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Mitsuru Hashida

Publications and source records attributed to Mitsuru Hashida.

At least 73 records · Page 4Linked to original sources
PubMed2004

Tissue and intrahepatic distribution and subcellular localization of a mannosylated lipoplex after intravenous administration in mice.

We have previously reported that, unlike a lipoplex and mannosylated (Man) lipoplex underwent gene transfer to liver nonparenchymal cells (NPC) that possess mannose receptors after intravenous administration in mice. In this study, the tissue, intrahepatic distribution, and subcellular localization of the lipoplex after intravenous administration were investigated. DC-Chol liposome was selected as a cationic liposomes. After administration of lipoplex and Man lipoplex, the high gene expression was observed in the lung and liver, respectively. After administration of [32P]Man lipoplex, about 80% of [32P]plasmid DNA (pDNA) was accumulated in the liver. As for the intrahepatic distribution, the NPC/parenchymal cells (PC) ratio of [32P]Man lipoplex was 9.64, whereas the NPC/PC ratio of [32P]lipoplex was 1.93. The radioactivity in the cytosolic fraction of liver homogenate of [111In]Man lipoplex was two-fold higher than that of [111In]lipoplex, indicating that Man liposomes facilitate the release of pDNA into the cytosolic space. However, a rapid sorting of the radioactivity from endosomes to lysosomes was observed with the [111In]Man lipoplex. Also, amplification of pDNA by PCR suggested that the Man lipoplex is more rapidly degraded within the intracellular vesicles than the lipoplex. These results suggested that modulation of its intracellular sorting could improve the transfection efficiency of Man lipoplex.

Animals↗
PubMed2004

Hepatic uptake of negatively charged particles in rats: possible involvement of serum proteins in recognition by scavenger receptor.

The mechanisms involved in the hepatic uptake of negatively charged carboxylated-polystyrene nanospheres with a size of 50 nm (CNS-50) were examined in rats. The liver perfusion experiments revealed that hepatic disposition of CNS-50 in the absence of serum could be partially ascribed to the direct recognition of the surface negative charge by scavenger receptors. On the other hand, the apparent negative charge of CNS-50 surface dramatically reduced in the presence of serum, because the adsorption of serum protein on their surface results in masking their intrinsic negative charge. However, hepatic disposition of CNS-50 in the presence of serum was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor for scavenger receptors, and the extent of inhibition by poly I was even larger than that in the absence of serum, suggesting that the serum proteins associated on CNS-50 surface could be recognized by scavenger receptors. These results indicate that not only the intrinsic negative charge but also serum proteins associated on the surface play an important role in hepatic uptake of negatively charged particles via scavenger receptors.

Animals↗
PubMed2004

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice.

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.

Animals↗
PubMed2004

Physicochemical, tissue distribution, and vasodilation characteristics of nitrosated serum albumin: delivery of nitric oxide in vivo.

Conjugates of nitric oxide (NO) to serum albumins are candidates for controlled delivery of NO in vivo, but their physicochemical and tissue distribution characteristics have hardly been examined yet. In this study, to achieve its in vivo delivery, bovine serum albumin (BSA) was reacted with sodium nitrite to obtain NO-BSA, which had 0.25-0.28 molecules of S-nitrosothiol/BSA. In addition to cystein, other amino acid residues were modified by the reaction. The conjugation had no significant effect on the molecular weight, but reduced the electric charge and induced reversible changes in the secondary structure of BSA. After intravenous injection in mice at a dose of 1 mg/kg, 111In-NO-BSA slowly disappeared from plasma in a similar manner to 111In-BSA, but showed greater accumulation in the liver and kidney. NO-BSA induced a transient decrease in arterial pressure after intravenous injection in rats at a dose of 100 mg/kg, and significantly increased the distribution of 111In-BSA to the lung in mice. These results indicate that NO is released from NO-BSA shortly after injection, and this NO decreases blood pressure and increases the distribution of macromolecules to the lung. These findings provide useful basic information for designing macromolecular NO donors able to achieve controlled delivery of NO.

