Yu Ishima, Kragh-Hansen Ulrich and Otagiri Masaki : S-Nitroso Adducts of Albumin Analogs: Characterization, Categorization, and Possible Future Therapeutic Applications, Nov. 2016.
Rie Mukai, Okuyama Hitomi, Uchimura Miku, Sakao Kozue, Matsuhiro Miyu, Ikeda-Imafuku Mayumi, Yu Ishima, Nishikawa Miyu, Ikushiro Shinichi and Akihiro Tai : The binding selectivity of quercetin and its structure-related polyphenols to human serum albumin using a fluorescent dye cocktail for multiplex drug-site mapping., Bioorganic Chemistry, Vol.145, 107184, 2024.
2.
Takeru Hirai, Mayumi Ikeda-Imafuku, Nanami Tasaka, Chuang Tuan Giam Victor, Xian Ming, Tatsuhiro Ishida, Takaaki Akaike and Yu Ishima : Human hair keratin responds to oxidative stress via reactive sulfur and supersulfides, Advances in Redox Research, Vol.10, 100091, 2024.
Taro Shimizu, Lila S Abu Amr, Yuka Kitayama, Ryo Abe, Haruka Takata, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Peritoneal B Cells Play a Role in The Production of Anti-Polyethylene Glycol (PEG) IgM Against Intravenously Injected siRNA-PEGylated Liposome Complexes, Biological & Pharmaceutical Bulletin, Vol.47, No.2, 469-477, 2024.
Kengo Yasuda, Hitoshi Maeda, Ryo Kinoshita, Yuki Minayoshi, Yuki Mizuta, Yuka Nakamura, Shuhei Imoto, Koji Nishi, Keishi Yamasaki, Mina Sakuragi, Teruya Nakamura, Mayumi Ikeda-Imafuku, Yasunori Iwao, Yu Ishima, Tatsuhiro Ishida, Yasuko Iwakiri, Masaki Otagiri, Hiroshi Watanabe and Toru Maruyama : Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticle for the Treatment of Hepatitis, ACS Nano, Vol.17, No.17, 16668-16681, 2023.
(要約)
Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.
Haruka Takata, Taro Shimizu, Rina Yamade, Nehal Ali Emam Elsadek Emam Elhewan, Sherif Abdallah Emam Emam, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Anti-PEG IgM production induced by PEGylated liposomes as a function of administration route, Journal of Controlled Release, Vol.360, 285-292, 2023.
(要約)
Modifying the surface of nanoparticles with polyethylene glycol (PEG) is a commonly used approach for improving the in vitro stability of nanoparticles such as liposomes and increasing their circulation half-lives. We have demonstrated that, in certain conditions, an intravenous (i.v.) injection of PEGylated liposomes (PEG-Lip) induced anti-PEG IgM antibodies, which led to rapid clearance of second doses in mice. SARS-CoV-2 vaccines, composed of mRNA-containing PEGylated lipid nanoparticles, have been widely administered as intramuscular (i.m.) injections, so it is important to determine if PEGylated formulations can induce anti-PEG antibodies. If the favorable properties that PEGylation imparts to therapeutic nanoparticles are to be widely applicable this should apply to various routes of administration. However, there are few reports on the effect of different administration routes on the in vivo production of anti-PEG IgM. In this study, we investigated anti-PEG IgM production in mice following i.m., intraperitoneal (i.p.) and subcutaneous (s.c.) administration of PEG-Lip. PEG-Lip appeared to induce anti-PEG IgM by all the tested routes of administration, although the lipid dose causing maximum responses varied. Splenectomy attenuated the anti-PEG IgM production for all routes of administration, suggesting that splenic immune cells may have contributed to anti-PEG IgM production. Interestingly, in vitro experiments indicated that not only splenic cells but also cells in the peritoneal cavity induced anti-PEG IgM following incubation with PEG-Lip. These observations confirm previous experiments that have shown that measurable amounts of PEG-Lip administered i.p., i.m. or s.c. are absorbed to some extent into the blood circulation, where they can be distributed to the spleen and/or peritoneal cavity, and are recognized by B cells, triggering anti-PEG IgM production. The results obtained in this study have important implications for developing efficient PEGylated nanoparticular delivery system.
Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.
Mohamed Ibrahim, Taro Shimizu, Hidenori ANDO, Yu Ishima, Helmy Omar Elgarhy, A Hatem Sarhan, K Amal Hussein and Tatsuhiro Ishida : Investigation of anti-PEG antibody response to PEG-containing cosmetic products in mice, Journal of Controlled Release, Vol.354, 260-267, 2023.
