Biomedical subjects

Hiroko Shibata

Publications and source records attributed to Hiroko Shibata.

18 recordsLinked to original sources

PubMed2006

[The preparatory education of electronic patient record for nursing students before practical nursing training].

Preparatory education has been provided for both nursing students and teachers to understand the electronic patient record (EPR) since 2004 when EPR was introduced to the hospitals where students are allocated to undertake their work experience. First, the training and management board contacted our medical information department for an appointment and sent us a working group. They taught the nursing training staff how to use EPR and how to assign students to the proper patient record in the EPR system. Second, as preparatory education for the students, they explained the procedure for the use of EPR and the protection of personal information. Students practiced with training in the EPR system, focusing on the functions which are used frequently in practical tasks. As a result of this preparatory education, students understood the protection of personal information very well, although their understanding of the operation and management of the equipment was relatively poor and adversely affected their practice. We need to review our education contents more often. We also need to examine the present state of understanding of EPR and the problems of teaching in practical nursing training.

Curriculum↗

PubMed2006

[The instruction of the patient educational method in clinical nursing training: the actual condition of a student guidance case about diabetic's nursing using the diabetes retinopathy model].

We taught fourth-year students in a university about patient education using the diabetes retinopathy model in adult nursing practice. We analyzed teacher's student guidance plans and the guidance process using a clinical training education analysis form from the viewpoint of qualitative interpretation. We evaluated the degree of achievement of the students' goal for practice and the training subject. Consequently, the teacher developed training guidance as a class.

Adult↗

PubMed2006

A novel method for construction of gene fragment library to searching epitopes.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.

Amino Acid Sequence↗

PubMed2006

Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.

The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.

Amino Acid Sequence↗

PubMed2006

Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system.

Significant research effort is currently focused on Protein Transduction Domains (PTDs) as potential intracellular drug delivery carriers. However, the application of this technology is limited because the transduction efficiencies are often insufficient for therapeutic purposes, even using HIV-1 Tat peptide. Here we describe a high-throughput screening method based on a phage display system for isolating novel PTDs with improved cell penetration activity. The screening method involves using protein synthesis inhibitory factor (PSIF) as cargo of PTD. Using this method, several Tat-PTD mutants of superior cell-penetrating activity were isolated. Interestingly, the amino acid sequence of the PTD mutants contained some characteristic residues, such as proline. Thus, our screening method may prove useful in determining the relationship between protein transduction and amino acid sequence.

Bacteriophages↗

PubMed2006

Promotion of optimized protein therapy by bioconjugation as a polymeric DDS.

In recent years, clinical applications of recombinantly produced bioactive proteins such as cytokines have attracted attention. However, since these recombinant proteins are rather unstable in vivo, their clinical use as therapeutic agents requires frequent administration at a high dosage. This regimen disrupts homeostasis and results in severe side effects. To overcome these problems, bioactive proteins have been conjugated with water-soluble synthetic (WSS) polymeric carriers. Chemical modification of a protein with a WSS polymeric carrier (bioconjugation) regulates tissue distribution, resulting in a selective increase in its desirable therapeutic effects and a decrease in undesirable side effects. Among several drug delivery system (DDS) technologies, bioconjugation has been recognized as one of the most efficient methods for improving therapeutic potency of proteins. However, for further enhancement of the therapeutic potency and safety of conjugated bioactive proteins, more precise regulation of the in vivo behavior of each protein is necessary for selective expression of its therapeutic effect. Therefore, alternative WSS polymeric modifiers in which new functions such as targeting and controlled release of drugs can be added are required for further development of bioconjugated drugs. Recently, we have synthesized a novel polymeric drug carrier, poly(vinylpyrrolidone-co-dimethyl maleic anhydride) [PVD], which was a powerful candidate drug carrier for cancer therapy. In this review, we introduce useful information that enabled us to design polymeric drug carriers and their application for protein therapy.

Antineoplastic Agents↗

PubMed2004

Functionalization of tumor necrosis factor-alpha using phage display technique and PEGylation improves its antitumor therapeutic window.

PURPOSE: In this study, the optimization of antitumor therapy with tumor necrosis factor-alpha (TNF-alpha) was attempted. EXPERIMENTAL DESIGN: Using the phage display technique, we created a lysine-deficient mutant TNF-alpha (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. RESULTS: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-alpha (wTNF-alpha). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-alpha and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-alpha, whereas the randomly mono-PEGylated wTNF-alpha had 6% of the bioactivity of the wTNF-alpha. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-alpha. CONCLUSIONS: These results indicated that this functionalized TNF-alpha may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.

Animals↗

PubMed2004

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen.

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.

