Yuki Fuchigami

Pharmacodynamic interactions between MDMA and concomitants in MDMA tablets on extracellular dopamine and serotonin in the rat brain.

3,4-methylenedioxymethamphetamine (MDMA) is a psychoactive stimulant abused by young people as the recreational drug ecstasy. Other compounds, either deliberately added or present as byproducts, are often found in MDMA tablets and can unexpectedly interact with each other. The aim of this study was to evaluate the pharmacodynamic effects of interactions caused by concomitants in MDMA tablets on extracellular dopamine and serotonin (5-HT) by microdialysis in the striatum of ethylcarbamate-anesthetized rats. Baseline levels of dopamine and 5-HT in the striatum were 16.5±7.7 and 3.5±1.7 nM (mean±standard deviation), respectively. After a single administration of MDMA (10 mg/kg, i.p.), a dramatic increase in extracellular dopamine (Cmax: 36.1-fold vs. baseline) and 5-HT levels (Cmax: 9.3-fold vs. baseline) was observed. When rats were co-administered with methamphetamine (1, 5 or 10 mg/kg) with MDMA, the dopamine levels induced by MDMA increased in a methamphetamine-dose-dependent manner (Cmax: 2.5-, 3.5-, and 3.8-fold vs. MDMA). A similar trend was observed in 5-HT levels (Cmax: 1.1-, 1.3-, and 1.8-fold vs. MDMA). In contrast, ketamine and caffeine showed synergistic effects on the monoamine levels induced by MDMA, whereas the individual administration of either of these compounds did not affect monoamine levels. Ketamine (1, 5 mg/kg) decreased the dopamine levels induced by MDMA (Cmax: 0.9- and 0.7-fold vs. MDMA) and increased the 5-HT levels induced by MDMA (Cmax: 1.4- and 1.6-fold vs. MDMA), and co-administration of caffeine (20 mg/kg) with MDMA increased dopamine levels (Cmax: 1.7-fold vs. MDMA). These results suggest that exposure to multiple drugs in addition to MDMA can have neurotoxic effects.

Ligand peptide-grafted PEGylated liposomes using HER2 targeted peptide-lipid derivatives for targeted delivery in breast cancer cells: The effect of serine-glycine repeated peptides as a spacer

Ligand peptide-grafted PEGylated liposomes have been widely studied for targeted drug delivery systems. Because ligand peptides are commonly grafted using PEG as a spacer on the surface of PEGylated liposomes, the interaction between ligand peptides and their corresponding receptors can be interrupted by steric hindrance of the PEG layer. Therefore, we aimed to develop ligand peptide-lipid derivatives to enhance the targeting efficiency of ligand peptide-grafted PEGylated liposomes, and designed a new ligand peptide-lipid derivatives having serine-glycine repeats (SG)n as a spacer based on the peptide length calculated by PyMol (v0.99). We selected KCCYSL (KCC) as the ligand peptide for binding to human epidermal growth factor receptor-2 (HER2). We synthesized new KCC-(SG)n-lipid derivatives (n=3, 5, 7) and evaluated their cellular association in breast cancer cells. KCC-(SG)n/PEGylated liposomes dramatically increased cellular association on HER2-positive breast cancer cells. The results suggest that KCC can be grafted on the surface of KCC-(SG)n/PEGylated liposomes prepared from KCC-(SG)n-lipid derivatives (n=3, 5, 7). In summary, we succeeded in developing KCC-(SG)n-lipid derivatives for the preparation of ligand peptide-grafted PEGylated liposomes.

Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

Abstract In this study, we demonstrate the low toxicity and highly efficient and spatially improved transfection of plasmid DNA (pDNA) with liposomal nanobubbles (bubble liposomes [BLs]) using ultrasound (US) irradiation in mice. Naked pDNA with BLs was intraperitoneally injected, followed by US irradiation. The injection volume, the duration of US irradiation, and the dose of BLs were optimized. Both BLs and US irradiation were essential to achieve high transgene expression from naked pDNA. We observed transgene expression in the entire peritoneal tissues, including the peritoneal wall, liver, spleen, stomach and small and large intestines. The area of transfection could be controlled with focused US irradiation. There were few changes in the morphology of the peritoneum, the peritoneal function or serum alanine aminotransferase levels, suggesting the safety of BLs with US irradiation. Using a tissue-clearing method, the spatial distribution of transgene expression was evaluated. BLs with US irradiation delivered pDNA to the submesothelial layer in the peritoneal wall, whereas transgene expression was restricted to the surface layer in the liver and stomach. Therefore, BLs with US irradiation could be an effective and safe method of gene transfection to the peritoneum.

