In vivo tracking transplanted cardiomyocytes derived from human induced pluripotent stem cells using nuclear medicine imaging.

Introduction: Transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a promising treatment for heart failure. Information on long-term cell engraftment after transplantation is clinically important. However, clinically applicable evaluation methods have not yet been established. Methods: In this study, to noninvasively assess transplanted cell engraftment, human SLC5A5, which encodes a sodium/iodide symporter (NIS) that transports radioactive tracers such as 125I, 18F-tetrafluoroborate (TFB), and 99mTc-pertechnetate (99mTcO4-), was transduced into human induced pluripotent stem cells (iPSCs), and nuclear medicine imaging was used to track engrafted human iPSC-CMs. Results: To evaluate the pluripotency of NIS-expressing human iPSCs, they were subcutaneously transplanted into immunodeficient rats. Teratomas were detected by 99mTcO4- single photon emission computed tomography (SPECT/CT) imaging. NIS expression and the uptake ability of 125I were maintained in purified human iPSC-CMs. NIS-expressing human iPSC-CMs transplanted into immunodeficient rats could be detected over time using 99mTcO4- SPECT/CT imaging. Unexpectedly, NIS expression affected cell proliferation of human iPSCs and iPSC-derived cells. Discussion: Such functionally designed iPSC-CMs have potential clinical applications as a noninvasive method of grafted cell evaluation, but further studies are needed to determine the effects of NIS transduction on cellular characteristics and functions.

Novel Self-Forming Nanosized DDS Particles for BNCT: Utilizing A Hydrophobic Boron Cluster and Its Molecular Glue Effect.

BNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.

Teratogenicity and Fetal-Transfer Assessment of the Retinoid X Receptor Agonist Bexarotene.

Bexarotene, a retinoid X receptor (RXR) agonist, is used to treat cutaneous T-cell lymphoma, and drug repositioning research has also been reported, despite warnings of teratogenicity. However, fetal transfer of bexarotene and its effect on rat fetal bone formation have not been examined. In this study, we conducted a detailed teratogenicity and fetal transferability assessment of bexarotene in rats. Repeated administration of bexarotene during pregnancy caused marked fetal atrophy and bone dysplasia. Although fetal transfer was not detectable by dynamic imaging of [11C]bexarotene by means of positron emission tomography, transfer to the fetus was confirmed by using a gamma counter. Similar levels were found in mother and fetus. In addition, we found that bexarotene was accumulated in the placenta. These findings will be useful for the toxicity assessment of bexarotene as well as for drug discovery research targeting RXR agonists, which are expected to have therapeutic effects in various diseases.

A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): "Lactosome" Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy.

"Theranostics," a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, "Lactosome" nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the "89Zr-labeled CPP and TPP-loaded Lactosome particles" and future directions based on important milestones and recent developments in this platform.

Novel single-chain variant of antibody against mesothelin established by phage library.

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.

Fluorine-18 (18F)-labeled retinoid x receptor (RXR) partial agonist whose tissue transferability is affected by other RXR ligands.

Bexarotene (1), a retinoid X receptor (RXR) agonist approved for the treatment of cutaneous T cell lymphoma (CTCL), was reported to migrate into baboon brain based on findings obtained by positron emission tomography (PET) with a 11C-labeled tracer. However, co-administration of non-radioactive 1 had no effect on the distribution of [11C]1, probably due to non-specific binding of 1 as a result of its high lipophilicity. Here, we report a fluorine-18 (18F)-labeled PET tracer [18F]6 derived from RXR partial agonist CBt-PMN (2), which has lower lipophilicity and weaker RXR-binding ability than [11C]1. The concomitant administration of 1 or 2 with [18F]6 with resulted in decreased accumulation of [18F]6 in liver, together with increased brain uptake and increased accumulation in kidney and muscle, as visualized by PET. A plausible explanation of these findings is the inhibition of [18F]6 uptake into the liver by concomitantly administered 1 or 2, leading to an increase in blood concentration of [18F]6 followed by increased accumulation in other tissues.

Synthesis of 11C-Labeled RXR Partial Agonist 1-[(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic Acid (CBt-PMN) by Direct [11C]Carbon Dioxide Fixation via Organolithiation of Trialkyltin Precursor and PET Imaging Thereof.

