In Vivo Molecular Imaging

Abstract

In vivo molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems. Among the methodologies used in in vivo molecular imaging, two methodologies are of great interest from the view of high sensitivity. One is nuclear medical imaging, and distribution and kinetics of a radiolabeled molecular probe are measured using positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The other is optical molecular imaging, and distribution and kinetics of a fluorescent probe are measured using a fluorescent imaging instrument. In this review, the development of imaging probes for these two methodologies is briefly discussed. In nuclear medical molecular imaging, based on structure–activity–biodistribution relationship studies for small molecule and the concept of “functional unit-binding multifunctional molecular probe” containing 3 functional units (target recognition unit, signal-releasing unit, linker unit) for peptides and proteins, we developed radiolabeled probes with high and specific accumulation to the target for neuroreceptors, β-amyloid plaques, and tau aggregates in the brain, tumors, atherosclerotic plaques, pancreatic β-cell, myocardial sympathetic nerves, and so on. We also discuss the progression of molecular imaging toward therapy (radiotheranotics). In in vivo optical molecular imaging, taking into account the characteristics of optical imaging, we designed tumor-specific optical imaging probes with characteristic imaging mechanism, including near-infrared (NIR) fluorescent probes and activatable probes. Furthermore, we developed a photoacoustic imaging probe, which enables highly sensitive and high-resolution imaging in deep tissues.

1. INTRODUCTION

The body carries on diverse functions through molecular interactions. Accordingly, to elucidate the biological functions in the living subject, “molecular imaging” is attractive because it enables in vivo imaging, characterization, and quantification of biological processes (events) taking place at the cellular and subcellular levels in humans and other living systems, and studies on molecular imaging are expected to contribute to clinical diagnosis, treatment, drug discovery, and basic research of life science as a methodology to explore and interpret biological phenomena from a new viewpoint.^1–5)

For this “molecular imaging,” several techniques have been considered such as radiation, optical, nuclear magnetic resonance, and ultrasound techniques.^6–9) Among these methodologies, two methodologies are of great interest from the view of high sensitivity. One is nuclear medical imaging, in which a radioactive molecular probe is administered into the body and its distribution and kinetics are measured by positron emission tomography (PET) and single-photon emission computed tomography (SPECT). This methodology enables imaging inside the entire body (whole-body imaging) because of unlimited depth penetration of radiation and quantitative molecular imaging. The other methodology is optical molecular imaging, in which a fluorescent probe is used. This technique does not involve exposure to ionizing irradiation and provides real-time imaging with high spatial resolution inexpensively, although the imaging region is limited to the body surface and whole-body imaging is not possible.

Thus since nuclear medical imaging and optical imaging have high sensitivity but exhibit different characteristics, the complementary use of these two technologies exploiting their individual characteristics enables extensive acquisition of in vivo information of biological events within intact living subjects. We performed research on the development of molecular imaging probes for nuclear medical imaging and optical imaging and applied these to the medical and pharmaceutical fields.

2. DEVELOPMENT AND APPLICATION OF PROBES FOR NUCLEAR MEDICAL MOLECULAR IMAGING

Only limited radioisotopes are used for molecule probes for nuclear medical molecular imaging because of their nuclear properties (type of decay, energy of photon, half-life). Among these radioisotopes, there are two groups. One is radioisotopes of essential elements constituting bioactive molecules and drugs, such as ¹¹C and ¹⁵O. The other is radioisotopes of non-constituent elements of bioactive molecules and drugs, which therefore cannot be used simply to replace one of the common constituent atoms in biologically interesting compounds, such as ¹⁸F, ¹²³I, and ^99mTc.

For the former, the development of synthesis methods with high radiochemical yield within a short time is an important factor because they have a very short half-life. For the latter, molecular design is important, because changes in the chemical structure caused by introduction of a foreign element induce changes in physiological activity and pharmacodynamics in the body. So, we have developed useful molecular probes by understanding the characteristics of these radioisotopes and designing the accumulation mechanism suitable for each target.

2.1. Molecular Imaging of Neuroreceptors

The basic requirements for radiolabeled probes for nuclear medical imaging of brain neuroreceptors include a high radiochemical yield of synthesis within a short time, high binding affinity and specificity to the target receptor, high blood–brain barrier permeability, rapid and quantitatively significant brain uptake following peripheral administration, and a high ratio of radioactivity accumulated in the target within a short time after injection, compared with in surrounding tissue.

Taking account of requirements described above, we designed molecules for nuclear medical imaging of brain neuroreceptors using ¹²³I, which is one of the most widely used radioisotopes for nuclear medical imaging because of its superior radiation properties (adequate energy of photon (159 keV) and half-life (13.2 h)) and high versatility.

These ¹²³I-labeled molecular probes include ¹²³I-2′-iodospiperone (2′-ISP),^{10–14) 123}I-2′-iododiazepam (2′-IDZ),^15–17) and ¹²³I-5-iodo-A85830 (5IA)^18–27) for imaging the dopamine D2 receptor (D2R), central benzodiazepine receptor (BZR), and nicotinic acetylcholine receptor (nAChR) in the brain, respectively. Especially, neuroreceptor imaging of the human brain with ¹²³I-2′-ISP was a world first as a ¹²³I-labeled molecular probe of neuroreceptors, and was evaluated as a pioneer study in this field^9–11) (Fig. 1). Quantitative analysis of the neuroreceptor distribution in human brain using these ¹²³I-labeled molecular probes led to the discovery of reduced D2R density in the cerebral basal ganglia in Parkinsonism (Fig. 1), reduction of nAChR density in the brain with Alzheimer’s disease (AD) and Parkinson’s disease, and upregulation of nAChR density in the brain in smokers^18,20,21) (Fig. 2).

Fig. 1. (A) Chemical Structure of 2′-Iodospiperone (2′-ISP); (B) SPECT Images (Transaxial Images) of ¹²³I-2′-ISP in Parkinson’s Disease, Showing Normal Uptake in the Basal Ganglia (Arrow) (Left) and in Parkinsonism, Showing Reduction of Uptake in the Basal Ganglia (Right)

(B) From ref. 14 with modification.

Fig. 2. (A) Drug Design of 5-Iodo-A85830 (5IA), as a Probe for SPECT Imaging of Nicotinic Acetylcholine Receptor (nAChR) in the Brain, Chemical Structure of ¹²³I-5IA and SPECT Images of Human Brain (Transaxial Images) 80 min Post-Injection of ¹²³I-5IA in a Normal Volunteer and Alzheimer’s Disease Patient; (B) Change of Cerebral nAChR Density in Smokers Measured by ¹²³I-5IA Imaging; (C) Cerebral nAChR Density in Normal Volunteers and Alzheimer’s Disease Patient Measured by ¹²³I-5IA Imaging

(A) From refs. 19, 21 with modification. (B) From ref. 23 with modification.