Kohki Tachibana, Kohshi Kusumoto, Mai Ogawa, Hidenori ANDO, Taro Shimizu, Yu Ishima, Tatsuhiro Ishida and Keiichiro Okuhira : FTY720 reduces lipid accumulation by upregulating ABCA1 through liver X receptor and sphingosine kinase 2 signaling in macrophages, International Journal of Molecular Sciences, Vol.23, 14617, 2022.
Yu Ishima, Nio Yamazaki, V Chuang, Taro Shimizu, Hidenori ANDO and Tatsuhiro Ishida : A maleimide-terminally modified PEGylated liposome induced the accelerated blood clearance independent of the production of anti-PEG IgM antibodies, Biological & Pharmaceutical Bulletin, Vol.45, No.10, 1518-1524, 2022.
Hidenori ANDO, Ai Ikeda, Maho Tagami, Nana Matsuo, Taro Shimizu, Yu Ishima, K Eshima and Tatsuhiro Ishida : Oral administration of sodium bicarbonate can enhance the therapeutic outcome of Doxil® via neutralizing the acidic tumor microenvironment, Journal of Controlled Release, Vol.350, 414-420, 2022.
(要約)
The pH of the tumor microenvironment in solid tumors is reported to be more acidic than that of normal tissues. The pH is controlled by over-expression of several transporters that are associated with the progression, angiogenesis, and metastasis of solid tumors. Antitumor effects of weak-base anticancer agents, such as doxorubicin (DXR), could be reduced in an acidic environment because of increases in the ionized form of the drug under these conditions, reducing its membrane penetrability. In our previous studies, we demonstrated that oral administration of sodium bicarbonate (NaHCO) can neutralize the acidic tumor microenvironment and enhance the effects of small molecule anticancer drugs. However, it is not known whether or not increasing the tumor pH by oral administration of NaHCO leads to enhanced antitumor effects of lipidic nanoparticle formulations of weak-base anticancer drugs, such as Doxil®. In this study, we investigated the antitumor efficacy of Doxil® in combination with oral administration of NaHCO in a Colon26 tumor-bearing mouse model. NaHCO clearly enhanced the tumor-growth inhibitory effect of Doxil® without exacerbating any systemic side effects. In vitro studies indicated that high levels of DXR were internalized into cells at neutral pH. These studies demonstrate that the neutralization of acidic tumor microenvironment by an oral administration of NaHCO could be a promising approach to enhance the therapeutic outcomes of Doxil®.
Yoshino Kawaguchi, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Development of a nanocarrier-based splenic B cell-targeting system for loading antigens in vitro, Biological & Pharmaceutical Bulletin, Vol.45, No.7, 926-933, 2022.
Taro Shimizu, Yoshino Kawaguchi, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Development of an antigen delivery system for a B cell-targeted vaccine as an alternative to dendritic cell-targeted vaccines, Chemical & Pharmaceutical Bulletin, Vol.70, No.5, 341-350, 2022.
Marwa Sayed El, Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Elhewan Emam Elsadek Emam Ali Nehal, Emam Emam Abdallah Sherif, Eman Alaaeldin, Amal Kamal, Hatem Sarhan, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : A mouse model for studying the effect of blood anti-PEG IgMs levels on the in vivo fate of PEGylated liposomes, International Journal of Pharmaceutics, Vol.615, 121539, 2022.
(要約)
The presence of anti-polyethylene glycol (PEG) antibodies in the systemic circulation might have potential implications for the therapeutic activity of PEGylated products in vivo in the clinic. In order to study the effect of pre-existing anti-PEG antibodies on the in vivo fate and the therapeutic efficiency of PEGylated therapeutics, we developed a BALB/c mouse model by virtue of the intraperitoneal (i.p.) inoculation of hybridoma cells (HIK-M09 and HIK-M11), secreting monoclonal anti-PEG IgM, mimicking the presence of pre-existing anti-PEG antibodies in the blood. In the model, the titers of anti-PEG IgM in the blood increased as a function of hybridoma cells numbers and time after i.p. inoculation. The in vivo levels of anti-PEG IgM decreased in a dose-dependent manner, following i.v. administration of empty PEGylated liposomes. C26 tumor-bearing mice with measurable levels of anti-PEG IgM, receiving i.v. injection of DiR-labeled empty PEGylated liposomes, showed lower levels of liposomal tumor accumulation and higher levels of liver and spleen accumulation, compared to C26 tumor-bearing mice without measurable anti-PEG IgM. This specifies that the presence of anti-PEG IgM in the murine circulation induced accelerated blood clearance of PEGylated liposomes and reduced their tumor accumulation. The biodistribution and antitumor efficacy of commercially available doxorubicin (DXR)-containing PEGylated liposomes, Doxil®, were scrutinized in the anti-PEG IgM mouse model. In C26 tumor-bearing mice having circulating anti-PEG IgM, at 24 h after injection almost no DXR was observed in blood and tumor, and increased DXR accumulation was observed in spleen and liver, compared to tumor-bearing mice with no circulating anti-PEG IgM. The antitumor efficacy of Doxil® was significantly compromised in the C26 tumor-bearing mice in the presence of anti-PEG IgM. These results demonstrate that the anti-PEG IgM mouse model could be a useful prognostic indicator for the therapeutic effectiveness of different formulations of PEGylated therapeutics in pre-clinical studies.