Animals↗

PubMed2004

Effective accumulation of poly(vinylpyrrolidone-co-vinyl laurate) into the spleen.

To optimize polymer-conjugated drugs as a polymeric drug delivery system, it is essential to design polymeric carriers with tissue-specific targeting capacity. Previously, we showed that polyvinylpyrrolidone (PVP) was the most suitable polymeric carrier for prolonging the blood-residency of drugs, and was one of the best parent polymers to design the polymeric carriers with targeting capacity. In this study, we synthesized some hydrophobic PVP derivatives, poly(vinylpyrrolidone-co-styrene) [poly(VP-co-S)] and poly(vinylpyrrolidone-co-vinyl laurate) [poly(VP-co-VL)], and assessed their biopharmaceutical properties after intravenous administration in mice. The elimination of hydrophobic PVP derivatives from blood was the same as PVP, and the plasma half-lives of poly(VP-co-S) were almost similar to that of poly(VP-co-VL). Poly(VP-co-VL) efficiently accumulated in the spleen, whereas poly(VP-co-S) effectively accumulated in the liver. The level of poly(VP-co-VL) in the spleen was about 20 times higher than PVP and poly(VP-co-S). These hydrophobic PVP derivatives did not show any cytotoxicity against endothelial cells in vitro. Thus, poly(VP-co-VL) may be a useful polymeric carrier for drug delivery to the spleen. This study will provide useful information to design optimal polymeric carriers with targeting capacity to the spleen and liver.

Animals↗

PubMed2004

Design of a pH-sensitive polymeric carrier for drug release and its application in cancer therapy.

PURPOSE: In this study, to optimize the polymeric drug delivery system for cancer chemotherapy, we developed a new pH-sensitive polymeric carrier, poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD], that could gradually release native form of drugs with full activity, from the conjugates in response to changes in pH. We examined the usefulness of PVD as a polymeric drug carrier. EXPERIMENTAL DESIGN: PVD was radically synthesized with vinylpyrrolidone and 2,3-dimethylmaleic anhydride, which is known to be a pH-reversible amino-protecting reagent. Conjugates between PVD and other drugs, such as Adriamycin (ADR), were prepared under the slightly basic conditions (pH 8.5). The drug-release pattern and the antitumor activity of PVD were examined. RESULTS: At pH 8.5, the release of the drugs from the conjugate was not observed. In contrast, PVD could release fully active drugs in the native form in response to the change in pH near neutrality, and gradually released drugs at neutral pH (7.0) and slightly acidic pH (6.0). The drug-release pattern in serum was almost similar to that observed during these physiological conditions. The PVD-conjugated ADR showed superior antitumor activity against sarcoma-180 solid tumor in mice, and it had less toxic side effects than free ADR. This enhancement in the antitumor therapeutic window may be due to not only the improvement of plasma half-lives and tumor accumulation of ADR, but also its controlled and sustained release from the conjugates in vivo. CONCLUSIONS: These results indicate that PVD is an effective polymeric carrier for optimizing cancer therapy.

Animals↗

PubMed2004

Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency.

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).

Animals↗

PubMed2004

Poly(vinylpyrrolidone-co-dimethyl maleic acid) as a novel renal targeting carrier.

Poly(vinylpyrrolidone-co-dimethyl maleic acid) (PVD) was found to have high renal-targeting capability and safety as a drug carrier. To optimize the renal drug delivery system using PVD, the relationship between the molecular weight of PVD and its renal accumulation were evaluated in mice by their intravenous injection. It was found that the molecular size of 6-8 kDa was associated with the highest renal accumulation. The specific bioactivity of PVD-conjugated superoxide dismutase (SOD) relative to that of unmodified SOD gradually decreased with an increase in the degree of modification to SOD with PVD6K. The conjugated SOD (L-PVD-SOD) with the molecular size of 73 kDa, which had comparable specific bioactivity with native SOD, showed longer plasma half-life than native SOD. About sixfold more L-PVD-SOD was distributed to the kidneys than native SOD 3 h after intravenous injection, whereas extensive PVD modification did not enhance the renal accumulation of SOD. This L-PVD-SOD effectively accelerated recovery from mercuric chloride-induced acute renal failure in vivo. These results suggest that L-PVD-SOD may be the optimal derivative as a potential therapeutic agent to various renal diseases.

Acute Kidney Injury↗

PubMed2004

The use of PVP as a polymeric carrier to improve the plasma half-life of drugs.