2.2. Molecular Imaging of β-Amyloid Plaques and Tau Aggregates in the Brain

The presence of senile plaques (SPs), composed of β-amyloid (Aβ) peptides, and of neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau proteins, are two neuropathological hallmarks of AD brain. Therefore detection of SPs and NFTs in living brain by nuclear medical imaging methodology could lead to presymptomatic detection of AD and elucidation of the pathology. Thus we designed molecular probes for nuclear medicinal imaging the distribution of aggregates of Aβ and hyperphosphorylated tau protein in the brain of a patient with AD, which satisfied the following requirements: 1) high blood–brain barrier permeability; 2) high and specific binding ability to Aβ and tau aggregates; and 3) rapid elimination from normal regions of the brain. Consequently, we succeeded in developing ¹⁸F-labeled pyridyl benzofuran derivative, ¹⁸F-FPYBF-2, as a probe for PET imaging of Aβ plaques^28–36) and ¹²³I-labeled benzo imidazopyridine (BIP) derivative, BIP-3, as a probe for SPECT imaging of tau aggregates^37–41) (Fig. 3). Then, we studied the ¹⁸F-FPYBF-2 probe in AD patients, and found that it could image Aβ plaques with significant differences in radioactivity accumulation in AD brains versus controls.

Fig. 3. Drug Design of Pyridyl Benzofuran Derivative, ¹⁸F-FPYBF-2, as a Probe for PET Imaging of Aβ Plaques in the Brain

Furthermore, since this FPYBF-2 emits fluorescence, it is also usable as a probe for fluorescent imaging, and was used in basic studies.³⁸⁾

2.3. Molecular Imaging of Tumors

For the diagnosis of cancer, we have developed various nuclear medical imaging probes targeting molecules expressed in cancer tissue: hypoxia-induced factor (HIF-1α), membrane type-1 matrix metalloproteinase (MT1-MMP), and various cancer-specific antigens and receptors.

For rational design of molecular imaging probes, we proposed a concept of “functional unit-binding multifunctional molecular probe”; that is, a compound containing 3 functional units: “targeting unit,” “signal unit,” and “linker unit” connecting the former 2 units, in which each unit does not affect the function of the other units (Fig. 4). Based on this concept, in the development of molecular probes for high-contrast successful images of HIF-1-active tumor microenvironments at the early period comparable to short half-life of radioisotopes used, we proposed a pretargeting approach, considering that HIF-1α is stable under hypoxic conditions and degraded under normoxic condition, biotin binds to streptavidin (SAV) (linker unit) with very high affinity and selectivity, and a radiolabeled small compound is cleared from the blood in a relatively short time compared with a higher-molecular weight compound. That is to say, we fused an amino acid sequence that increases cell membrane permeability and facilitates the transport of protein into cells—the protein transduction domain (PTD) and monomeric SAV to oxygen-dependent degradation domain (ODD) of HIF-1α (targeting unit) to produce PTD-ODD-SAV (POS). After in vivo administration of POS, it degrades in a manner similar to HIF-1α in normoxic tissues and is retained inside the cells in hypoxic tissues. After allowing sufficient time for POS to degrade in normal tissues, ¹²³I-labeled biotin derivative (¹²³I-IBB) (signal unit) is administered. ¹²³I-IBB enters cells by passive diffusion and binds to the SAV moiety of the POS retained in hypoxic cells; this does not occur in normoxic tissues. Therefore pretargeting POS followed by ¹²³I-IBB administration enables specific imaging of HIF-1-active hypoxic microenvironments. Indeed, PET imaging clearly delineated HIF-1α-positive tumors, reducing the time taken from probe administration to image acquisition by 4 fold^42–45) (Fig. 5).

Fig. 4. Concept of “Functional Unit-Binding Multifunctional Molecular Probe”

Fig. 5. (A) Principle Underlying the Imaging of HIF-1α-Active Tumor Microenvironments, Using Pretargeted POS and ¹²³I-IBB (Left); (B) Planar Image of Tumor-Bearing Mice Acquired in the Pretargeting Method

(A) PTD enables the delivery of POS to all tissues. In normoxic tissues, POS degrades in a manner similar to HIF-1α. In contrast, in hypoxic tissues, POS escapes degradation and is retained inside the cells. After allowing sufficient time for POS to degrade in normal tissues, ¹²³I-IBB is administered. ¹²³I-IBB enters cells by passive diffusion and binds to the SAV moiety of the POS retained in hypoxic cells; this does not occur in normoxic tissues. Therefore pretargeting POS followed by ¹²³I-IBB administration enables specific imaging of HIF-1α active hypoxic microenvironments. (B) Images were acquired 6 h after injection of ¹²³I-IBB in the pretargeting method. (B) From ref. 43.

We also developed probes using anti-MT1-MMP antibody or its reduced-molecular-weight form for the target recognition unit and succeeded in imaging of MT1-MMP.^46–51) In addition, we performed research on structure–activity–distribution relations of an asymmetric urea compound that binds to prostate-specific membrane antigen (PSMA) expressed in prostate cancer cell membrane, and developed a nuclear medical molecular imaging probe for PSMA-positive tumor.^52–54) We are currently preparing a clinical study using this probe.

Based on data on cancer-selective distribution obtained by these imaging probes, research on development of internal radiation therapeutic compounds labeled with α- or β-ray-emitting radioisotopes are underway.

2.4. Molecular Imaging of Atherosclerosis

Aiming at in vivo evaluation of vulnerable atherosclerotic plaque playing an important role in thrombus formation causing myocardial and cerebral infarction, focusing on involvement of macrophages (Mφ) in rupture of vulnerable atherosclerotic plaque, we investigated relations between Mφ density and accumulation of a glucose derivative, ¹⁸F-fluorodeoxy glucose (FDG), in atherosclerotic lesions, and found a strong correlation, indicating the usefulness of ¹⁸F-FDG as a probe for imaging vulnerable atherosclerotic plaque, evaluating therapeutic effect of drugs clinically, and developing new drugs that can stabilize vulnerable plaques.^55,56)

We also developed molecular probes targeting a scavenger receptor, lectin-like oxidized LDL receptor-1 (LOX-1), and tissue factor.^57–64) Currently, atherosclerotic plaques are diagnosed based on the grade of vascular stenosis measured on angiography. However, vulnerability of plaque cannot be judged on angiography because the vascular lumen volume is not related to plaque stability. Nuclear medical molecular imaging enables researchers to visualize specific molecular processes in vivo, which could be used for imaging in vivo biological properties of atherosclerotic plaques beyond morphological information. Thus nuclear imaging probes developed at our laboratory are expected to be useful as potential imaging probes for predicting lesions prone to spontaneous rupture and monitoring the effects of timely treatment in patients with advanced atherosclerosis.

To detect coronary arterial vulnerable plaque using a detection method other than imaging, we developed a catheter-type ultra-small radiation detection system using a plastic detector.^65,66) This device was inserted in canine coronary artery and was able to detect point sources fixed on its surface with good resolution. Thus this detector in combination with charged-particle-emitting radiolabeled probe may be useful for the endovascular detection of small lesions such as vulnerable coronary plaques.

2.5. Molecular Imaging of Pancreatic β-Cells

A method for molecular imaging of pancreatic β-cell mass measurement is needed to enhance our understanding of the pathogenesis of diabetes, facilitate the early diagnosis of this disease, and promote the development of novel therapeutics. We designed and synthesized various derivatives of Exendin-4 and Exendin(9–39), which bind to the GLP-1 receptor selectively expressed at a high level in pancreatic β cells, as molecular imaging probes targeting pancreatic β cells.^67–69) That is, compounds in which ¹⁸F-fluorobenzoyl group and ¹²³I-iodobenzoyl group were introduced into the lysine residue at position 12 of Exendin-4 and Exendin(9–39) through polyethylene glycol, were successfully developed as molecular probes for PET and SPECT of pancreatic β cells, respectively. In addition, since an increase in the number of pancreatic β cells in insulinoma has been reported, imaging of insulinoma using these probes is also being investigated.

