Seigo Ishino

Targeting of Lectinlike Oxidized Low-Density Lipoprotein Receptor 1 (LOX-1) with 99mTc-Labeled Anti–LOX-1 Antibody: Potential Agent for Imaging of Vulnerable Plaque

Lectinlike oxidized low-density lipoprotein (LDL) receptor 1 (LOX-1), a cell surface receptor for oxidized LDL, has been implicated in vascular cell dysfunction related to plaque instability, which could be a potential target for an atherosclerosis imaging tracer. In this study, we designed and prepared 99mTc-labeled anti–LOX-1 monoclonal IgG and investigated its usefulness as an atherosclerosis imaging agent. Methods: Anti–LOX-1 monoclonal IgG and control mouse IgG2a were labeled with 99mTc after derivatization with 6-hydrazinonicotinic acid to yield 99mTc-LOX-1-mAb and 99mTc-IgG2a, respectively. Myocardial infarction–prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (atherosclerosis model) and control rabbits were injected intravenously with these probes, and in vivo planar imaging was performed. At 24 h after the injection, the aortas were removed, and radioactivity was measured. Autoradiography and histologic studies were performed with serial aortic sections. Results: The level of 99mTc-LOX-1-mAb accumulation was 2.0-fold higher than the level of 99mTc-IgG2a accumulation in WHHLMI rabbit aortas, and the level of 99mTc-LOX-1-mAb accumulation in WHHLMI rabbit aortas was 10.0-fold higher than the level of 99mTc-LOX-1-mAb accumulation in control rabbit aortas. In vivo imaging clearly visualized the atherosclerotic aortas of WHHLMI rabbits. Autoradiography and histologic studies revealed that regional 99mTc-IgG2a accumulation was independent of the histologic grade of the lesions; however, regional 99mTc-LOX-1-mAb accumulation was significantly correlated with LOX-1 expression density and the vulnerability index. The highest level of 99mTc-LOX-1-mAb accumulation, expressed as {radioactivity in region of interest (Bq/mm2)/[injected radioactivity (Bq)/animal body weight (g)]} × 102, was found in atheromatous lesions (3.8 ± 1.1 [mean ± SD]), followed in decreasing order by fibroatheromatous lesions (2.0 ± 1.0), collagen-rich lesions (1.6 ± 0.8), and neointimal lesions (1.4 ± 0.7). Conclusion: The level of 99mTc-LOX-1-mAb accumulation in grade IV atheroma was higher than that in neointimal lesions or other, more stable lesions. Nuclear imaging of LOX-1 expression with 99mTc-LOX-1-mAb may be a useful means for predicting atheroma at high risk for rupture.

Comparison of Contrast Agents for Atherosclerosis Imaging Using Cultured Macrophages: FDG Versus Ultrasmall Superparamagnetic Iron Oxide

Various noninvasive imaging methods have been developed to evaluate atherosclerotic plaques. Among them, 18F-FDG PET and MR imaging with ultrasmall superparamagnetic iron oxide particles (USPIO) have been used to quantify plaque inflammation. Both methods are based on the efficient uptake of FDG and USPIO by macrophages in atherosclerotic lesions. Differently polarized macrophages have been reported to have different characteristics that are involved in the pathologic development of atherosclerosis. M1 polarized macrophages are considered the more proatherogenic phenotype than M2 polarized macrophages. However, little is known regarding the association between macrophage polarization and FDG or USPIO accumulation. In this study, we investigated intracellular FDG and USPIO accumulation in M1 and M2 polarized macrophages. Methods: THP-1 macrophages were differentiated into M1 and M2 polarized macrophages. Under optimal glucose conditions, we investigated the 3H-labeled FDG uptake in M1 and M2 polarized macrophages. We then investigated intracellular USPIO uptake by M1 and M2 macrophages. Results: We found that M1 polarization, compared with M2 polarization, results in increased intracellular accumulation of FDG. To elucidate the mechanism by which FDG was preferentially accumulated in M1 macrophages, we examined messenger RNA expressions of glucose transporters (GLUTs) and hexokinases, which have pivotal roles in glucose uptake, and glucose-6-phosphatase (G6Pase), which catalyzes the reverse reaction of hexokinase. In M1 macrophages, GLUT-1, GLUT-3, hexokinase 1, and hexokinase 2 were upregulated and G6Pase was downregulated. In contrast to FDG, M1 polarization resulted in decreased intracellular accumulation of USPIO. We found that scavenger receptor A and CD11b, which are involved in USPIO binding and uptake, were significantly downregulated by M1 polarization. Conclusion: Compared with M2, proatherogenic M1 macrophages preferentially accumulated FDG but not USPIO, suggesting that FDG PET is a useful method for the detection of proinflammatory M1 macrophages.

