Kodai Nishi

Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial Infarction in Mice

Rationale: Stem cell therapy faces several challenges. It is difficult to grow, preserve, and transport stem cells before they are administered to the patient. Synthetic analogs for stem cells represent a new approach to overcome these hurdles and hold the potential to revolutionize regenerative medicine. Objective: We aim to fabricate synthetic analogs of stem cells and test their therapeutic potential for treatment of acute myocardial infarction in mice. Methods and Results: We packaged secreted factors from human bone marrow–derived mesenchymal stem cells (MSC) into poly(lactic-co-glycolic acid) microparticles and then coated them with MSC membranes. We named these therapeutic particles synthetic MSC (or synMSC). synMSC exhibited a factor release profile and surface antigens similar to those of genuine MSC. synMSC promoted cardiomyocyte functions and displayed cryopreservation and lyophilization stability in vitro and in vivo. In a mouse model of acute myocardial infarction, direct injection of synMSC promoted angiogenesis and mitigated left ventricle remodeling. Conclusions: We successfully fabricated a synMSC therapeutic particle and demonstrated its regenerative potential in mice with acute myocardial infarction. The synMSC strategy may provide novel insight into tissue engineering for treating multiple diseases.

Transport mechanism of 11C-labeled L- and D-methionine in human-derived tumor cells

INTRODUCTION S-methyl-(11)C-labeled l- and d-methionine ((11)C-l- and d-MET) are useful as radiotracers for tumor imaging. However, it is not known whether the transport mechanism of (11)C-d-MET is the same as that for (11)C-l-MET, which is transported by the amino acid transport system L. In this study, we investigated the transport mechanism of (11)C-l- and d-MET by analyzing the expression of transport system genes in human-derived tumor cells. METHODS The expression of transport system genes in human-derived tumor cells was quantitatively analyzed. The mechanism of MET transport in these cells was investigated by incubating the cells with [S-methyl-(3)H]-l-MET ((3)H-l-MET) or [S-methyl-(3)H]-d-MET ((3)H-d-MET) and the effect of 2-amino-2- norbornane-carboxylic acid, a system L transport inhibitor, or α-(methylamino)isobutyric acid, a system A transport inhibitor, on their transport was measured. The transport and metabolic stability of [S-methyl-(14)C]-l-MET ((14)C-l-MET) and (3)H-d-MET was also analyzed using bearing mice with H441 or PC14 tumor cells. RESULTS (3)H-d-MET was mainly transported by both systems L and alanine-serine-cysteine (ASC), while system L was involved in (3)H-l-MET transport. There was a high correlation between both (3)H-l-MET and (3)H-d-MET uptake and the expression of amino acid transport system genes. In the in vivo study, H441-cell accumulation of (3)H-d-MET was higher than that of (14)C-l-MET. Hepatic and renal accumulation of (3)H-d-MET was lower than that of (14)C-l-MET. CONCLUSION The transport mechanism of (3)H-d-MET was different from that of (3)H-l-MET. Since (3)H-d-MET has high metabolic stability, its accumulation reflects the transporter function of system L and ASC.

Comparison of the transcellular transport of FDG and D-glucose by the kidney epithelial cell line, LLC-PK1.

ObjectivesAlmost all D-glucose (GLU) filtered through the glomerulus is reabsorbed by the renal proximal tubules, whereas a high portion of 2-[18F]fluoro-2-deoxy-D-glucose [(18F)FDG] is excreted through the urine. However, [18F]FDG is not entirely excreted in the urine suggesting that it may be partially reabsorbed by the proximal tubules. The purpose of this study was to compare the time course of transcellular transport of administered [14C] labeled FDG ([14C]FDG) with that of [14C] labeled GLU ([14C]GLU) using the kidney epithelial cell line, LLC-PK1. MethodsTranscellular transport of [14C]FDG and [14C]GLU by LLC-PK1 cells was measured in Na+-containing or Na+-free Dulbeccos phosphate-buffered saline [PBS(+) and PBS(−), respectively] in the presence or absence of phlorizin, phloretin, probenecid, or tetraethylammonium bromide inhibitors that predominantly inhibit sodium-dependent glucose transporters (SGLTs), sodium-independent glucose transporters, organic anion transporters, and organic cation transporters, respectively. ResultsWhen assayed in PBS(+), less [14C]FDG than [14C]GLU was reabsorbed by the proximal tubular cells over the entire incubation time. Reabsorption of [14C]FDG was mediated mainly by SGLT at early time points in the incubation, whereas high reabsorption of [14C]GLU was mediated by both SGLT and glucose transporter over 90 min of incubation. Secretion of [14C]FDG also tended to be slightly higher than that of [14C]GLU over 90 min of incubation. ConclusionTranscellular transport of [14C]FDG over time by LLC-PK1 cells was clarified. The polarized distribution of transcellular transporters of [14C]FDG and [14C]GLU in LLC-PK1 cells differs.