2.6. Molecular Imaging of Myocardial Sympathetic Nerve Function

The probability of developing cardiac diseases such as cardiomyopathy, heart failure, arrhythmia, and sudden death is high in diabetic patients. Using a rat diabetes model, we investigated changes of the density of norepinephrine transporter (NET), which is located presynaptically on noradrenergic nerve terminals and plays a critical role in regulation of the synaptic norepinephrine (NE) concentration via the reuptake of NE. Consequently, we found that NET density decreases before changes in heart muscle morphology and blood flow in diabetes.^70–73) Furthermore, we developed an ¹²³I-labeled compound with high and selective affinity for NET in vitro and in vivo as a molecular imaging probe for evaluating cardiac sympathetic nervous function.^74–79)

2.7. Progression of Molecular Imaging toward Therapy

Recently, the field of theranostics has attracted a great deal of attention. It is a combination of diagnosis and therapeutics and focuses on patient-centered care. Theranostics using radioisotopes is called radiotheranotics, and the use of radioisotopes of elements with similar chemical characters, such as ^99mTc and ^186/188Re, facilitates seamless progression of diagnosis and therapeutics because both radioisotopes can coordinate with the same chelating group and both compounds produced have the same biodistribution characteristics. Thus based on the molecular design concept of “functional unit-binding multifunctional molecular probe,” we selected bisphosphonate with high affinity for bone as the target recognition unit and synthesized compounds in which the chelating group (signal release unit) with ^99mTc or ^186/188Re is independent from the region involved in affinity for bone (target recognition unit). The compound labeled with ^99mTc was useful for imaging bone tumors, and the compound labeled with ^186/188Re inhibited tumor growth and palliated pain induced by bone metastasis significantly.^80–88)

Furthermore, considering that the biocompatible hydrophilic polymer, poly(oxazoline) (POZ), self-aggregates thermo-responsively, we synthesized a POZ derivative labeled with radioisotope emitting β-ray, ⁹⁰Y, with transition temperature (25°C) lower than body temperature. When the thermo-responsive ⁹⁰Y-labeled POZ derivative was injected intratumorally in tumor-bearing mice, it showed significantly higher retention of radioactivity in the tumor tissues via intratumoral aggregation for a prolonged period. Consequently, ⁹⁰Y-labeled POZ derivative significantly suppressed tumor growth and prolonged animal survival rate in a dose-dependent manner. Therefore this therapeutic strategy using ⁹⁰Y-labeled POZ derivatives may be promising for brachytherapy, a typical radiotherapy using “seeds” containing therapeutic radioisotopes.⁸⁹⁾

We also developed an injectable ¹⁵O oxygen obtained after ¹⁵O–O₂ gas circulation into artificial lung and estimated changes in oxygen metabolism after occluding the middle cerebral artery in hypertensive rats using it. Consequently, we found that hypertension intensifies metabolic disturbances after the onset of strokes.^90–93) Thus injectable ¹⁵O–O₂ could be used reliably to estimate oxygen metabolism in an infarction rat model with PET. Furthermore, we developed molecular probes for nuclear medicinal imaging targeting β-secretase in the brain, acetylcholine esterase, cyclooxygenase-2 (COX-2), epidermal growth factor receptor (EGFR), and hepatic organic anion transporter (OATP1B1).^94–103)

2.8. Synthetic Method and Instrument for Computer-Controlled Automatic Synthesis

Synthesis of nuclear medical molecular imaging probes, mainly molecular probes for PET, requires rapidity, high reaction yield of a trace amount, and high radiochemical yield. Thus we designed and developed an effective synthetic method for probes labeled with short half-life radioisotopes.^104–106) For clinical use of these molecular imaging probes, we designed and developed a computer-controlled automatic synthesizer.¹⁰⁷⁾

3. DEVELOPMENT AND APPLICATION OF PROBES FOR OPTICAL MOLECULAR IMAGING

Optical molecular imaging can conveniently and safely offer pronounced spatial and temporal resolution. Thus in vivo fluorescence imaging using a near infrared fluorescence probe has attracted attention not only as a clinical diagnostic method but also as a useful method to analyze whole biological phenomena. Unlike nuclear medical molecular imaging, it has activatability (specificity), that is, the optical signal is turned ON only in the target tissue, exploiting the characteristic that ON/OFF of the signal can be controlled depending on the environmental conditions. This characteristic enables reduction of background signal and specific imaging of only the target. Thus rationally using the characteristics of these optical compounds, we developed molecular probes for optical imaging of each target.

3.1. Near Infrared Fluorescent Imaging

In in vivo optical imaging, absorption and scattering of light by biological tissue and autofluorescence reduce quantitative precision and sensitivity. Therefore it is necessary to use a fluorescence probe with wavelength of emission and excitation in the near infrared region (700–900 nm) for which the influences of molecules existing in the body are small. Indocyanine green (ICG) is clinically used as a safe fluorescent dye; however, there are problems with ICG, such as low quantum yield and optical stability. Thus we designed and synthesized a novel cyanine near infrared fluorescent probe, IC7-1, by introducing a cyclohexene structure and a low hydrophilic functional group into the basic skeleton of ICG, tricarbocyanine structure, to improve the structural stability and quantum yield (Fig. 6). IC7-1 has also attracted attention as a fluorescent probe for in vivo fluorescence imaging because its maximum emission and fluorescence wavelengths are present in the near infrared region (λ_ex=830 nm, λ_em=858 nm), and the quantum yield is about 2 times that of ICG, showing that the fluorescence characteristics are appropriate for in vivo imaging.^108,109)

Fig. 6. (A) Chemical Structure of IC7-1-Bu; (B) In Vivo Fluorescence Imaging of Tumor-Bearing Mouse Administered IC7-1-Bu

(B) From ref. 109 with modification.

Since IC7-1 is a lipid-soluble neutral molecule with quaternary ammonium in the fluorophore and a carboxyl group in the side chain, an IC7-1 derivative was synthesized, in which a region of the side chain considered to have a small spectroscopic influence was derivatized and cationized, and a butyl group was introduced into the side chain so as to increase the affinity for plasma albumin, which is effective for efficient delivery of the probe to cancer tissue through the enhanced permeability and retention (EPR) effect. Using this probe (IC7-1-Bu), clear fluorescence imaging of tumors in tumor-transplanted mice was successfully performed (Fig. 6).