The retinoid X receptor (RXR) partial agonist 1-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (1; CBt-PMN, Emax = 75%, EC50 = 143 nM) is a candidate for treatment of central nervous system (CNS) diseases such as Alzheimer's and Parkinson's diseases based on reports that RXR-full agonist 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) shows therapeutic effects on these disease in rodent models. Here, we synthesized carbon-11-labeled ([11C]1) as a tracer for positron emission tomography (PET) and used it in a PET imaging study to examine the brain uptake and biodistribution of 1. We found that 11CO2 fixation after tin-lithium exchange at -20 °C afforded [11C]1. This methodology may also be useful for synthesizing 11CO2H-PET tracer derivatives of other compounds bearing π-rich heterocyclic rings. A PET/CT imaging study of [11C]1 in mice indicated 1 is distributed to the brain and is thus a candidate for treatment of CNS diseases.

Anti-high Mobility Group Box 1 Antibody Ameliorates Albuminuria in MRL/lpr Lupus-Prone Mice.

We evaluated the efficacy of a neutralizing anti-high mobility group box 1 (HMGB1) monoclonal antibody in MRL/lpr lupus-prone mice. The anti-HMGB1 monoclonal antibody (5 mg/kg weight) or class-matched control immunoglobulin G2a (IgG2a) was administered intravenously twice a week for 4-15 weeks. Urine albumin was monitored, and histological evaluation of the kidneys was conducted at 16 weeks. Lymphadenopathies were evaluated by 1-(2'-deoxy-2'-[18F]fluoro-ß-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed tomography (PET/CT) at 12 weeks. Following 4-week treatment, [18F]FAC-PET/CT showed similar accumulation in cervical and axillary lymph nodes at 12 weeks of age. However, anti-HMGB1 monoclonal antibody sufficiently inhibited the increase in albuminuria compared to an isotype control following 15-week treatment. Complement deposition was also improved; however, there were no significant differences in IgG deposition and renal pathological scores between the two groups. Anti-double-stranded DNA (dsDNA) antibody titers and cytokine and chemokine levels were also unaltered. Although there were no significant differences in glomerular macrophage infiltration, neutrophil infiltration was significantly decreased by the anti-HMGB1 monoclonal antibody. Antagonizing HMGB1 treatment suppressed HMGB1 translocation from nuclei in the kidney and suppressed neutrophil extracellular traps. The anti-HMGB1 monoclonal antibody demonstrated therapeutic potential against albuminuria in lupus nephritis by inhibiting neutrophil recruitment and neutrophil extracellular traps.

The Function of ß2-glycoprotein I in Angiogenesis and Its in Vivo Distribution in Tumor Xenografts.

Intact ß2-glycoprotein I (iß2GPI) is a glycoprotein that regulates coagulation and fibrinolysis. Nicked ß2GPI (nß2GPI) possesses an angiogenic property at a relatively low concentration, and an antiangiogenic property at a high concentration. Here we investigated the functions of ßi 2GPI and nß2GPI in vascular endothelial growth factor (VEGF)-A-induced endothelial cell proliferation and tube formation. We used noninvasive PET imaging to analyze the ｉｎ　ｖｉｖｏ distribution of intravenously injected ß2GPI variants in tumor lesions in mice. iß2GPI was incubated with plasmin to obtain nß2GPI, and its N-terminal sequence was analyzed. nß2GPI had at least one other cleavage site upstream of the ß2GPI's domain V, whereas the former plasmin-cleavage site locates between K317 and T318. Both of intact and nicked ß2GPI significantly inhibited the VEGF-A-induced cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs). PET imaging visualized considerably distributed intensities of all tested ß2GPI variants in tumor lesions of pancreatic tumor cell-xenografts. These results indicate that ß2GPI may be physiologically and pathophysiologically important in the regulation of not only coagulation and fibrinolysis, but also angiogenesis.

Positron emission tomography to elucidate pharmacokinetic differences of regioisomeric retinoid x receptor agonists.

RXR partial agonist NEt-4IB (2a, 6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: EC50 = 169 nM, E max = 55%) showed a blood concentration higher than its E max after single oral administration at 30 mg/kg to mice, and repeated oral administration at 10 mg/kg/day to KK-A(y) mice afforded antitype 2 diabetes activity without the side effects caused by RXR full agonists. However, RXR full agonist NEt-3IB (1a), in which the isobutoxy and isopropyl groups of 2a are interchanged, gave a much lower blood concentration than 2a. Here we used positron emission tomography (PET) with tracers [(11)C]1a, [(11)C]2a and fluorinated derivatives [(18)F]1b, [(18)F]2b, which have longer half-lives, to examine the reason why 1a and 2a exhibited significantly different blood concentrations. As a result, the reason for the high blood concentration of 2a after oral administration was found to be linked to higher intestinal absorbability together with lower biliary excretion, compared with 1a.