Ionic Liquid-Mediated Hydrofluorination of o-Azaxylylenes Derived from 3-Bromooxindoles

The hydrofluorination reaction of 3-bromooxindole using mild HF reagents in an ionic liquid is described. This transformation can operate at room temperature to give a series of 3-substituted 3-fluorooxindole derivatives including racemic BMS 204352 (MaxiPost). The mechanistic study about interactions between HF and 3-butyl-1-methylimidazolium tetrafluoroborate [bmim][BF4] is also discussed on the basis of energy calculations.

Differences in accumulation and the transport mechanism of l- and d-methionine in high- and low-grade human glioma cells.

INTRODUCTION Although [S-methyl-11C]-labeled L-methionine and D-methionine (11C-L-MET and 11C-D-MET) are useful radiotracers for positron emission tomography imaging of brain tumors, it is not known whether the accumulation and transport mechanisms underlying uptake of 11C-D-MET and 11C-L-MET are the same. 11C-L-MET is mainly taken up by the amino acid transport system L. We evaluated accumulation and the transport mechanism of D-MET in high- and low-grade human glioma cells in vitro. METHODS The expression of transport system genes in high- (A172 and T98G) and low-grade (SW1088 and Hs683) glioma cells was quantitatively analyzed. Accumulation of [S-methyl-3H]-L-MET (3H-L-MET) and [S-methyl-3H]-D-MET (3H-D-MET) in these cells was compared during 60min of incubation. The transport mechanism of 3H-L-MET and 3H-D-MET was investigated by incubating the cells with these compounds and examining the effect of the inhibitors 2-amino-2-norbornane-carboxylic acid or α-(methylamino) isobutyric acid. RESULTS Absolute expression levels of system L and system alanine-serine-cysteine (ASC) in high-grade glioma cells were higher than in low-grade cells. In high-grade glioma cells, expression of system ASC genes was higher than that of system L genes. 3H-D-MET, which is transported by systems L and ASC, accumulated at higher levels than 3H-L-MET at all incubation times because 3H-D-MET is more sensitive to system ASC than 3H-L-MET. Conversely, in low-grade glioma cells with lower expression of system L and ASC, 3H-D-MET accumulated at higher levels than 3H-L-MET in early incubation times because 3H-D-MET may be more sensitive to system ASC than system L. CONCLUSION 3H-D-MET was mainly transported by systems L and ASC and sensitive to system ASC, whereas 3H-L-MET was transported by system L in human glioma cells. In vitro, the accumulation of 3H-D-MET was significantly higher than that of 3H-L-MET during the entire incubation time in high-grade glioma cells, and in early incubation times in low-grade glioma cells.

Pharmacokinetic alteration of(99m)Tc-MAG3 using serum protein binding displacement method

INTRODUCTION When a radiopharmaceutical is simultaneously administered with a medicine that has high affinity for the same plasma protein, the radiopharmaceutical is released at higher concentrations in blood, leading to enhanced transfer into target tissues. This is known as the serum protein binding displacement method. In this study, we investigated the pharmacokinetic alteration of technetium-99m-labeled mercaptoacetylglycylglycylglycine ((99m)Tc-MAG3) using the serum protein binding displacement method. METHODS Rat and human serum protein binding rates of (99m)Tc-MAG3 were measured by ultrafiltration with or without displacers of human serum albumin (HSA) binding sites I and II (200μM and 400μM loading). Male Wistar rats were injected with (99m)Tc-MAG3 (740kBq/0.3mL saline) via the tail vein, and biodistribution was assessed at 2, 5, 10 and 15min. Dynamic whole-body images were obtained for (99m)Tc-MAG3 (11.1MBq/0.3mL saline)-injected rats, with or without HSA displacers. RESULTS (99m)Tc-MAG3 strongly bound to HSA (87.37%±2.13%). Using HSA site I displacers, the free fraction of (99m)Tc-MAG3 increased significantly (1.20 to 1.47 times) when compared with controls. For biodistribution and imaging, rapid blood clearance was observed with bucolome (BCL) loading, which is an HSA site I displacer. With BCL loading, peak times for rat renograms were respectively shifted from 240s to 110s, and from 170s to 120s. CONCLUSIONS We found that (99m)Tc-MAG3 bound to the HSA binding site I. It was confirmed that pharmacokinetic distribution of (99m)Tc-MAG3 is altered by presence of BCL, which leads to increases in the free fraction of (99m)Tc-MAG3, and BCL produced rapid blood clearance and fast peak times on rat renograms. The serum protein binding displacement method using (99m)Tc-MAG3 and BCL, a safe displacer for humans, may be applicable to clinical study and lead to better diagnostic images with shorter waiting times and lower radiation doses for patients.