3.2. Activatable Optical Imaging

Fluorescent molecular probes useful for in vivo fluorescence molecular imaging require: 1) the presence of excitement and maximum fluorescence wavelengths in the near infrared region with good biological permeability; 2) efficient delivery to the target tissue; 3) high target/non-target fluorescence signal intensity (S/N) ratio; and 4) high safety. IC7-1, a novel near infrared fluorescent agent developed in 3.1., has fluorescence characteristics appropriate for in vivo imaging, as described above, but further improvement of IC-7-1 was required for higher specificity. Considering that Lactosome is a polydepsipeptide micelle composed of polysarcosine (PSar) and poly-L-lactic acid (PLLA) with ability to escape from capture by the reticuloendothelial system and can be made to target tumors though the EPR effect, we designed the specific activatable probe, hIC7L, in which IC-7-1 was encapsulated in lactosome. That is to say, this probe can be obtained in a self-quenched form because it is encapsulated in lactosomes and dequenched to emit optical signals on degradation of the probe in lysosomes following cellular internalization (Fig. 7). Indeed, using this probe, clear fluorescence imaging of tumors in tumor-transplanted mice was obtained.^110–112)

Fig. 7. (A) Concept of Off-On Activatable Fluorescence Imaging Probe (MT1-hIC7L); (B) In Vivo Fluorescence Imaging of Tumor-Bearing Mouse Administered MT1-hIC7L (B)

The quenched fluorescence of MT1-hIC7L due to high concentration of dyes encapsulated is activated following internalization and degradation in a tumor cell expressing MT1-MMP. (B) From ref. 112.

Furthermore, we developed a switching-type probe for Aβ aggregates. This was designed to bind to the β sheet structure in Aβ aggregates and fix its molecular structure, through which fluorescence markedly increases, enabling in vivo optical imaging of Aβ aggregates in a mouse AD model.

3.3. Fluorescence Sensor of Intracellar Metal Ions

We developed fluorescent compounds comprising pyiridine and quinolizine derivatives as a low-molecule-weight zinc fluorescence sensor of intracellular zinc ions.^113–116)

4. DEVELOPMENT AND APPLICATION OF PROBES FOR PHOTOACOUSTIC IMAGING

Photoacoustic imaging (PAI) using the photoacoustic effect of endogenous biomolecules or exogenous contrast agents has emerged as an attractive new imaging modality that has developed rapidly over recent years because PAI can detect PA signals from depths of up to 5 cm with high contrast and spatial resolution. Thus the development of effective PA contrast agents is urgently needed.

For PA imaging, near-infrared (NIR) light in the wavelength range 650–900 nm is irradiated since this wavelength range is relatively penetrative through tissues enabling researchers to visualize deep tissues. In our previous studies, we developed a symmetrical NIR cyanine dye for use as a probe in fluorescence imaging of tumors. Thus we evaluated its usefulness for tumor imaging with PAI and demonstrated that it accumulated in tumors effectively to allow clear PAI of tumors in vivo. However, a second laser irradiation for serial observation of dye biodistribution greatly diminished the PA signals detected in tumors. Thus to obtain high photostability, we designed and synthesized a stabilized PAI probe using IC7-1 as a lead compound and found that it was a potentially useful PAI probe for in vivo sequential tumor imaging.

Furthermore, the appeal of nanomedicines for preclinical and clinical studies over the last decades motivated us to investigate the possibility of nanoparticles and water-soluble polymers as carriers for PAI probes targeting tumors, because they demonstrate increased half-life in the blood circulation and high accumulation in well-vascularized tumors via the EPR effect. Thus we designed iron oxide particles, human serum albumin, polyethylene glycol, and POZ derivatives, which were conjugated with ICG as tumor-targeted PAI probes. In particular, POZ conjugated with ICG derivative showed clear PAI of tumors in tumor-transplanted mice (Fig. 8).^117–123)

Fig. 8. (A) Chemical Structure of Poly(oxazoline) (POZ) Derivatives Conjugated with Indocyanine Green (ICG) Derivative; (B) Photoacoustic Imaging of Tumor Bearing Mouse before and at 24 h after Probe Administration Administered with POZ-ICG Derivative

Dotted circles and arrows indicate tumor regions and blood vessels, respectively. (B) From ref. 123 with slight modification.

5. DEVELOPMENT AND APPLICATION OF PROBES FOR OTHER MOLECULAR IMAGING

Magnetic resonance imaging (MRI) is also one of the medical imaging techniques, which is widely used for various diagnostic applications including noninvasive morphologic diagnosis that use high spatial resolution without exposing the patients to ionizing radiation. MRI plays an essential role in diagnosis, staging, and follow-up on many diseases including cancer. To use MRI in functional molecular imaging coupled with anatomical information, MR contrast agents that can selectively accumulate in the targeted tissue/region and produce intense MR signals are required. Based on the concept of “functional unit-binding multifunctional molecular probe,” we succeeded in developing some probes for MRI, such as a novel probe for in vivo functional MRI with an on/off switching system, pre-targeting strategy, and nanoparticles.^124,125)

6. CONCLUSION

In vivo molecular imaging has rapidly developed. In this review, I discussed our laboratory’s research on in vivo molecular imaging. Especially, I focused on the development of molecular imaging probes for nuclear medical imaging, optical imaging, and photoacoustic imaging. Each of the various imaging methodologies has advantages and disadvantages, and therefore a combination of these methodologies is useful. Further development of each methodology in in vivo molecular imaging and multimodality molecular imaging with modalities combining each molecular imaging methodology should accelerate the elucidation of biological processes in human diseases, the accuracy of diagnosis, and the discovery and development of new effective therapies.

This review of the author’s work was written by the author upon receiving the 2017 Pharmaceutical Society of Japan Award.

Acknowledgments

I would like to express sincere gratitude to my supervisor, Dr. Hisashi Tanaka (Emeritus Professor, Kyoto University), Dr. Akira Yokoyama (Emeritus Professor, Kyoto University), and Dr. Kanji Torizuka (Emeritus Professor, Kyoto University) for their great guidance and suggestions. I sincerely thank all the collaborators contributing to our research described herein, many of whom are cited in this review. I also express cordial thanks to all the members of our research group. This work was partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) KAKENHI, a Grant from the New Energy and Industrial Technology Development Organization (NEDO), Japan, a Grant from the Practical Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED), a Grant from the Innovative Techno-Hub for Integrated Medical Bio-imaging Project of the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a Grant from the Sankyo Foundation of Life Science, a Grant from the SGH Foundation and a Grant from the Smoking Research Foundation.

Conflict of Interest

The author declares no conflict of interest.