Peptide-Based Cancer-Targeted DDS and Molecular Imaging

Targeting cancer cell-surface receptors is an attractive approach for cancer treatment and diagnosis. Peptides having high binding affinities to receptors overexpressed in cancer cells are useful because of their simple structure, low immunogenicity, and easy, cost-effective chemical synthesis. A number of peptide ligands have been developed for cancer cell-surface receptors and applied to nanoparticles with anticancer drugs, genes, small interfering RNAs (siRNAs), and molecular imaging agents. In particular, recent findings have revealed that peptide-modified PEGylated liposome-encapsulated drugs are effective in cancer-targeted therapy and cancer cell-specific imaging. This review discusses peptide-modified nanoparticles for drug delivery systems (DDS) and molecular imaging, focusing on peptide ligands for somatostatin receptors, integrin, transferrin receptor, human epidermal growth factor 2 (HER2), etc. In addition, methods to improve binding affinity or endosomal escape with spacer peptides and stimuli (internal and external) are discussed.

Quantitative and antioxidative behavior of Trolox in rats' blood and brain by HPLC-UV and SMFIA-CL methods.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10(3) antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0-360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood-brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified.

Long-term in vivo gene expression in mouse kidney using φC31 integrase and electroporation

Abstract Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes. Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ϕC31 integrase. Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5 µg of pORF-luc/attB as a donor plasmid and 20 µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression. Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ϕC31 integrase-transfected cells had less genomic DNA damage caused by integrase expression. Discussion and conclusion: These results demonstrated that the ϕC31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.

Optimization of renal transfection using a renal suction-mediated transfection method in mice

Abstract Background: We previously developed a suction-mediated transfection method in mice. Purpose: The purpose of this study was to optimize the suction-mediated transfection conditions using a pressure-controlled computer system for efficient and safe kidney-targeted gene delivery in mice. Methods: Naked pCMV-Luc was injected into the tail vein in mice, and then the right kidney was suctioned by a device of the suction pressure-controlled system. The effects of renal transfection conditions, such as the suction pressure degree, suction pressure waveform and device area were evaluated by measuring luciferase expression. In addition, renal injury was examined. Results: The renal suction-mediated transfection method at −30 kPa showed high transgene expression. The renal suction waveform did not affect the transfection activity. Under the optimized conditions, the high transgene expression was mostly observed at the renal suctioned site. The transfection conditions used did not induce histological defects or increases in two renal injury biomarkers (Kidney injury molecule-1 mRNA and Clusterin mRNA). Discussion and conclusion: We have clarified the transfection conditions for efficient and safe transfection in the kidney using the suction-mediated transfection method in mice.

Effect of pH and Additives on the Compatibility between Vancomycin and Furosemide Injections

　The physicochemical compatibility between injections of different agents is very important. An injection of the antibiotic vancomycin (VCM) is acidic and its standard pH range is 2.5-4.5. In clinical treatments, VCM injections are often used with Lasix® (furosemide) injections. The Lasix® injection is alkaline and its standard pH range is 8.6-9.6. Therefore, mixing VCM injections with Lasix® injections may cause compatibility problems. We evaluated the effect of pH on the compatibility between VCM (original and two generic) and Lasix® injections. Compatibility was not observed in non-pH-adjusted VCM with Lasix® injections, but white crystals appeared when VCM injections adjusted to pH 2.5 experimentally were mixed with a Lasix® injection, suggesting that the acidic condition of VCM injections cause compatibility. However, the residual rates of VCM did not change after 24 h in all mixtures. We analyzed the crystals by mass spectrometry and 1H-NMR, and identified them to comprise furosemide.