Animals↗
PubMed2004

Two- and three-dimensional QSAR of carrier-mediated transport of beta-lactam antibiotics in Caco-2 cells.

In this study, we investigated whether such a topological descriptor-based approach is suitable for predicting the carrier-mediated transport of 20 beta-lactam antibiotics that are substrates of peptide transporters. To select the molecular descriptors that can effectively predict a targeted property in QSAR analysis, the genetic algorithm-combined partial least squares approach was used. The feasibility of the two-dimensional (2D)-QSAR approach was compared with that of comparative molecular field analysis (CoMFA). The logarithm of the uptake values of 20 beta-lactam antibiotics in Caco-2 cells obtained from the literature ranged from -1.15 to 1.09 (nmol/cm2/2 h). When preliminary leave-one-out cross-validated partial least squares analyses implemented in the SYBYL/CoMFA program were conducted, the r2pred was 0.759 and the standard error of prediction (s) was 0.373. However, the 2D-QSAR approach based on Molconn-Z descriptors gave a better predictability (r2pred = 0.923, s = 0.211), where 14 descriptors were selected and the optimal number of principal components was 4. Considering that the 2D-topological descriptors are less computationally intensive and practically completely automated, the simple 2D-QSAR model is also of great importance in drug discovery settings.

Anti-Bacterial Agents↗
PubMed2004

Enhanced hepatocyte-selective in vivo gene expression by stabilized galactosylated liposome/plasmid DNA complex using sodium chloride for complex formation.

In this study, we demonstrated that the presence of an essential amount of sodium chloride (NaCl) during the formation of cationic liposome/plasmid DNA complexes (lipoplexes) stabilizes the lipoplexes according to the surface charge regulation (SCR) theory. Fluorescence resonance energy transfer analysis revealed that cationic liposomes in an SCR lipoplex (5 and 10 mM NaCl solution in lipoplex) increased fusion. Also, aggregation of SCR lipoplexes was significantly delayed after exposure to saline (150 mM NaCl) as a model of physiological conditions. After intraportal administration, the hepatic transfection activity of galactosylated SCR lipoplexes (5 and 10 mM NaCl solution in lipoplex) was approximately 10- to 20-fold higher than that of galactosylated conventional lipoplexes in mice. The transfection activity in hepatocytes of galactosylated SCR lipoplexes was significantly higher than that of conventional lipoplexes, and preexposure to competitive asialoglycoprotein-receptor blocker significantly reduced the hepatic gene expression, suggesting that hepatocytes are responsible for high hepatic transgene expression of the galactosylated SCR lipoplexes. Pharmacokinetic studies both in situ and in vivo demonstrated a higher tissue binding affinity and a greater expanse of intrahepatic distribution by galactosylated SCR lipoplexes. Moreover, enhanced transfection activity of galactosylated SCR lipoplexes was observed in HepG2 cells, and investigation of confocal microscopic images showed that the release of plasmid DNA in the cell was markedly accelerated. These characteristics partly explain the mechanism of enhanced in vivo transfection efficacy by galactosylated SCR lipoplexes. Hence, information in this study will be valuable for the future use, design, and development of ligand-modified lipoplexes for in vivo applications.

Animals↗
PubMed2004

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice.

Bovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 x 10(5) colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35000 units/kg of body weight) significantly (P < 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (P < 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (P < 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.

Animals↗
PubMed2004

Biodistribution characteristics of galactosylated emulsions and incorporated probucol for hepatocyte-selective targeting of lipophilic drugs in mice.

