Takashi Temma

GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic beta-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [(125)I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [(125)I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [(125)I]BH-exendin(9-39) injection into transgenic mice with pancreatic beta-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic beta-cell imaging.

Development of Injectable O-15 Oxygen and Estimation of Rat OEF

Cerebral metabolic rate for oxygen (CMRO2) and cerebral oxygen extraction fraction (OEF) are some of the most fundamental parameters to characterize the pathophysiologic status of cerebral tissue. Although O-15-labeled gases inhalation method is performed in clinical studies, application of the inhalation method on small animals requires too many intensive procedures. On this basis, the development of a new method to measure CMRO2 and OEF in small animals is of interest. This study was aimed at developing a method to assess CMRO2 and OEF using intravenously injectable oxygen (injectable 15O–O2) for small animals such as rats. Injectable 15O–O2, 72 MBq/mL of radioactivity, was obtained after 15O–O2 gas circulation into the artificial lung. OEF after injection of injectable 15O–O2 was calculated using the same equation as that applied to the bolus inhalation of 15O–O2 gas method. Values of 44 ± 4.5 mL · min−1 · 100 g−1 of CBF and 0.54 ± 0.11 of OEF were obtained (n = 13). This OEF value was well accordance with OEF evaluated by arterial-venous difference of oxygen concentration (0.57 ± 0.13). This method is useful to study the CMRO2 and OEF in small animals using an animal positron emission tomography system. It may accelerate the basic research of several cerebral perfusion diseases.

Micelle-based activatable probe for in vivo near-infrared optical imaging of cancer biomolecules

UNLABELLED Near-infrared (NIR: 800-1000 nm) fluorescent probes, which activate their fluorescence following interaction with functional biomolecules, are desirable for noninvasive and sensitive tumor diagnosis due to minimal tissue interference. Focusing on bioavailability and applicability, we developed a probe with a self-assembling polymer micelle, a lactosome, encapsulating various quantities of NIR dye (IC7-1). We also conjugated anti-HER2 single chain antibodies to the lactosome surface and examined the probes capacity to detect HER2 in cells and in vivo. Micelles encapsulating 20mol% IC7-1 (hIC7L) showed 30-fold higher fluorescence (λem: 858 nm) after micelle denaturation compared to aqueous buffer. Furthermore, antibody modification allowed specific activation of the probe (HER2-hIC7L) following internalization by HER2-positive cells, with the probe concentrating in lysosomes. HER2-hIC7L intravenously administered to mice clearly and specifically visualized HER2-positive tumors by in vivo optical imaging. These results indicate that HER2-hIC7L is a potential activatable NIR probe for sensitive tumor diagnosis. FROM THE CLINICAL EDITOR Near-infrared probes that activate their fluorescence following interaction with specific biomolecules are desirable for noninvasive and sensitive tumor detection due to minimal tissue interference. This team of authors developed a probe termed hIC7L and demonstrate its potential in HER2 tumor diagnosis.

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.

PET O-15 cerebral blood flow and metabolism after acute stroke in spontaneously hypertensive rats.

Hypertension is a major stroke risk factor and is correlated with worse outcome after stroke. Thus, the effects of hypertension on cerebral hemodynamics and metabolism within an hour after stroke must be evaluated in detail. Cerebral blood flow (CBF), oxygen extraction fraction (OEF), cerebral metabolic rate for oxygen (CMRO2) and cerebral metabolic rate for glucose (CMRglc) were measured 1 h after the occlusion of the right middle cerebral artery (MCA) in male spontaneously hypertensive rats (SHR) and male normotensive Wistar Kyoto rats (WKY). Physiological responses were determined by positron emission tomography (PET) using 15O-H2O and radiolabeled 15O-O2 blood (methodology previously developed in this laboratory) and by autoradiography (ARG) using 18F-FDG. The right hemisphere of SHR showed lower CBF values than the left hemisphere after stroke (right: 0.17+/-0.07 mL/min/g; left: 0.29+/-0.08 mL/min/g), CMRO2 (right: 2.55+/-0.80 mL/min/100 g; left: 4.11+/- 0.84 mL/min/100 g) and CMRglc (right: 52.4+/-16.2 mg/min/100 g; left: 65.6+/-10.2 mg/min/100 g). WKY rats exhibited significant decreases only in CBF and CMRO2. These results suggest greater underlying physiologic disturbances in SHR. Also, the occlusion significantly reduced CBF in both hemispheres of SHR compared with WKY, suggesting a disturbance of the autoregulatory mechanism in SHR. In summary, our results indicate that hypertension intensifies metabolic disturbances after the onset of stroke, at least in the first hour. Therefore, we suggest that hypertension not only increases the incidence of stroke but also exacerbates stroke-mediated damage.