99mTc-Annexin A5 for noninvasive characterization of atherosclerotic lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits

PurposeApoptosis is commonly observed in advanced atherosclerotic lesions. 99mTc-annexin A5 (99mTc-annexin V) has been proposed as a potential tracer for imaging apoptosis in atherosclerotic plaques. Accordingly, we determined the usefulness of 99mTc-annexin A5 as an atherosclerosis imaging tracer in a rabbit model (myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits; WHHLMI rabbits) of spontaneous atherosclerosis.MethodsThe WHHLMI and control rabbits were injected intravenously with 99mTc-annexin A5. After in vivo planar imaging, the radioactivity in the aorta was measured. Autoradiography, TUNEL staining, Azan-Mallory staining and immunohistological studies were performed serially throughout the aorta.Results99mTc-Annexin A5 accumulation in the aorta of the WHHLMI rabbits was 5.6-fold higher than in that of control rabbits. Autoradiography showed heterogeneous multifocal accumulation of 99mTc-annexin A5 in WHHLMI rabbits. 99mTc-Annexin A5 accumulation was highest in the atheromatous lesions (6.2 ± 2.5, %ID × BW/mm2 × 103), followed in decreasing order by neointimal (4.9 ± 1.3), fibroatheromatous (4.5 ± 1.9), and collagen-rich lesions (3.3 ± 1.4). The regional 99mTc-annexin A5 accumulation was significantly correlated with the TUNEL-positive cell density, macrophage density and “vulnerability index,” an index of the morphological destabilized characteristics. The in vivo imaging clearly visualized the atherosclerotic lesions in WHHLMI rabbits.ConclusionThe present study in WHHLMI rabbits showed higher 99mTc-annexin A5 accumulation in grade IV atheroma than in other more stable lesions. 99mTc-Annexin A5 may be useful in identifying atheroma that is at higher risk for rupture and possibly in assessing the response to anti-atherosclerotic therapy.

Tissue Factor Detection for Selectively Discriminating Unstable Plaques in an Atherosclerotic Rabbit Model

Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation in vivo. TF expression is closely related to plaque vulnerability, and high TF expression is shown in macrophage-rich atheromatous lesions, making TF a potential target for detecting atheromatous lesions in vivo. Thus, we prepared 99mTc-labeled anti-TF-monoclonal antibody (TF-mAb) IgG as a molecular probe and evaluated its usefulness to achieve TF-specific imaging using myocardial infarction–prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. Methods: Anti-TF-mAb was created using a standard hybridoma technique and was labeled by 99mTc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain 99mTc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with 99mTc-TF-mAb. Twenty-four hours after the injection, the aorta was removed and radioactivity was measured. Autoradiography and histologic studies were performed using serial aorta sections. Subclass matched antibody (IgG1) was used as a negative control. Results: HYNIC-TF-mAb showed 93% immunoreactivity of the anti-TF-mAb. The radioactivity accumulation in WHHLMI aortas was 6.1-fold higher than that of control rabbits. Autoradiograms showed a heterogeneous distribution of radioactivity in the intima of WHHLMI aortas. Regional radioactivity accumulation was positively correlated with TF expression density (R = 0.64, P < 0.0001). The highest radioactivity accumulation in percentage injected dose × body weight/mm2 × 102 was found in atheromatous lesions (5.2 ± 1.9) followed by fibroatheromatous (2.1 ± 0.7), collagen-rich (1.8 ± 0.7), and neointimal lesions (1.8 ± 0.6). In contrast, 99mTc-IgG1 showed low radioactivity accumulation in WHHLMI aortas that was independent of the histologic grade of lesions. Conclusion: The TF-detecting ability and preferential accumulation in atheromatous lesions of 99mTc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.