PURPOSE: Galactosylated emulsions containing cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a "homing device" were developed for hepatocyte-selective drug targeting. The targeting efficiency of galactosylated emulsions was evaluated by a distribution study in mice. METHODS: Soybean oil/EggPC/cholesterol (Chol) (weight ratio, 70:25: 5) (bare) emulsions and soybean oil/EggPC/Gal-C4-Chol (weight ratio, 70:25:5) (Gal) emulsions were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [14C]probucol as a model lipophilic drug was incorporated in the emulsions or EggPC/Chol/Gal-C4-Chol (Gal) liposomes. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. RESULTS: After intravenous injection, Gal-emulsions were rapidly eliminated from the blood and accumulated in the liver, in contrast to the bare-emulsions. The liver uptake clearance of Gal-emulsions was 3.2- and 1.2-times greater than that of bare-emulsions and Gal-liposomes, respectively. The uptake ratio in liver parenchymal cells (PC) and nonparenchymal cells (NPC) of Gal-emulsions was higher than that of Gal-liposomes, being 7.4 and 3.0, suggesting that Gal-emulsions are an effective PC-selective carrier. The hepatic uptake of Gal-emulsions, but not that of bare-emulsions, was significantly inhibited by the pre-dosing of not only lactoferrin but also Gal-liposomes, suggesting asialoglycoprotein receptor-mediated endocytosis. Furthermore, [14C]probucol incorporated in Gal-emulsions was efficiently delivered to the liver compared with Gal-liposomes. CONCLUSION: Gal-emulsions have been proven to be an alternative carrier for hepatocyte-selective drug targeting.

Animals↗
PubMed2004

Exogenous expression of interferon-beta in cultured brain microvessel endothelial cells.

Brain microvessel endothelial cells (BMECs) make up the blood-brain barrier (BBB) and regulate the passage of therapeutic proteins as well as drugs from the cerebrovasucular circulation to the brain. In the present study, we transferred mouse or human interferon-beta (IFN-beta) gene via cationic liposomes into primary cultures of bovine BMECs developed as an in vitro model of the BBB. The gene-transferred BMECs secreted transiently a substantial amount of IFN activity more efficiently during the growth phase than at confluence. This was suggested to be due to a difference in the potential for plasmid incorporation between growing and confluent BMECs in a series of cell association experiments with (32)P-labelled plasmid DNA. Furthermore, when BMEC monolayers in Transwell plates were transfected with the IFN-beta-expression vectors from the upper side, IFN-beta was predominantly detected in the upper compartments, suggesting polarized secretion of the transgene products in BMEC monolayers. These findings provide important basic information about therapeutic secretory protein gene delivery to BMECs.

Animals↗
PubMed2004

Expression and visualization of a human interferon-beta-enhanced green fluorescent protein chimeric molecule in cultured cells.

We have constructed a recombinant cDNA encoding the chimeric protein between human IFN-beta (HuIFN-beta) and enhanced green fluorescent protein (EGFP) to elucidate the intracellular localization of IFN-beta. Transient expression of the chimeric molecule, HuIFN-beta-EGFP, in L cells demonstrated that the chimeric molecule secreted from the cells had an intact biological activity as far as antiviral effect was concerned. Immunostaining of the transfected cells using anti-HuIFN-beta antibody demonstrated that green-fluorescence was co-localized with the IFN signal and its profile was similar to IFN signals in the cells transfected with HuIFN-beta expressing plasmid DNA. These results indicate that the HuIFN-beta-EGFP chimeric gene was expressed as a chimeric protein and the chimera was transported via the regular secretory pathway in the cells. In other cell types, the fluorescence derived from the chimeric protein was also seen on cytoplasmic vesicular structures. These results suggest that HuIFN-beta-EGFP will be a useful tool to investigate the intracellular trafficking processes of HuIFN-beta in a variety of cell types.

Animals↗
PubMed2004

In silico approaches for predicting ADME properties of drugs.