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• 21) Mamede M, Ishizu K, Ueda M, Mukai T, Iida Y, Fukuyama H, Saga T, Saji H. Quantification of human nicotinic acetylcholine receptors with ¹²³I-5IA SPECT. J. Nucl. Med., 45, 1458–1470 (2004).
• 22) Iida Y, Ogawa M, Ueda M, Tominaga A, Kawashima H, Magata Y, Nishiyama S, Tsukada H, Mukai T, Saji H. Evaluation of 5-¹¹C-methyl-A-85380 as an imaging agent for PET investigations of brain nicotinic acetylcholine receptors. J. Nucl. Med., 45, 878–884 (2004).
• 23) Mamede M, Ishizu K, Ueda M, Mukai T, Iida Y, Kawashima H, Fukuyama H, Togashi K, Saji H. Temporal change in human nicotinic acetylcholine receptor after smoking cessation: 5IA SPECT study. J. Nucl. Med., 48, 1829–1835 (2007).
• 24) Oishi N, Hashikawa K, Yoshida H, Ishizu K, Ueda M, Kawashima H, Saji H, Fukuyama H. Qunatification of nicotinic acetylcholine receptors in Parkinson’s disease with ¹²³I-5IA SPECT. J. Neurol. Sci., 256, 52–60 (2007).
• 25) Ueda M, Iida Y, Kitamura Y, Kawashima H, Ogawa M, Magata Y, Saji H. 5-Iodo-A-85380, aspecific ligand for α4β2 nicotinic acetylcholine receptors, prevents glutamate neurotoxicity in rat cortical cultured neurons. Brain Res., 1199, 46–52 (2008).
• 26) Ueda M, Iida Y, Tominaga A, Yoneyama T, Ogawa M, Magata M, Nishimura H, Kuge Y, Saji H. Nicotinic acetylcholine receptors expressed in the ventralposterolateral thamic nucleus play an important role in anti-allodynic effects. Br. J. Pharmacol., 159, 1201–1210 (2010).
• 27) Ueda M, Iida Y, Yoneyama T, Kawai T, Ogawa M, Magata Y, Saji H. In vivo relationship between thalamic nicotinic acetylcholine receptor occupancy rates and antiallodynic effects in a rat model of neuropathic pain: persistent agonist binding inhibits the expression of antiallodynic effects. Synapse, 65, 77–83 (2011).
• 28) Ono M, Hayashi S, Kimura H, Kawashima H, Nakayama M, Saji H. Push-pull benzothiazole derivatives as probes for detecting beta-amyloid plaques in Alzheimer’s brains. Bioorg. Med. Chem., 17, 7002–7007 (2009).
• 29) Cheng Y, Ono M, Kimura H, Kagawa S, Nishii R, Kawashima H, Saji H. Fluorinated benzofuran derivatives for PET imaging of β-amyloid plaques in Alzheimer’s disease brains. ACS Med Chem Lett, 1, 321–325 (2010).
• 30) Ono M, Cheng Y, Kimura H, Cui M, Kagawa S, Nishii R, Saji H. Novel ¹⁸F-Labeled Benzofuran derivatives with improved properties for positron emission tomography (PET) imaging of β-amyloid plaques in Alzheimer’s brains. J. Med. Chem., 54, 2971–2979 (2011).
• 31) Cui M, Ono M, Kimura H, Liu BL, Saji H. Synthesis and structure-affinity relationships of novel dibenzylideneacetone derivatives as probes for β-amyloid plaques. J. Med. Chem., 54, 2225–2240 (2011).
• 32) Ono M, Saji H. Molecular approaches to the treatment, prophylaxis, and diagnosis of Alzheimer’s disease: Novel PET/SPECT imaging probes for diagnosis of Alzheimer’s disease. J. Pharmacol. Sci., 118, 338–344 (2012).
• 33) Ono M, Cheng Y, Kimura H, Watanabe H, Matsumura K, Yoshimura M, Iikumi S, Okamoto Y, Ihara M, Takahashi R, Saji H. Development of novel ¹²³I-labeled pyridyl benzofuran derivatives for SPECT imaging of β-amyloid plaques in Alzheimer’s disease. PLOS ONE, 8, e74104 (2013).
• 34) Yoshimura M, Ono M, Matsumura K, Watanabe H, Kimura H, Cui M, Nakamoto Y, Togashi K, Okamoto Y, Ihara M, Takahashi R, Saji H. Structure–activity relationships and in vivo evaluation of quinoxaline derivatives for PET imaging of β-amyloid plaques. ACS Med. Chem. Lett., 4, 596–600 (2013).
• 35) Ono M, Saji H. Recent advances in molecular imaging probes for β-amyloid plaques. Med. Chem. Commun., 6, 391–402 (2015).
• 36) Watanabe H, Ono M, Iikuni S, Kimura H, Okamoto Y, Ihara M, Saji H. Synthesis and biological evaluation of ¹²³I-labeled pyridyl benzoxazole derivatives: novel β-amyloid imaging probes for single-photon emission computed tomography. RSC Adv., 5, 1009–1015 (2015).
• 37) Matsumura K, Ono M, Hayashi S, Kimura H, Okamoto Y, Ihara M, Takahashi R, Mori H, Saji H. Phenyldiazenyl benzothiazole derivatives as probes for in vivo imaging of neurofibrillary tangles in Alzheimer’s disease brains. Med. Chem. Commun., 2, 596–600 (2011).
• 38) Ono M, Hayashi S, Matsumura K, Kimura H, Okamoto Y, Ihara M, Takahashi R, Mori H, Saji H. Rhodamine and thiohydantoin derivatives for detectiong tau pathology in Alzheimer’s brain. ACS Chem. Neurosci., 2, 269–275 (2011).
• 39) Matsumura K, Ono M, Kimura H, Ueda M, Nakamoto Y, Togashi K, Okamoto Y, Ihara M, Takahashi R, Saji H. ¹⁸F-Labeled phenyldiazenyl benzothiazole for in vivo imaging of neurofibrillary tangles in Alzheimer’s disease brains. ACS Med. Chem. Lett., 3, 58–62 (2012).
• 40) Matsumura K, Ono M, Kitada A, Watanabe H, Yoshimura M, Iikuni S, Kimura H, Okamoto Y, Ihara M, Saji H. Structure–activity relationship study of heterocyclic phenylethenyl and pyridinylethenyl derivatives as tau-imaging agents that selectively detect neurofibrillary tangles in Alzheimer’s disease brains. J. Med. Chem., 58, 7241–7257 (2015).
• 41) Cui M, Ono M, Watanabe H, Kimura H, Liu B, Saji H. Smart near-infrared fluorescence probes with donor-acceptor structure for in vivo detection of β-amyloid deposits. J. Am. Chem. Soc., 136, 3388–3394 (2014).
• 42) Kudo T, Ueda M, Kuge Y, Mukai T, Tanaka S, Masutani M, Kiyono Y, Kizaka-Kondoh S, Hiraoka M, Saji H. Imaging of HIF-1-active tumor hypoxia using a protein effectively delivered to and specifically stabilized in HIF-1-active tumor cells. J. Nucl. Med., 50, 942–949 (2009).
• 43) Ueda M, Kudo T, Kuge Y, Mukai T, Tanaka T, Konishi H, Miyano A, Ono M, Kizaka-Kondoh S, Hiraoka M, Saji H. Rapid detection of hypoxia-inducible factor-1-active tumours: pretaregeted imaging with a protein degrading a mechanism similar to hypoxia-inducible factor-1α. Eur. J. Nucl. Med. Mol. Imaging, 37, 1566–1574 (2010).
