inducible factor-1-active regions in a tumor: comparison among single-photon emission computed tomography/X-ray computed tomography imaging, autoradiography, and immunohistochemistry.

To image hypoxia‐inducible factor‐1 (HIF‐1)‐active tumors, we previously developed a chimeric protein probe ([123/125I]IPOS) that is degraded in the same manner as HIF‐1α under normoxic conditions. In the present study, we aim to show that the accumulation of radioiodinated POS reflects the expression of HIF‐1. In vivo single‐photon emission computed tomography (SPECT)/X‐ray CT (CT) imaging, autoradiography, and double‐fluorescent immunostaining for HIF‐1α and pimonidazole (PIMO) were carried out 24 h after the injection of [125I]IPOS. Tumor metabolite analysis was also carried out. A tumor was clearly visualized by multi‐pinhole, high‐resolution SPECT/CT imaging with [125I]IPOS. The obtained images were in accordance with the corresponding autoradiograms and with the results of ex vivo biodistribution. A metabolite analysis revealed that 77% of the radioactivity was eluted in the macromolecular fraction, suggesting that the radioactivity mainly existed as [125I]IPOS in the tumors. Immunohistochemistry revealed that the HIF‐1α‐positive areas and PIMO‐positive areas were not always identical, only some of the regions were positive for both markers. The areas showing [125I]IPOS accumulation were positively and significantly correlated with the HIF‐1α‐positive areas (R = 0.75, P < 0.0001). The correlation coefficient between [125I]IPOS‐accumulated areas and HIF‐1α‐positive areas was significantly greater than that between the [125I]IPOS‐accumulated areas and the PIMO‐positive areas (P < 0.01). These findings indicate that [125I]IPOS accumulation reflects HIF‐1 expression. Thus, [123/125I]IPOS can serve as a useful probe for the molecular imaging of HIF‐1‐active tumors. (Cancer Sci 2011; 102: 2090–2096)

5-S-GAD, a novel radical scavenging compound, prevents lens opacity development

The ability of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD)-a novel catechol derivative isolated from an insect as an antibacterial substance-to scavenge free radicals and prevent cataract progression was examined. 5-S-GAD scavenged 1,1-diphenylpicrylhydrazyl (DPPH) and superoxide anions (O(2)(*)(-)), and inhibited lipid peroxidation. It also significantly inhibited the onset of glucocorticoid-induced lens opacification in chick embryos. These effects of 5-S-GAD were stronger than those of N-acetylcarnosine and TEMPOL, which are reported to be effective radical scavengers in the prevention of cataract progression. 5-S-GAD clearly delayed the maturation of cataracts induced by diamide in cultured lenses of rats. Daily instillation of 5-S-GAD retarded the development of lens opacity in galactose-fed rats. Biochemical analysis of the lenses revealed that 20-kDa proteins, presumably consisting of alpha-crystallin, were the most susceptible to oxidative stress, which leads to the carbonylation of the side chains of these proteins. alpha-Crystallin carbonylation induced by diamide or galactose was notably inhibited by 5-S-GAD in a dose-dependent manner. Our results show that 5-S-GAD prevents acute lens opacification in these short-term experimental models, possibly in part by virtue of its antioxidative property, and 5-S-GAD is expected to have long-term pharmaceutical effects.

In Vivo Visualization of Heterogeneous Intratumoral Distributionof Hypoxia-Inducible Factor-1α Activity by the Fusion of High-Resolution SPECT and Morphological Imaging Tests

Purpose. We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α (HIF) activity in tumor tissues in vivo. Methods. We synthesized of 125I-IPOS, a 125I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of 125I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of 125I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtained in vivo SPECT-CT fusion images were compared with ex vivo images of excised tumors. Fusion imaging with MRI was also examined. Results. 125I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of 125I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of 125I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of 125I-IPOS. Conclusion. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of 125I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activity in vivo.