Biological Characterization of TAK-901, an Investigational, Novel, Multitargeted Aurora B Kinase Inhibitor

Protein kinases Aurora A, B, and C play essential roles during mitosis and cell division, are frequently elevated in cancer, and represent attractive targets for therapeutic intervention. TAK-901 is an investigational, multitargeted Aurora B kinase inhibitor derived from a novel azacarboline kinase hinge-binder chemotype. TAK-901 exhibited time-dependent, tight-binding inhibition of Aurora B, but not Aurora A. Consistent with Aurora B inhibition, TAK-901 suppressed cellular histone H3 phosphorylation and induced polyploidy. In various human cancer cell lines, TAK-901 inhibited cell proliferation with effective concentration values from 40 to 500 nmol/L. Examination of a broad panel of kinases in biochemical assays revealed inhibition of multiple kinases. However, TAK-901 potently inhibited only a few kinases other than Aurora B in intact cells, including FLT3 and FGFR2. In rodent xenografts, TAK-901 exhibited potent activity against multiple human solid tumor types, and complete regression was observed in the ovarian cancer A2780 model. TAK-901 also displayed potent activity against several leukemia models. In vivo biomarker studies showed that TAK-901 induced pharmacodynamic responses consistent with Aurora B inhibition and correlating with retention of TAK-901 in tumor tissue. These preclinical data highlight the therapeutic potential of TAK-901, which has entered phase I clinical trials in patients within a diverse range of cancers. Mol Cancer Ther; 12(4); 460–70. ©2013 AACR.

Effect of Exercise and Calorie Restriction on Tissue Acylcarnitines, Tissue Desaturase Indices, and Fat Accumulation in Diet-Induced Obese Rats

Both exercise and calorie restriction interventions have been recommended for inducing weight-loss in obese states. However, there is conflicting evidence on their relative benefits for metabolic health and insulin sensitivity. This study seeks to evaluate the differential effects of the two interventions on fat mobilization, fat metabolism, and insulin sensitivity in diet-induced obese animal models. After 4 months of ad libitum high fat diet feeding, 35 male Fischer F344 rats were grouped (n = 7 per cohort) into sedentary control (CON), exercise once a day (EX1), exercise twice a day (EX2), 15% calorie restriction (CR1) and 30% calorie restriction (CR2) cohorts. Interventions were carried out over a 4-week period. We found elevated hepatic and muscle long chain acylcarnitines with both exercise and calorie restriction, and a positive association between hepatic long chain acylcarnitines and insulin sensitivity in the pooled cohort. Our result suggests that long chain acylcarnitines may not indicate incomplete fat oxidation in weight loss interventions. Calorie restriction was found to be more effective than exercise in reducing body weight. Exercise, on the other hand, was more effective in reducing adipose depots and muscle triglycerides, favorably altering muscle/liver desaturase activity and improving insulin sensitivity.