• 44) Ueda M, Kudo T, Mutou Y, Umeda IO, Miyano A, Ogawa K, Ono M, Fujii H, Kizaka-Kondoh S, Hiraoka M, Saji H. 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. Cancer Sci., 102, 2090–2096 (2011).
• 45) Ueda M, Ogawa K, Miyano A, Ono M, Kizaka-Kondoh S, Saji H. Development of an oxygen-sensitive degradable peptide probe for the imaging of hypoxia-inducible factor-1-active regions in tumors. Mol. Imaging Biol., 15, 713–721 (2013).
• 46) Temma T, Sano K, Kuge Y, Kamihashi J, Takai N, Ogawa Y, Saji H. Development of a radiolabeled probe for detecting membrane type-1 matrix metalloproteinase on malignant tumors. Biol. Pharm. Bull., 32, 1272–1277 (2009).
• 47) Sano K, Temma T, Kuge Y, Kudo T, Kamihashi J, Zhao S, Saji H. Radioimmunodetection of membrane type-1 matrix metalloproteinase relevant to tumor malignancy with a pre-targeting method. Biol. Pharm. Bull., 33, 1589–1595 (2010).
• 48) Kondo N, Temma T, Shimizu Y, Watanabe H, Higano K, Takagi Y, Ono M, Saji H. Miniaturized antibodies for imaging membrane type-1 matrix metalloproteinase in cancers. Cancer Sci., 104, 495–501 (2013).
• 49) Temma T, Hanaoka H, Yonezawa A, Kondo N, Sano K, Sakamoto T, Seiki M, Ono M, Saji H. Investigation of a MMP-2 activity-dependent anchoring probe for nuclear imaging of cancer. PLOS ONE, 9, e102180 (2014).
• 50) Kondo N, Temma T, Deguchi J, Sano K, Ono M, Saji H. Development of PEGylated peptide probes conjugated with ¹⁸F-labeled BODIPY for PET/optical imaging of MT1-MMP activity. J. Control. Release, 220 (Pt A), 476–483 (2015).
• 51) Kondo N, Temma T, Shimizu Y, Ono M, Saji H. Radioiodinated peptidic imaging probes for in vivo detection of membrane type-1 matrix metalloproteinase in cancers. Biol. Pharm. Bull., 38, 1375–1382 (2015).
• 52) Harada N, Kimura H, Ono M, Saji H. Preparation of asymmetric urea derivatives that target prostate-specific membrane antigen for SPECT imaging. J. Med. Chem., 56, 7890–7901 (2013).
• 53) Harada N, Kimura H, Onoe S, Watanabe H, Matsuoka D, Arimitsu K, Ono M, Saji H. Synthesis and biological evaluation of novel ¹⁸F-labeled probes targeting prostate-specific membrane antigen for positron emission tomography of prostate cancer. J. Nucl. Med., 57, 1978–1984 (2016).
• 54) Kimura H, Sampei S, Matsuoka D, Harada N, Watanabe H, Arimitsu K, Ono M, Saji H. Development of ^99mTc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging. Bioorg. Med. Chem., 24, 2251–2256 (2016).
• 55) Ogawa M, Ishino S, Mukai T, Asano D, Teramoto N, Watabe H, Kudomi N, Shiomi M, Magata Y, Iida H, Saji H. ¹⁸F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study. J. Nucl. Med., 45, 1245–1250 (2004).
• 56) Ogawa M, Magata Y, Kato T, Hatano K, Ishino S, Mukai T, Shiomi M, Ito K, Saji H. Application of [¹⁸F]FDG-PET for monitoring the therapeutic effect of anti-inflammatory drugs on stabilization of vulnerable atherosclerotic plaques. J. Nucl. Med., 47, 1845–1850 (2006).
• 57) Ishino S, Mukai T, Kume N, Asano D, Ogawa M, Kuge Y, Minami M, Kita T, Shiomi M, Saji H. Lectin-like oxidized LDL receptor-1 (LOX-1) expression is associated with atherosclerotic plaque instability-analysis in hypercholesterolemic rabbits. Atherosclerosis, 195, 48–56 (2007).
• 58) Ishino S, Mukai T, Kuge Y, Kume N, Ogawa M, Takai N, Kamihashi J, Shiomi M, Minami M, Kita T, Saji H. Tageting of lectinlike oxidized low-density lipoprotein receptor (LOX-1) with ^99mTc-labeled anti-LOX-1 antibody: potential agent for imaging of vulnerable plaque. J. Nucl. Med., 49, 1677–1685 (2008).
• 59) Kuge Y, Kume N, Ishino S, Takai N, Ogawa Y, Mukai T, Minami M, Shiomi M, Saji H. Prominent lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1) expression in atherosclerotic lesions is associated with tissue factor expression and apoptosis in hypercholesterolemic rabbits. Biol. Pharm. Bull., 31, 1475–1482 (2008).
• 60) Ishino S, Kuge Y, Takai N, Tamaki N, Strauss HW, Blankenberg FG, Shiomi M, Saji H. ^99mTc-Annexin A5 for noninvasive characterization of atherosclerotic lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits. Eur. J. Nucl. Med. Mol. Imaging, 34, 889–899 (2007).
• 61) Kuge Y, Takai N, Ishino S, Takai N, Temma T, Shiomi M, Saji H. Distribution profiles of membrane type-1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) in rabbit atherosclerosis: comparison with plaque instability analysis. Biol. Pharm. Bull., 30, 1634–1640 (2007).
• 62) Doue T, Ohtsuki K, Ogawa K, Ueda M, Azuma A, Saji H, Strauss HW, Matsubara H. Cardioprotective effects of erythropoietin in rats subjected to ischemia–reperfusion injury: assessment of infact size with ^99mTc-Annexin V. J. Nucl. Med., 49, 1694–1700 (2008).
• 63) Kuge Y, Takai N, Ogawa Y, Temma T, Zhao Y, Nishigori K, Ishino S, Kamihashi J, Kiyono Y, Shiomi M, Saji H. Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques. Eur. J. Nucl. Med. Mol. Imaging, 37, 2093–2104 (2010).
• 64) Temma T, Ogawa Y, Kuge Y, Ishino S, Takai N, Nishigori K, Shiomi M, Ono M, Saji H. Tissue factor detection for selectively discriminating unstable plaques in an atherosclerotic rabbit model. J. Nucl. Med., 51, 1979–1986 (2010).
• 65) Mukai T, Nohara R, Ogawa M, Ishino S, Kambara N, Kataoka K, Kanoi T, Saito K, Motomura T, Konishi J, Saji H. A catherer-based radiation detector for endovascular detection of atheromatous plaques. Eur. J. Nucl. Med. Mol. Imaging, 31, 1299–1303 (2004).
• 66) Hosokawa R, Kambara N, Ohba M, Mukai T, Ogawa M, Motomura H, Kume N, Saji H, Kita T, Nohara R. A catheter-based intravascular radiation detector of vulnerable plaques. J. Nucl. Med., 47, 863–867 (2006).
• 67) Mukai E, Toyoda K, Kimura H, Kawashima H, Fujimoto H, Ueda M, Temma T, Hirao K, Nagakawa K, Saji H, Inagaki N. GLP-1 receptor antagonist as a potential probe for pancreatic beta-cell imaging. Biochem. Biophys. Res. Commun., 389, 523–526 (2009).