To achieve an optimum drug delivery such as targeting or controlled release utilizing bioconjugation with polymeric modifier, the conjugate between drugs and polymeric modifiers must be designed to show desirable pharmacokinetic characteristics in vivo. In this study, we assessed the biopharmaceutical properties of various nonionic water-soluble polymers as polymeric drug carriers. Polyvinylpyrrolidone (PVP) showed the longest mean resident time (MRT) after i.v. injection of all nonionic polymers with the same molecular size. In fact, tumor necrosis factor-alpha (TNF-alpha) bioconjugated with PVP (PVP-TNF-alpha) circulated longer than TNF-alpha bioconjugated with polyethylene glycol (PEG-TNF-alpha) with the same molecular size. Each nonionic polymeric modifier showed a different tissue distribution. Dextran was accumulated in the spleen and liver. Polydimethylacrylamide (PDAAm) tended to distribute in the kidney. However, PVP showed the minimum volume of tissue distribution. These results suggested that PVP is the most suitable polymeric modifier for prolonging the circulation lifetime of a drug and localizing the conjugated drug in blood.

Animals↗

PubMed2004

The targeting of anionized polyvinylpyrrolidone to the renal system.

We reported that the co-polymer composed of vinylpyrrolidone and maleic acid selectively distributed into the kidneys after i.v. injection. To further optimize the renal drug delivery system, we assessed the renal targeting capability of anionized polyvinylpyrrolidone (PVP) derivatives after intravenous administration in mice. The elimination of anionized PVP derivatives from the blood decreased with increasing anionic groups, and the clearance of carboxylated PVP and sulfonated PVP from the blood was almost similar. But carboxylated PVP efficiently accumulated in the kidney, whereas sulfonated PVP was rapidly excreted in the urine. The renal levels of carboxylated PVP were about five-fold higher than sulfonated PVP. Additionally, carboxylated PVP was effectively taken up by the renal proximal tubular epithelial cells in vivo after i.v. injection. These anionized PVP derivatives did not show any cytotoxicity against renal tubular cells and endothelial cells in vitro. Thus, these carboxylated and sulfonated PVPs may be useful polymeric carriers for drug delivery to the kidney and bladder, respectively.

Animals↗

PubMed2004

Development of novel drug delivery system (DDS) technologies for proteomic-based drug development.

With the success of human genome projects, the focus of life science research has shifted to the functional and structural analyses of proteins, such as disease proteomics. These structural and functional analyses of expressed proteins in the cells and/or tissues are expected to contribute to the identification of therapeutically applicable proteins for various diseases. Thus, pharmaco-proteomic based drug development for protein therapies is most noticed currently. However, there is a clinical difficulty to use almost bioactive proteins, because of their very low stability and pleiotropic actions in vivo. To promote pharmaco-proteomic based drug development for protein therapies to various diseases, we have attempted to establish a system for creating functional mutant proteins (muteins) with desired properties, and to develop a site-specific polymer-conjugation system for further improving the therapeutic potency of proteins. In this review, we are introducing our original protein-drug innovation systems mentioned above.

Animals↗

PubMed2004

Selective enhancer of tumor vascular permeability for optimization of cancer chemotherapy.

Clinical approach using tumor necrosis factor-alpha (TNF-alpha) as selective destruction against tumor endothelial cells and selective enhancer of tumor vascular permeability for effective accumulation of antitumor chemotherapeutic agents has attracted attention. However, the clinical application of TNF-alpha as a systemic antitumor agent has been limited because of toxic side-effects. To systemically use TNF-alpha as an antitumor agent and the selective enhancer of tumor vascular permeability, we assessed the usefulness of PEGylated TNF-alpha (PEG-TNF-alpha). PEG-TNF-alpha at a dose of 1000 JRU showed marked hemorrhagic necrosis in S-180 tumors without side-effects due to selective destruction of tumor vasculature, whereas wild-type TNF-alpha at a dose of 10,000 JRU showed a little hemorrhagic necrosis with severe side-effects. PEG-TNF-alpha induced the enhancement of tumor vascular permeability. The permeability was increased at 1 h, after an i.v. injection of PEG-TNF-alpha and returned to the basal level at 2 h. In addition, high molecular weight of PEG (molecular weight; 500K) accumulated in tumor tissue as well as low molecular weight of PEG (molecular weight; 12K). On the other hand, PEG-TNF-alpha didn't affect the permeability of normal tissue and inflammation site. This data suggested that PEG-TNF-alpha was useful agent as selective enhancer of tumor vascular permeability with safe.

Animals↗

PubMed2003

Combination effects of complement regulatory proteins and anti-complement polymer.

We previously reported the development of a "cytomedicine" that consists of cells trapped in alginate-poly-L-lysine-alginate (APA) microcapsules and agarose microbeads. The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system. However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low. Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system. We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine. Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack. These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.

Animals↗

PubMed2003

A novel cytomedical vehicle capable of protecting cells against complement.

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.

Alginates↗