(キーワード)
Animals / Immunoglobulin M / Liposomes / Mice / Mice, Inbred BALB C / Polyethylene Glycols / Tissue Distribution
Yoshiaki Tanaka, Hitomi Okuyama, Miyu Nishikawa, Shin-ichi Ikushiro, Mayumi Ikeda, Yu Ishima, Yuichi Ukawa, Kenichi Oe, Junji Terao and Rie Mukai : 8-Prenylnaringenin tissue distribution and pharmacokinetics in mice and its binding to human serum albumin and cellular uptake in human embryonic kidney cells., Food Science & Nutrition, Vol.10, No.4, 1070-1080, 2022.
(要約)
8-Prenylnaringenin (8-PN), a hop flavonoid, is a promising food substance with health benefits. Compared with nonprenylated naringenin, 8-PN exhibits stronger estrogenic activity and prevents muscle atrophy. Moreover, 8-PN prevents hot flushes and bone loss. Considering that prenylation reportedly improves the bioavailability of flavonoids, we compared the parameters related to the bioavailability [pharmacokinetics and tissue distribution in C57/BL6 mice, binding affinity to human serum albumin (HSA), and cellular uptake in HEK293 cells] of 8-PN and its mother (non-prenylated) compound naringenin. C57/BL6 mice were fed an 8-PN or naringenin mixed diet for 22 days. The amount of 8-PN (nmol/g tissue) in the kidneys (16.8 ± 9.20), liver (14.8 ± 2.58), muscles (3.33 ± 0.60), lungs (2.07 ± 0.68), pancreas (1.80 ± 0.38), heart (1.71 ± 0.27), spleen (1.36 ± 0.29), and brain (0.31 ± 0.09) was higher than that of naringenin. A pharmacokinetic study in mice demonstrated that the of 8-PN (50 mg/kg body weight) was lower than that of naringenin; however, the plasma concentration of 8-PN 8 h after ingestion was higher than that of naringenin. The binding affinity of 8-PN to HSA and cellular uptake in HEK293 cells were higher than those of naringenin. 8-PN bioavailability features assessed in mouse or human model experiments were obviously different from those of naringenin.
M Mostafa Mahmoud, Elhewan Emam Elsadek Emam Ali Nehal, Emam Emam Abdallah Sherif, Hidenori ANDO, Taro Shimizu, H Abdelkader, Yu Ishima, U Aly Farghaly, A H Sarhan and Tatsuhiro Ishida : Using Bio-Layer Interferometry to evaluate anti-PEG antibody-mediated complement activation, Biological & Pharmaceutical Bulletin, Vol.45, No.1, 129-135, 2022.
Rie Ando-Matsuoka, Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Noriyuki Maeda, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : I.p.-injected cationic liposomes are retained and accumulate in peritoneally disseminated tumors, Journal of Controlled Release, Vol.341, 524-532, 2022.
Hidenori ANDO, Takashi Mochizuki, Amr Abu Ali Ahmed Selim Lila, Shunsuke Akagi, Kenji Tajima, Kenji Fujita, Taro Shimizu, Yu Ishima, Tokuo Matsushima, Takatomo Kusano and Tatsuhiro Ishida : Doxorubicin embedded into nanofibrillated bacterial cellulose (NFBC) produces a promising therapeutic outcome for peritoneally metastatic gastric cancer in mice models via intraperitoneal direct injection, Nanomaterials, Vol.11, 1697, 2021.
Emam Emam Abdallah Sherif, Elhewan Emam Elsadek Emam Ali Nehal, Lila Selim Ahmed Ali Abu Amr, Haruka Takata, Yoshino Kawaguchi, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Anti-PEG IgM production and accelerated blood clearance phenomenon after the administration of PEGylated exosomes in mice, Journal of Controlled Release, Vol.334, 327-334, 2021.