• 68) Kimura H, Matsuda H, Fujimoto H, Arimitsu K, Toyoda K, Mukai E, Nakamura H, Ogawa Y, Takagi M, Ono M, Inagaki N, Saji H. ¹⁸F-Labeled mitiglinide derivatives as positron emission tomography tracers for β-cell imaging. Bioorg. Med. Chem., 22, 285–291 (2014).
• 69) Kimura H, Matsuda H, Ogawa Y, Fujimoto H, Toyoda K, Fujita N, Arimitsu K, Hamamatsu K, Yagi Y, Ono M, Inagaki N, Saji H. Development of ¹¹¹In-labeled Exendin(9–39) derivatives for single-photon emission computed tomography imaging of insulinoma. Bioorg. Med. Chem., 25, 1406–1412 (2017).
• 70) Kiyono Y, Iida Y, Kawashima H, Ogawa M, Tamaki N, Nishimura H, Saji H. Norepinephrine transporter density as a causative factor in alterations in MIBG myocardial uptake in NIDDM model rats. Eur. J. Nucl. Med. Mol. Imaging, 29, 999–1005 (2002).
• 71) Kiyono Y, Kanegawa N, Kawashima H, Iida Y, Kinoshita T, Tamaki N, Nishimura H, Ogawa M, Saji H. Age-related changes of myocardial norepinephrine transporter density in rats: implications for differential cardiac accumulation of MIBG in aging. Nucl. Med. Biol., 29, 679–684 (2002).
• 72) Kiyono Y, Kajiyama S, Fujiwara H, Kanegawa N, Saji H. Influence of the polyol pathway on norepinephrine transporter reduction in diabetic cardiac sympathetic nerves: implications for heterogeneous accumulation of MIBG. Eur. J. Nucl. Med. Mol. Imaging, 32, 438–442 (2005).
• 73) Kiyono Y, Yamashita T, Doi H, Kuge Y, Katsura T, Inui K, Saji H. Is MIBG a substrate of P-glycoprotein? Eur. J. Nucl. Med. Mol. Imaging, 34, 448–452 (2007).
• 74) Kiyono Y, Kanegawa N, Kawashima H, Fujiwara H, Iida Y, Nishimura H, Saji H. A new norepinephrine transporter imaging agent for cardiac sympathetic nervous function imaging: radioiodinated (R)-N-methyl-3-(2-iodophenyl)-3-phenylpropanamine. Nucl. Med. Biol., 30, 697–706 (2003).
• 75) Kiyono Y, Kanegawa N, Kawashima H, Kitamura Y, Iida Y, Saji H. Evaluation of radioiodinated (R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine as a ligand for brain norepinephrine transporter imaging. Nucl. Med. Biol., 31, 147–153 (2004).
• 76) Kanegawa N, Kiyono Y, Kimura H, Sugita T, Kajiyama S, Kawashima H, Ueda M, Kuge Y, Saji H. Synthesis and evaluation of radioiodinated (S,S)-2-(alpha-(2-iodophenoxy)benzyl)morpholine for imaging brain norepinephrine transporter. Eur. J. Nucl. Med. Mol. Imaging, 33, 639–647 (2006).
• 77) Kanegawa N, Kiyono Y, Kimura H, Sugita T, Kajiyama S, Kawashima H, Ueda M, Kuge Y, Saji H. Synthesis and evaluation of radioiodinated (S,S)-2-(α-(2-idodphenoxy)benzyl)morpholine for imaging brain norepinephrine transporter. Eur. J. Nucl. Med. Mol. Imaging, 34, 636–647 (2007).
• 78) Kiyono Y, Sugita T, Ueda M, Kawashima H, Kanegawa N, Kuge Y, Fujibayashi Y, Saji H. Evaluation of radioiodinated (2S,αS)-2-(α-(2-iodophenoxy)benzyl)morpholine as a radioligand for imaging of norepinephirine transporter in the heart. Nucl. Med. Biol., 35, 213–218 (2008).
• 79) Kanegawa N, Kiyono Y, Sugitaa T, Kuge Y, Fujibayasi Y, Saji H. Norepinephrine transporter imaging in the brain of a rat model of depression using radioiodinated (2S,αS)-2-(α-(2-iodophenoxy)benzyl) morpholine. Mol. Imaging, 11, 280–285 (2012).
• 80) Ogawa K, Mukai T, Arano Y, Ono M, Hanaoka H, Ishino S, Hashimoto K, Nishimura H, Saji H. Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals. Bioconjug. Chem., 16, 751–757 (2005).
• 81) Ogawa K, Mukai T, Arano Y, Otaka A, Ueda M, Uehara T, Magata Y, Hashimoto K, Saji H. Rhenium-186-monoaminemonoamidedithiol-conjugated bisphosphonate derivatives for bone pain palliation. Nucl. Med. Biol., 33, 513–520 (2006).
• 82) Ogawa K, Mukai T, Inoue Y, Ono M, Saji H. Development of a novel technetium-99m-chelate-conjugated bisphosphonate with high affinity for bone as a bone scintigraphic agent. J. Nucl. Med., 47, 2042–2047 (2006).
• 83) Ogawa K, Mukai T, Asano D, Kawashima H, Kinuya S, Shiba K, Hashimoto K, Mori H, Saji H. Therapeutic effects for the palliation of metastatic bone pain of a rhenium-186 complex-conjugated bisphosphonate in an animal model. J. Nucl. Med., 48, 122–127 (2007).
• 84) Ogawa K, Saji H. Advances in drug design of radiometal-based imaging agents for bone disorders. Int. J. Mol. Imaging, 2011, 537687 (2011).
• 85) Ogawa K, Kawashima H, Shiba K, Washiyama K, Yoshimoto M, Kiyono Y, Ueda M, Mori H, Saji H. Development of [⁹⁰Y]DOTA-conjugated bisphosphonate for treatment of painful bone metastases. Nucl. Med. Biol., 36, 129–135 (2009).
• 86) Ogawa K, Kawashima H, Kimuya S, Shiba K, Onoguchi M, Kimura H, Hashimoto K, Odani A, Saji H. Preparation and evaluation of ^186/188Re-labeled antibody (A7) for radioimmunotherapy with rhenium(I) tricarbonyl core as a chelate site. Ann. Nucl. Med., 17, 129–135 (2009).
• 87) Ogawa K, Mukai T, Kawai K, Takamura N, Hanaoka H, Hashimoto K, Shiba K, Mori H, Saji H. Usefulness of competitive inhibitors of protein binding for improving the pharmacokinetics of ¹⁸⁶Re-MAG₃-conjugated bisphosphonate (¹⁸⁶Re-MAG₃-HBP), an agent for treatment of painful bone metastases. Eur. J. Nucl. Med. Mol. Imaging, 36, 115–121 (2009).
• 88) Kurihara K, Ueda M, Hara I, Hara E, Sano K, Makino A, Ozeki E, Yamamoto F, Saji H, Togashi K, Kimura S. Inflammation-induced synergetic enhancement of nanoparticle treatments with DOXIL^® and ⁹⁰Y-Lactosome for orthotopic mammary tumor. J. Nanopart. Res., 18, 137 (2016).
• 89) Sano K, Kanada Y, Kanazaki K, Ding N, Ono M, Saji H. Brachytherapy with intratumoral injections of radiometal-labeled polymers that thermo-responsively self-aggregate in tumor tissues. J. Nucl. Med., jnumed.117.189993 (2017).
• 90) Magata Y, Temma T, Iida H, Ogawa M, Mukai T, Iida Y, Morimoto T, Konishi J, Saji H. Development of injectable O-15 oxygen and estimation of rat OEF. J. Cereb. Blood Flow Metab., 23, 671–676 (2003).