Glucose uptake of the muscle and adipose tissues in diabetes and obesity disease models: evaluation of insulin and β3-adrenergic receptor agonist effects by 18F-FDG

ObjectiveOne of the major causes of diabetes and obesity is abnormality in glucose metabolism and glucose uptake in the muscle and adipose tissue based on an insufficient action of insulin. Therefore, many of the drug discovery programs are based on the concept of stimulating glucose uptake in these tissues. Improvement of glucose metabolism has been assessed based on blood parameters, but these merely reflect the systemic reaction to the drug administered. We have conducted basic studies to investigate the usefulness of glucose uptake measurement in various muscle and adipose tissues in pharmacological tests using disease-model animals.MethodsA radiotracer for glucose, 18F-2-deoxy-2-fluoro-d-glucose (18F-FDG), was administered to Wistar fatty rats (type 2 diabetes model), DIO mouse (obese model), and the corresponding control animals, and the basal glucose uptake in the muscle and adipose (white and brown) tissues were compared using biodistribution method. Moreover, insulin and a β3 agonist (CL316,243), which are known to stimulate glucose uptake in the muscle and adipose tissues, were administered to assess their effect. 18F-FDG uptake in each tissue was measured as the radioactivity and the distribution was confirmed by autoradiography.ResultsIn Wistar fatty rats, all the tissues measured showed a decrease in the basal level of glucose uptake when compared to Wistar lean rats. On the other hand, the same trend was observed only in the white adipose tissue in DIO mice, while brown adipose tissue showed increments in the basal glucose uptake in this model. Insulin administration stimulated glucose uptake in both Wistar lean and fatty rats, although the responses were inhibited in Wistar fatty rats. The same tendency was shown also in control mice, but clear increments in glucose uptake were not observed in the muscle and brown adipose tissue of DIO mice after insulin administration. β3 agonist administration showed the similar trend in Wistar lean and fatty rats as insulin, while the responses were inhibited in the adipose tissues of Wistar fatty rats.ConclusionA system to monitor tissue glucose uptake with 18F-FDG enabled us to detect clear differences in basal glucose uptake between disease-model animals and their corresponding controls. The responses in the tissues to insulin or β3 agonist could be identified. Taken as a whole, the biodistribution method with 18F-FDG was confirmed to be useful for pharmacological evaluation of anti-diabetic or anti-obesity drugs using disease-model animals.

Investigation of Fat Metabolism during Antiobesity Interventions by Magnetic Resonance Imaging and Spectroscopy.

The focus of current treatments for obesity is to reduce the body weight or visceral fat, which requires longer duration to show effect. In this study, we investigated the short-term changes in fat metabolism in liver, abdomen, and skeletal muscle during antiobesity interventions including Sibutra mine treatment and diet restriction in obese rats using magnetic resonance imaging, magnetic resonance spectroscopy, and blood chemistry. Sibutramine is an antiobesity drug that results in weight loss by increasing satiety and energy expenditure. The Sibutramine-treated rats showed reduction of liver fat and intramyocellular lipids on day 3. The triglycerides (TG) decreased on day 1 and 3 compared to baseline (day 0). The early response/nonresponse in different fat depots will permit optimization of treatment for better clinical outcome rather than staying with a drug for longer periods.

Abstract 5217: Evaluation of the therapeutic efficacy of MEK inhibitor (TAK-733) using [18F]FDG-PET in the human lung xenograft model A549