Development of High-Functionality and -Quality Lipids with RGD Peptide Ligands: Application for PEGylated Liposomes and Analysis of Intratumoral Distribution in a Murine Colon Cancer Model

High-functionality and -quality (HFQ) lipids have a discrete molecular weight and good water dispersibility and can be produced by solid-phase peptide synthesis. Therefore, HFQ lipids are a promising material for the preparation of ligand-grafted PEGylated liposomes. Recently, we have reported serine-glycine repeated peptides ((SG) n) as a spacer of HFQ lipids and to substitute a conventional PEG spacer. We demonstrated the advantage of using (SG) n spacers for peptide ligand presentation on the liposomal surface in vitro; however, the use of (SG) n spacers in ligand-grafted PEGylated liposomes in vivo has not been validated. The aim of this study was to validate the in vivo targeting ability of HFQ lipid-grafted PEGylated liposomes. We synthesized lipids containing GRGDS (RGD-(SG) n-lipid) to target integrin αvβ3 and prepared RGD-(SG) n/PEGylated liposomes. Subsequently, their cellular uptake characteristics in murine colon carcinoma (Colon-26) cells were evaluated. Two-color imaging of liposomes and tumor blood vessels following tissue clearing was performed to examine the spatial intratumoral distribution of liposomes. RGD-(SG)5/PEGylated liposomes were selectively associated with the cells in vitro. In vivo analysis of intratumoral distribution following tissue clearing revealed the superior targeting ability of RGD-(SG)5/PEGylated liposomes compared with that of conventional RGD-PEG2000/PEGylated liposomes for both tumor tissues and tumor blood vessels. We successfully synthesized RGD-HFQ lipids to prepare RGD-grafted PEGylated liposomes for the efficient targeting of integrin αvβ3-expressing cells. To the best of our knowledge, this is the first report of the intratumoral distribution of ligand-grafted PEGylated liposomes by two-color imaging following tissue clearing.

Efficient gene transfection to the brain with ultrasound irradiation in mice using stabilized bubble lipopolyplexes prepared by the surface charge regulation method

Introduction We previously developed anionic ternary bubble lipopolyplexes, an ultrasound-responsive carrier, expecting safe and efficient gene transfection. However, bubble lipopolyplexes have a low capacity for echo gas (C3F8) encapsulation (EGE) in nonionic solution such as 5% glucose. On the other hand, we were able to prepare bubble lipopolyplexes by inserting phosphate-buffered saline before C3F8 encapsulation. Surface charge regulation (SCR) by electrolytes stabilizes liposome/plasmid DNA (pDNA) complexes by accelerated membrane fusion. Considering these facts, we hypothesized that SCR by electrolytes such as NaCl would promote C3F8 encapsulation in bubble lipopolyplexes mediated by accelerated membrane fusion. We defined this hypothesis as SCR-based EGE (SCR-EGE). Bubble lipopolyplexes prepared by the SCR-EGE method (SCR-EGE bubble lipopolyplexes) are expected to facilitate the gene transfection because of the high amount of C3F8. Therefore, we applied these methods for gene delivery to the brain and evaluated the characteristics of transgene expression in the brain. Methods First, we measured the encapsulation efficiency of C3F8 in SCR-EGE bubble lipopolyplexes. Next, we applied these bubble lipopolyplexes to the mouse brain; then, we evaluated the transfection efficiency. Furthermore, three-dimensional transgene distribution was observed using multicolor deep imaging. Results SCR-EGE bubble lipopolyplexes had a higher C3F8 content than conventional bubble lipopolyplexes. In terms of safety, SCR-EGE bubble lipopolyplexes possessed an anionic potential and showed no aggregation with erythrocytes. After applying SCR-EGE bubble lipopolyplexes to the brain, high transgene expression was observed by combining with ultrasound irradiation. As a result, transgene expression mediated by SCR-EGE bubble lipopolyplexes was observed mainly on blood vessels and partially outside of blood vessels. Conclusion The SCR-EGE method may promote C3F8 encapsulation in bubble lipopolyplexes, and SCR-EGE bubble lipopolyplexes may be potent carriers for efficient and safe gene transfection in the brain, especially to the blood vessels.