• 91) Temma T, Magata Y, Kuge Y, Shimonaka S, Sano K, Katada Y, Kawashima H, Mukai T, Watabe H, Iida H, Saji H. Estimation of oxygen metabolism in a rat model of permanent ischemia using positron emission tomography with injectable ¹⁵O–O₂. J. Cereb. Blood Flow Metab., 26, 1577–1583 (2006).
• 92) Temma T, Kuge Y, Sano K, Kamihashi J, Obokata N, Kawashima H, Magata Y, Saji H. PET O-15 cerebral blood flow and metabolism after acute stroke in spontaneously hypertensive rats. Brain Res., 1212, 18–24 (2008).
• 93) Temma T, Iida H, Hayashi T, Teramoto N, Ohta Y, Kudomi N, Watabe H, Saji H, Magata Y. Quantification of regional myocardial oxygen metabolism in normal pigs using positron emission tomography with injectable ¹⁵O–O₂. Eur. J. Nucl. Med. Mol. Imaging, 37, 377–385 (2010).
• 94) Kawai T, Kawashima H, Kuge Y, Saji H. Synthesis and evaluation of ¹¹C-labeled naphthalene derivative as a novel non-peptidergic probe for the β-secretase (BACE1) imaging in Alzheimer’s disease brain. Nucl. Med. Biol., 40, 705–709 (2013).
• 95) Kuge Y, Takai N, Ishino S, Temma T, Shiomi M, Saji H. Distribution profiles of membrane type-1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) in rabbit atherosclerosis: comparison with plaque instability analysis. Biol. Pharm. Bull., 30, 1634–1640 (2007).
• 96) Kuge Y, Obokata N, Kimura H, Katada Y, Temma T, Sugimoto Y, Aita K, Seki K, Tamaki N, Saji H. Synthesis and evaluation of a radioiodinated lumiracoxib derivative for the imaging of cyclooxygenase-2 expression. Nucl. Med. Biol., 36, 869–876 (2009).
• 97) Kimura H, Kawai T, Hamashima Y, Kawashima H, Miura K, Nakaya Y, Hirasawa M, Arimitsu T, Kajimoto T, Ohmomo Y, Ono M, Node M, Saji H. Synthesis and evaluation of (−)- and (+)-[¹¹C]galanthamine as PET tracers for cerebral acetylcholinesterase imaging. Bioorg. Med. Chem., 22, 285–291 (2014).
• 98) Kuge Y, Takai N, Ogawa Y, Temma T, Zhao Y, Nishigori K, Ishino S, Kamihashi J, Kiyono Y, Shiomi M, Saji H. Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques. Eur. J. Nucl. Med. Mol. Imaging, 37, 2093–2104 (2010).
• 99) Makino A, Arai T, Hirata M, Ono M, Ohmomo Y, Saji H. Development of novel PET probes targeting phosphatidylinositol 3-kinase (PI3K) in tumors. Nucl. Med. Biol., 43, 101–107 (2016).
• 100) Kimura H, Okuda H, Ishiguro M, Arimitsu K, Makino A, Nishii R, Miyazaki A, Yagi Y, Watanabe H, Kawasaki I, Ono M, Saji H. ¹⁸F-Labeled pyrido[3,4-d]pyrimidine as an effective probe for imaging of L858R-mutant epidermal growth factor receptor. ACS Med. Chem. Lett., 8, 418–422 (2017).
• 101) Ono M, Kitada A, Watanabe H, Miyazaki A, Kimura H, Saji H. Synthesis and preliminary characterization of radioiodinated benzofuran-3-yl-(indol-3-yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase-3β (GSK-3β) in the brain. J. Labelled Comp. Radiopharm., 59, 317–321 (2016).
• 102) Yagi Y, Kimura H, Arimitsu K, Ono M, Maeda K, Kusuhara H, Kajimoto T, Sugiyama Y, Saji H. The synthesis of [¹⁸F]pitavastatin as a tracer for hOATP using the Suzuki coupling. Org. Biomol. Chem., 13, 1113–1121 (2015).
• 103) Kimura H, Yagi Y, Arimitsu K, Maeda K, Ikejiri K, Takano J, Kusuhara H, Kagawa S, Ono M, Sugiyama Y, Saji H. Radiosynthesis of novel pitavastatin derivative ([¹⁸F]PTV-F1) as a tracer for hepatic OATP using a one-pot synthetic procedure. J. Labelled Comp. Radiopharm., 59, 565–575 (2016).
• 104) Kimura H, Mori D, Harada N, Ono M, Ohmomo Y, Kajimoto T, Saji H. Microwave-assisted synthesis of organometallic complexes of ^99mTc(CO)₃ and Re(CO)₃: its application to radiopharmaceuticals. Chem. Pharm. Bull., 60, 79–85 (2012).
• 105) Kimura H, Yagi Y, Ohneda N, Odajima H, Ono M, Saji H. Development of a resonant-type microwave reactor and its application to the synthesis of PET radiopharmaceuticals. J. Labelled Comp. Radiopharm., 57, 680–686 (2014).
• 106) Kawashima H, Kimura H, Nakaya Y, Tomatsu K, Arimitsu K, Nakanishi H, Ozeki E, Kuge Y, Saji H. Application of microreactor to the preparation of C-11-labeled compounds via O-[¹¹C]methylation with [¹¹C]CH₃I: rapid synthesis of [¹¹C]raclopride. Chem. Pharm. Bull., 63, 737–740 (2015).
• 107) Kimura H, Tomatsu K, Saiki H, Arimitsu K, Ono M, Kawashima H, Iwata R, Nakanishi H, Ozeki E, Kuge Y, Saji H. Continuous-flow synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate using a single microfluidic chip. PLOS ONE, 11, e0159303 (2016).
• 108) Shimizu Y, Temma T, Hara I, Yamahara R, Ozeki E, Ono M, Saji H. Development of novel nanocarrier-based near-infrared optical probes for in vivo tumor imaging. J. Fluoresc., 22, 719–727 (2012).
• 109) Onoe S, Temma T, Shimizu Y, Ono M, Saji H. Investigation of cyanine dyes for in vivo optical imaging of altered mitochondrial membrane potential in tumors. Cancer Med., 3, 775–786 (2014).
• 110) Shimizu Y, Temma T, Sano K, Ono M, Saji H. Development of membrane type-1 matrix metalloproteinase-specific activatable fluorescent probe for malignat tumor detection. Cancer Sci., 102, 1897–1903 (2011).
• 111) Shimizu Y, Temma T, Hara I, Makino A, Yamahara R, Ozeki EI, Ono M, Saji H. Micelle-based activatable probe for in vivo near-infrared optical imaging of cancer biomolecules. Nanomedicine, 10, 187–195 (2014).
• 112) Shimizu Y, Temma T, Hara I, Makino A, Kondo N, Ozeki EI, Ono M, Saji H. In vivo imaging of membrane type-1 matrix metalloproteinase with a novel activatable near-infrared fluorescence probe. Cancer Sci., 105, 1056–1062 (2014).
• 113) Hagimori M, Mizuyama N, Yokota K, Morinaga O, Yamaguchi Y, Saji H, Tominaga Y. Synthesis of fluorescent pyrrolo[3,4-b]quinolizine derivatives and evaluation as a protein-labeling probe. Heterocycles, 83, 1983–1988 (2011).
• 114) Hagimori M, Uto T, Mizuyama N, Temma T, Ymaguchi Y, Tominaga Y, Saji H. Fluorescence ON/OFF switching Zn²⁺ sensor based on pyridine–pyridone scaffold. Sensora and Actuators B, 181, 823–828 (2013).
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