Introduction: TAK-733 is a novel, potent, selective, non-ATP competitive, allosteric inhibitor of MEK kinase, which has a potential to inhibit cancer proliferation and survival. [18F]Fluorodeoxyglucose ([18F]FDG) is an imaging biomarker for glucose metabolism detectable using positron emission tomography (PET) and widely used in clinical cancer diagnosis and is increasingly used to assess response to anti-cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of TAK-733 using [18F]FDG-PET in nude rats bearing A549 (human lung carcinoma) xenografts. Methods: A549 tumor fragments were subcutaneously implanted in female RNU nude rats. Treatments began when the mean estimated tumor mass for all groups was 250mg (Day 1). TAK-733 was orally administered at 0 (vehicle), 1, 3 and 10 mg/kg daily for two weeks (n = 8 / group). PET scans were performed before treatment (Day 0) and on Days 2, 4, 7, 10 and 14. PET image acquisition was performed for 13 min at 1.5 hr after [18F]FDG injection (400 μCi). Tracer accumulations in tumor tissue were quantified as mean standard uptake value (SUVmean) and percentage of injected dose (%ID). Results: TAK-733 showed dose-dependent inhibition of tumor growth and [18F]FDG uptake in tumor tissue. TAK-733, at 10mg/kg, produced a statistically significant difference in tumor mass (Day 14) compared to the vehicle group (31% T/C) and in SUVmean (Day 2) compared to the vehicle group. The statistical significance for the 10mg/kg did not persist however, and was only observed at a single time point for tumor mass and SUVmean. However, a significant response in SUVmean (Day 2) occurred before significant tumor growth inhibition (Day 14). In this treatment group, the SUVmean value on Day 2 (1.38 ± 0.26) was lower than that observed at pre-treatment, Day 0 (1.60 ± 0.30), and statistically lower compared to the SUVmean values in the vehicle (1.54 ± 0.46) and 1mg/kg groups (1.53 ± 0.29) on Day 2. The SUVmean value in the vehicle and 1 and 3 mg/kg groups showed an increase over time. On Day 14, the SUVmean value at 10 mg/kg (1.45 ± 0.33) was statistically significantly lower compared to the vehicle group (2.16 ± 0.33). TAK-733 produced a dose-dependent %ID response. 10mg/kg TAK-733 produced the lowest %ID for every imaging time point, while the vehicle group had the highest %ID at every imaging time point. There were no statistically significant differences in %ID response. However, %ID did not show statistically significant decreased values compared to the vehicle group, suggesting that SUVmean may be a more sensitive parameter for [18F]FDG uptake than %ID. Conclusion: [18F]FDG-PET enabled an early indication of later tumor growth in response to TAK-733 treatment in this model. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5217.

Abstract C193: Biological evaluation of novel multi‐targeted Aurora‐B kinase inhibitor TAK‐901 in xenograft models

Aurora kinases play an essential role in cell division, specifically orchestrating several intricate steps during the mitotic phase of the cell cycle. The human genome encodes for 3 Aurora kinase genes, designated Aurora A, B, and C. Aurora A localizes to centrosomes and spindle poles and is required for mitotic spindle assembly, whereas Aurora B is a chromosome passenger protein required for phosphorylation of histone H3, chromosome segregation, and cytokinesis. Expression levels of Aurora A and B kinases are frequently elevated in human cancers making them attractive targets for therapeutic intervention. TAK‐901 is an Aurora B kinase inhibitor with multi‐targeted inhibitory activity against other kinases important in malignancy. In the present study, we investigated the pharmacodynamic effects and in vivo activity of TAK‐901 in human tumor xenograftmodels. In solid tumor xenograft models, potent dose‐dependent antitumor activity was observed against HCT116 (colon), H460 (large cell lung) and A2780 (ovary) cancer models by twice daily intravenous dosing on 2 consecutive days per week for 3 cycles. TAK‐901 also displayed potent activity against several leukemia models such as subcutaneously implanted MV4‐11 and HL60 (AML). In the A2780, HCT116, and HL60 models, TAK‐901 induced tumor regression at higher dose levels, including complete regression of A2780 tumors at 30 and 45 mg/kg/injection. When dosed in combination with chemotherapeutic agents, such as irinotecan in the HCT116 model or daunorubicin in the HL60 model, TAK‐901 produced additive effects. Following intravenous administration of 20 and 40 mg/kg TAK‐901 in the A2780 xenograft model, histone H3 phosphorylation was completely suppressed for at least 6 hours and in the 40 mg/kg treated tumors did not return to control levels during the 12 hour time course. After multiple doses of TAK‐901, polyploidy was observed in the tumor samples. TAK‐901 drug levels remained constant throughout the time course in tumor tissues whereas plasma levels declined steadily. These in vivo biomarker studies demonstrate that TAK‐901 induces pharmacodynamic responses consistent with Aurora B kinase inhibition, which correlates with effective retention of TAK 901 in tumor tissues. Taken together, these preclinical data emphasize the therapeutic potential of TAK‐ 901 in the treatment of diverse human malignancies. TAK‐901 is currently under investigation in Phase I clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C193.