Combinatorial chemistry and high-throughput screening have increased the possibility of finding new lead compounds at much shorter time periods than conventional medicinal chemistry. However, too much promising drug candidates often fail because of unsatisfactory ADME properties. In silico ADME studies are expected to reduce the risk of late-stage attrition of drug development and to optimize screening and testing by looking at only the promising compounds. To this end, many in silico approaches for predicting ADME properties of compounds from their chemical structure have been developed, ranging from data-based approaches such as quantitative structure-activity relationship (QSAR), similarity searches, and 3-dimensional QSAR, to structure-based methods such as ligand-protein docking and pharmacophore modelling. In addition, several methods of integrating ADME properties to predict pharmacokinetics at the organ or body level have been studied. In this article, we briefly summarize in silico ADME approaches.

Animals↗
PubMed2004

Effect of polycyclic aromatic hydrocarbons on generation and efflux of glutathione conjugates in primary cultured alveolar epithelial cells.

This study was initiated to functionally characterize multidrug resistance associated protein (MRP)-mediated transport across the lung epithelium. Alveolar type II cells were isolated from rabbit lung and cultured on Transwell until forming a tight monolayers. After the cell monolayer was preloaded with monochlorobimane (mBCl) that is metabolized to a fluorescent glutathione conjugate (mBCl-SG), amount of mBCl-SG exported to apical and basal compartments were measured periodically. mBCl-SG was more preferentially exported in the apical direction than in the basolateral direction. Efflux of mBCl-SG from alveolar epithelial cells was significantly inhibited by a MRP inhibitor MK-571. Pharmacokinetic analysis of efflux profiles revealed that increased efflux of mBCl-SG by B[a]P is not due to enhanced MRP activity but simply due to an elevated level of mBCl-SG in the cells. Elevation of the intracellular level of mBCl-SG corresponded well to that of reduced GSH caused by B[a]P pretreatment.

Algorithms↗
PubMed2003

Targeted and sustained drug delivery using PEGylated galactosylated liposomes.

To achieve a sustained and targeted delivery of liposomes to liver parenchymal cells (PC), we modified distearoyl-L-phosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) (DSPC/Chol) liposomes with a galactosylated cholesterol derivative (Gal-C4-Chol), and polysorbate (Tween) 20 or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG(x)-DSPE). After intravenous injection, DSPC/Chol/Gal-C4-Chol (60:35:5) (Gal) liposomes were rapidly eliminated from the blood circulation and mostly recovered in the liver. The blood elimination of DSPC/Chol/Gal-C4-Chol/Tween 20 (55:35:5:5) (Tween 20-Gal) liposomes was slightly reduced as compared to Gal-liposomes. In contrast, a significant reduction in the blood elimination was observed with DSPC/Chol/Gal-C4-Chol/PEG(2000)-DSPE (59:35:5:1) (PEG(2000)-Gal) liposomes. Hepatic uptake of DSPC/Chol/Gal-C4-Chol/PEG(350)-DSPE (59:35:5:1) (PEG(350)-Gal) liposomes was intermediate between PEG(2000)-Gal-liposomes and Tween 20-Gal-liposomes. The uptake of PEG(350)-Gal-liposomes by liver PC was 7.7-fold higher than that by non-parenchymal cells (NPC). These results suggest that PEG(350)-DSPE can control the delivery rate of Gal-liposomes to liver PC without losing its targeting capability.

Animals↗
PubMed2003

Mechanistic and empirical modeling of skin permeation of drugs.

The skin forms a barrier to the external environment, maintaining body fluids within our system and excluding harmful substances, while the skin is a site of administration of drugs for topical and systemic chemotherapy. It is an important issue to predict the rate at which drugs or other xenobiotics penetrate the skin. In this article, we review modeling approaches for predicting skin permeation of compounds, including both mechanistic and empirical approaches. Mechanistic approaches can give us much information on understanding of skin permeation of the compounds, such as structure-permeability relationship, contribution of each barrier step, mechanism of penetration enhancers, and in vivo-in vitro relationship. On the other hand, empirical modeling can overcome any inaccuracies of mechanistic models caused by the existence of uncertainties and, therefore, give us better predictions from the practical point of view. Artificial neural networks are being available for empirical modeling of complex skin transport phenomenon.