Development of a 68Ge/68Ga Generator System Using Polysaccharide Polymers and Its Application in PET Imaging of Tropical Infectious Diseases

Gallium-68 (68Ga) is a positron emitter for clinical positron emission tomography (PET) applications that can be produced by a 68Ge/68Ga generator without cyclotron. However, commercially available 68Ge/68Ga generator systems require multiple steps for the preparation of 68Ga radiopharmaceuticals and are sometimes plagued by metallic impurities in the 68Ga eluent. We developed a 68Ge/68Ga generator system using polysaccharide-based adsorbents and direct application of the generator-eluted 68Ga-citrate to PET imaging of tropical infectious diseases. N-Methylglucamine (MG) as a 68Ge-adsorbing unit (Sepha-MGs) was introduced to a series of Sephadex G-10, G-15, G-25, G-50, and G-75. In the batch method, over 97% of the 68Ge in the solution was adsorbed onto the Sepha-MG series within 15 min. In particular, 68Ge was effectively adsorbed on the Sepha(15)-MG packed columns and 70–80% of the 68Ga was eluted by 1 mL of 0.1 M trisodium citrate with low 68Ge contamination (<0.001%). The chemical form of the generator-eluted 68Ga solution was identified as 68Ga-citrate. In PET studies, affected regions in mice infected with Leishmania and severe fever with thrombocytopenia syndrome virus were clearly visualized using the 68Ga-citrate. Sepha-MGs are useful adsorbents for 68Ge/68Ga generator systems with high 68Ga elution efficiency and minimal 68Ge breakthrough. These results indicated that eluted 68Ga-citrate can be directly used for PET imaging of infectious sites in mice. This novel generator system may be useful for straightforward PET imaging of infection in clinical practice.

Activity of N,N′-dialkyl-2-trifluoromethylthioimidazolium salts as phase-transfer catalyst for the alkylation of active methylene compounds

We herein report the synthesis of N,N′-dialkyl-2-trifluoromethylthioimidazolium salts from thioureas using nucleophilic trifluoromethylating reagents. The activity of imidazolium salts as phase-transfer catalysts under solid–liquid phase conditions was investigated for reactions such as the alkylation of active methylene compounds.

3,3-Dibromo-2-trifluoromethyl acrylic acid ethyl ester: a versatile platform for the stereoselective preparation of functionalized-α-trifluoromethyl α,β-unsaturated lactones and trifluoromethyl pyrazolinones

We herein describe a method for synthetic routes to multi-functionalized-α-trifluoromethyl α,β-unsaturated lactones and trifluoromethyl pyrazolinones. This involves a tandem stereoselective functionalization of 3,3-dibromo-2-trifluoromethyl acrylic acid ethyl ester and intramolecular cyclization reaction to afford precursors for a Suzuki–Miyaura cross-coupling reaction with arylboronic acids.

N-Isopropyl-p-iodoamphetamine Hydrochloride Is Predominantly Metabolized by CYP2C19

[123I]N-Isopropyl-p-iodoamphetamine hydrochloride ([123I]IMP) is clinically used to evaluate blood flow in the brain on single photon emission-computed tomography. This is a rare radiopharmaceutical that undergoes metabolism. The first step is reported to be [123I]p-iodoamphetamine formation. The drug-metabolizing enzyme(s) involved remain(s) unclear. This study examined the roles of human cytochrome P450 (P450) in the metabolism of nonradiolabeled IMP with the use of human liver microsomes (HLM) and recombinant human CYP1A1, -1A2, -1B1, -2A6, -2B6, -2C8, -2C9, -2C19, -2D6, -2E1, -3A4, and -3A5. Disappearance of IMP was examined because p-iodoamphetamine was not available. IMP (0.5 μM) time-dependently disappeared when HLM and NADPH-generating system were added to the reaction mixture. (S)-Mephenytoin (1 mM) inhibited the IMP disappearance by approximately 90%. The disappearance of IMP was predominantly catalyzed by recombinant CYP2C19, with Km and Vmax of 8.6 μM and 9.7 nmol · min−1 · nmol P450−1, respectively. IMP disappearance in CYP2C19-deficient HLM (CYP2C19*2/*2) was approximately 30% of that in the presence of HLM harboring wild-type CYP2C19, indicating that IMP is polymorphically metabolized by CYP2C19. High-performance liquid chromatography of the incubation mixture of IMP and CYP2C19 revealed an unidentified peak. As the area of the IMP peak decreased, the area of this unidentified peak increased in a time-dependent fashion. The peak was also detectable on incubation of IMP with HLM. Mass spectrometry revealed that the molecular weight of a compound in this unidentified peak was the same as that of p-iodoamphetamine. Thus, we demonstrated that IMP was predominantly metabolized by CYP2C19 to form p-iodoamphetamine.