Estimation of oxygen metabolism in a rat model of permanent ischemia using positron emission tomography with injectable15O-O2

The threshold of cerebral blood flow (CBF) into infarction in rats has been indicated to be similar to that in patients. However, CBF does not reflect metabolic function, and so estimations of oxygen metabolism have been required. Here, we estimated changes in oxygen metabolism after occluding the right middle cerebral artery (MCA) in rats using an injectable 15O-O2 we developed. A decrease in CBF (left: 0.67±0.22 mL/min/g, right: 0.44±0.17 mL/min/g, P < 0.05) and compensatory increase in the oxygen extraction fraction (OEF) (left: 0.42±0.13, right: 0.50±0.19, P < 0.05) were observed at 1-h after occlusion. In contrast, a marked decrease in CBF and the cerebral metabolic rate for oxygen and a collapse of the compensatory OEF mechanism were found at 24 h after occlusion. Injectable 15O-O2 could be used to reliably estimate oxygen metabolism in an infarction rat model with positron emission tomography.

Development of anti-HER2 fragment antibody conjugated to iron oxide nanoparticles for in vivo HER2-targeted photoacoustic tumor imaging

UNLABELLED Photoacoustic (PA) imaging is a promising imaging modality that provides biomedical information with high sensitivity and resolution. Iron oxide nanoparticles (IONPs) have been regarded as remarkable PA contrast agents because of their low toxicity and biodegradable properties. However, IONP delivery is restricted by its modest leakage and retention in tumors. In this study, we designed IONPs (20nm, 50nm, and 100nm) conjugated with anti-HER2 moieties [whole IgG, single-chain fragment variable (scFv), and peptide] for HER2-targeted PA tumor imaging. The binding affinity, cellular uptake, and in vivo biodistribution were examined. We propose 20-nm anti-HER2 scFv-conjugated IONPs (SNP20) as a novel PA contrast agent. SNP20 demonstrated high affinity and specific binding to HER2-expressing cells; it selectively visualized HER2-positive tumors in PA imaging studies. These data indicate that SNP20 is a potential PA contrast agent for imaging of HER2-expressing tumors. FROM THE CLINICAL EDITOR Iron oxide nanoparticles have been demonstrated to be good contrast agents for tumor imaging. They may also be useful in photoacoustic (PA) imaging, which can provide high sensitivity data and image resolution. The authors here coupled iron oxide nanoparticles with anti-HER2 antibody fragment and showed significant retention of these nanoparticles in tumors. This combination may provide another option for enhanced imaging of tumors.

Investigation of cyanine dyes for in vivo optical imaging of altered mitochondrial membrane potential in tumors

Mitochondrial membrane potential (Δψm) alteration is an important target for cancer diagnosis. In this study, we designed a series of near‐infrared fluorescent cationic cyanine dyes with varying alkyl chain lengths (IC7‐1 derivatives) to provide diverse lipophilicities and serum albumin‐binding rates, and we evaluated the usefulness of these derivatives for in vivo Δψm imaging. IC7‐1 derivatives with side chains from methyl to hexyl (IC7‐1‐Me to IC7‐1‐He) were synthesized, and their optical properties were measured. Cellular uptake and intracellular distribution were investigated with depolarized HeLa cells from carbonyl cyanine m‐chlorophenylhydrazone (CCCP) treatment using a spectrofluorometer and a fluorescence microscope. Serum albumin‐binding rates were evaluated using albumin‐binding inhibitors. In vivo optical imaging was performed with HeLa cell xenograft mice following intravenous administration of IC7‐1 derivatives with or without warfarin and CCCP as in vivo blocking agents. IC7‐1 derivatives showing maximum excitation and emission wavelengths at 823 nm and ~845 nm, respectively, were synthesized. IC7‐1‐Me to ‐Bu showed fluorescence in mitochondria that decreased with CCCP treatment in a concentration‐dependent manner, which showed that IC7‐1‐Me to ‐Bu successfully indicated Δψm. Tumors were clearly visualized after IC7‐1‐Bu administration. Treatment with warfarin or CCCP significantly decreased IC7‐1‐Bu fluorescence in the tumor region. In summary, IC7‐1‐Bu exhibited fluorescence localized to mitochondria dependent on Δψm, which enabled clear in vivo tumor imaging via serum albumin as a drug carrier for effective tumor targeting. Our data suggest that IC7‐1‐Bu is a promising NIR probe for in vivo imaging of the altered Δψm of tumor cells.

Development of human serum albumin conjugated with near-infrared dye for photoacoustic tumor imaging

Abstract. Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2=0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.

Miniaturized antibodies for imaging membrane type-1 matrix metalloproteinase in cancers

Since membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1‐MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a 99mTc‐labeled anti‐MT1‐MMP monoclonal IgG (99mTc‐MT1‐mAb) as an MT1‐MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1‐scFv) and a dimer of two molecules of scFv (MT1‐diabody), as the basic structures of MT1‐MMP imaging probes followed by in vitro and in vivo evaluation with an 111In radiolabel. Phage display screening successfully provided MT1‐scFv and MT1‐diabody, which had sufficiently high affinity for MT1‐MMP (KD = 29.8 and 17.1 nM). Both 111In labeled miniaturized antibodies showed higher uptake in MT1‐MMP expressing HT1080 cells than in non‐expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20‐fold higher tumor to blood radioactivity ratios (T/B ratio), an index for in vivo imaging, than 99mTc‐MT1‐mAb 6 h post‐administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1‐MMP‐positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1‐MMP in cancer cells.