Administration, Cutaneous↗
PubMed2003

Disposition and gene expression characteristics in solid tumors and skeletal muscle after direct injection of naked plasmid DNA in mice.

Previous studies have suggested that direct injection of naked plasmid DNA (pDNA) into solid tumors can be a useful method for in vivo gene transfer into tumor cells. To gain more insight into this approach, we studied the disposition and gene expression characteristics of naked pDNA after intratumoral injection by direct comparison with those after intramuscular injection in mice. pDNA encoding reporter genes were directly injected into subcutaneous solid tumor models and skeletal muscles. Biodistribution studies using radiolabeled pDNA showed that the elimination of pDNA from the injection site was relatively fast and a part of the pDNA was absorbed from the lymphatic system after both local injections. Confocal microscopic studies using fluorescein-labeled pDNA demonstrated that pDNA distributed efficiently throughout the muscle tissue whereas pDNA localization in the tumor tissue was restricted. Characterization of gene expression clarified the variation in expression level between tumor preparations and some factors affecting the expression level in the tumor. Reporter gene expression was significantly inhibited by simultaneous administration of some polyanions in both cases, suggesting that a specific mechanism may be involved in the naked pDNA uptake by muscle and tumor cells. These findings provide useful information for direct naked pDNA delivery into solid tumors.

Animals↗
PubMed2003

QSAR analysis of interstudy variable skin permeability based on the "latent membrane permeability" concept.

A number of QSAR models for skin permeability have been proposed, but these models lack consistency due to interspecies and interlaboratory differences. This study was initiated to extract an essential QSAR from the multiplicity of data sets of skin permeability by using a novel statistical approach. Ten data sets were collected from the literature, which include a total of 111 permeability coefficients in human, hairless mouse, or hairless rat skin for 94 structurally diverse compounds. Following a Potts and Guy's approach, the octanol/water partition coefficient and molecular weight were chosen as molecular descriptors. All of the data sets were analyzed simultaneously, assuming that all of the sets share a latent, common factor as far as the structure/permeability relationship is concerned. Despite the fact that the degree-of-freedom for the present analysis was limited compared with that for individual regression analyses, the determination coefficients (R2) were high enough for all the 10 data sets, with an average R2 of 0.815 (average R2 = 0.825 for individual analyses). Thus, skin permeability of compounds can be well explained from the log P and M.W., where the ratio of the contribution to skin permeability was approximately 1:1.

Animals↗
PubMed2003

Intravenous fate of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes.

The objective of this study was to assess the in vivo fate of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA)-based polyplexes after intravenous administration into mice. Circulation kinetics and tissue distribution in terms of plasmid localization and transfection efficiency were assessed. To gain more insight into the observed biodistribution and gene expression profile, the interaction of pDMAEMA-based polyplexes with blood components (erythrocytes and albumin) was investigated in vitro. In the case of i.v. injection of positively charged polyplexes at a dose of 30 microg DNA most of the radioactivity was found in the lungs and the liver 60 min after injection. In the case of pDMAEMA/DNA polyplexes with a negative charge, uptake occurred mainly by the liver. Administration of positively charged complexes at a 30 microg DNA dose resulted in reporter gene expression primarily in the lungs. Injection of negatively charged complexes and naked plasmid did not result in luciferase expression in any of the organs examined. In vitro turbidity experiments showed the induction of a charge dependent aggregation process upon addition of albumin to the polyplexes pointing out to the involvement of aggregate formation in the dominant lung uptake of the positively charged polyplexes. Also, incubations of polyplexes after pre-incubation with a physiological concentration of albumin with washed erythrocytes confirmed that polyplexes induce the formation of extremely large structures. This paper underlines the need for the design of systems with reduced interaction with blood components to promote the delivery of DNA to target tissues outside the lungs.

Animals↗