Novel Indocyanine Green–Phytate Colloid Technique for Sentinel Node Detection in Head and Neck Mouse Study

Objective Sentinel node navigation surgery using real-time, near-infrared imaging with indocyanine green is becoming popular by allowing head and neck surgeons to avoid unnecessary neck dissection. The major drawback of this method is its quick migration through the lymphatics, limiting the diagnostic time window and undesirable detection of downstream nodes. We resolved this problem by mixing indocyanine green (ICG) with phytate colloid to retard its migration and demonstrated its feasibility in a nude mouse study. Study Design Experimental prospective animal study. Settings Animal laboratory. Subjects and Methods Indocyanine green at 3 concentrations was tested to determine the optimal concentration for sentinel lymph node detection in a mouse model. Effect of indocyanine green with phytate colloid mixture solutions was also analyzed. Indocyanine green or mixture solution at different mixing ratios were injected into the tongue of nude mice and near-infrared fluorescence images were captured sequentially for up to 48 hours. The brightness of fluorescence in the sentinel lymph node and lymph nodes further downstream were assessed. Results Indocyanine green concentration >50 μg/mL did not improve sentinel lymph node detection. The addition of phytate colloid to indocyanine green extended the period when sentinel lymph node was detectable. Second echelon lymph nodes were not imaged in mice injected with the mixture, while these were visualized in mice injected with indocyanine green alone. Conclusion This novel technique of ICG–phytate colloid mixture allows prolonged diagnostic time window, prevention of downstream subsequent nodes detection, and improved accuracy for the detection of true sentinel lymph nodes.

Predominant phosphatase in the ocular lens regulated by physiological concentrations of magnesium and calcium.

A magnesium-dependent phosphatase with a molecular weight of about 55 kDa was found in the lens of chicken embryo, mouse, rabbit and bovine. It appears to be unique to the lens and, when activated by magnesium, accounts for the majority of the phosphatase activity in the lens. Phosphatases in the lens were separated by using high-resolution gel permeation column chromatography, and phosphatase activity was determined with p-nitrophenyl phosphate (pNPP) as a substrate. The 55-kDa phosphatase showed very low basal activity, but the activity was increased concentration-dependently by magnesium ion (Mg(2+)), and at 1 mM Mg(2+), this enzyme accounted for over 50% of the total phosphatase activity in the lens. Calcium potently inhibited the magnesium-activated phosphatase activity in a dose-dependent (IC(50); about 50 microM), uncompetitive manner. The phosphatase activity was high in the acidic pH range, with an optimum pH value of 5.5. The characteristics of the lens Mg(2+)-dependent phosphatase, such as substrate specificity and sensitivity to various phosphatase inhibitors, showed little similarity to those of any reported phosphatase. The Mg(2+) and Ca(2+) concentrations that were found to be effective in this study are similar to physiological concentrations in the lens; therefore it is likely that these ions physiologically regulate the 55-kDa phosphatase activity in the lens.

Antitumor effects of eribulin depend on modulation of the tumor microenvironment by vascular remodeling in mouse models

We previously reported that eribulin mesylate (eribulin), a tubulin‐binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin‐induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post‐erlotinib treatment H1650 (PE‐H1650) xenograft model. Furthermore, infiltrating CD11b‐positive immune cells were significantly increased in multiple eribulin‐treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.

Multi-head gamma camera system with CdZnTe semiconductor detectors

The purpose of our research is to develop a new brain single photon emission CT (SPECT) system with a semiconductor detector. For the detector we adopted CdZnTe semiconductor modular detectors to improve the energy resolution and spatial resolution. The size of a module was 39 × 39 mm2 (16 × 16 pixels) and the thickness of the CdZnTe crystal was 5 mm. With this modular detector we made three detector units each consisting of ten CdZnTe modules. In each detector unit we set 5 × 2 (=10) detector modules, and thus the maximum active field became 195 × 78 mm2. And to increase the sensitivity we used a multi-pinhole collimator with a hole size of 2 mmφ and the thickness of 1 cm. These pinholes were located on the surface of the hexagonal gantry around the object to be reconstructed. In this paper we evaluated the performance of our proposed system with several phantoms (a cold channel phantom, hot rod phantom and brain phantom). The results obtained with the experiments confirmed the validity of the proposed brain SPECT system.