(要約)
Recently, there is an increasing interest in exosomes or extracellular vesicles as potential candidates for delivering RNAs, proteins, genes, and anticancer agents. Engineering of exosome properties is rapidly evolving as a means of expanding exosome applications. PEGylation of exosomes is a technique used to improve their in vivo stability, circulation half-lives, and sometimes to allow the binding targeting ligands to the exosome exterior. According to FDA guidelines for the development of PEGylated proteins, immunological responses to PEGylated molecules and particles should be examined. In this study, we prepared PEGylated exosomes and investigated the production of anti-PEG IgM antibodies after single i.v. injections in mice. In addition, we monitored blood concentrations and tumor accumulation of a second dose of PEGylated exosomes administered after the initial dose. Single injections of PEGylated exosomes in mice induced anti-PEG IgM production in a T cell-dependent manner. The anti-PEG IgM production decreased when the injection dose of PEGylated exosomes was further increased. Anti-PEG IgM induced by injection of PEGylated exosomes decreased blood concentrations of a second dose of PEGylated exosomes and suppressed their tumor accumulation in a C26 murine colorectal cancer model. Initial injection doses of either PEGylated liposomes or PEGylated ovalbumin (PEG-OVA), both of them induced anti-PEG IgM production, also decreased the blood concentration of PEGylated exosomes. Interestingly, anti-PEG IgM induced by injection of PEGylated exosomes did not affect the blood concentration of PEG-OVA. These results imply the importance of monitoring anti-PEG IgM when repeat PEGylated exosome doses are required and/or when PEGylated exosomes are used together with other PEGylated therapeutics.
Milad Reda Qelliny, Taro Shimizu, Nehal Ali Emam Elsadek Emam Elhewan, Sherif Abdallah Emam Emam, Haruka Takata, Zeinab M. A. Fathalla, Amal K. Hussein, Khaled A. Khaled, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Incorporating gangliosides into PEGylated cationic liposomes that complexed DNA attenuates anti-PEG antibody production, but not anti-DNA antibody production in mice, Molecular Pharmaceutics, Vol.18, No.6, 2406-2415, 2021.
(要約)
Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.
(キーワード)
Animals / Antibody Formation / Cations / Clodronic Acid / DNA / Gangliosides / Gene Transfer Techniques / Genetic Therapy / Immunoglobulin M / Liposomes / Male / Mice / Phagocytes / Plasmids / Polyethylene Glycols
Haruka Takata, Taro Shimizu, Yoshino Kawaguchi, Hiro Ueda, Nehal Ali Emam Elsadek Emam Elhewan, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Nucleic acids delivered by PEGylated cationic liposomes in systemic lupus erythematosus-prone mice: a possible exacerbation of lupus nephritis in the presence of pre-existing anti-nucleic acid antibodies, International Journal of Pharmaceutics, Vol.601, 120529, 2021.
Taro Shimizu, Mizuki Awata, Lila Selim Ahmed Ali Abu Amr, Chihiro Yoshioka, Yoshino Kawaguchi, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Complement activation induced by PEG enhances humoral immune responses against antigens encapsulated in PEG-modified liposomes, Journal of Controlled Release, Vol.329, 1046-1053, 2021.
Kenji Tsukigawa, Shuhei Imoto, Keishi Yamasaki, Koji Nishi, Toshihiko Tsutsumi, Shoko Yokoyama, Yu Ishima and Masaki Otagiri : Synthesis and In Vitro Assessment of pH-Sensitive Human Serum Albumin Conjugates of Pirarubicin, Pharmaceuticals, Vol.14, No.1, 22, 2020.
Takuma Takayama, Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Yuki Kanazawa, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Adjuvant antitumor immunity contributes to the overall antitumor effect of PEGylated liposomal doxorubicin (Doxil®) in C26 tumor-bearing immunocompetent mice, Pharmaceutics, Vol.12, No.10, 990, 2020.
Elhewan Emam Elsadek Emam Ali Nehal, Emam Emam Abdallah Sherif, Lila Selim Ahmed Ali Abu Amr, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Pegfilgrastim (PEG-G-CSF) induces anti-polyethylene glycol (PEG) IgM via a T cell-dependent mechanism, Biological & Pharmaceutical Bulletin, Vol.43, No.9, 1393-1397, 2020.
(要約)
Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.
Elhewan Emam Elsadek Emam Ali Nehal, Eri Hondoh, Taro Shimizu, Haruka Takata, Lila Selim Ahmed Ali Abu Amr, Emam Emam Abdallah Sherif, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Impact of pre-existing or induced anti-PEG IgM on the pharmacokinetics of peginterferon alfa-2a (Pegasys®) in mice, Molecular Pharmaceutics, Vol.17, No.8, 2964-2970, 2020.
(要約)
PEGylation had been used successfully to improve the circulation half-lives and some physicochemical properties of protein therapeutics. However, anti-polyethylene glycol (anti-PEG) antibodies, either pre-existing or treatment-induced, can negatively affect the pharmacokinetics and pharmacological efficacy of PEGylated proteins. We have examined anti-PEG immune responses in mice for peginterferon alfa-2a (Pegasys), a clinically approved PEGylated protein therapeutic, at both the recommended dose (equivalent to 3 μg/kg in mice) and at higher doses (150 μg/kg) for single or repeated subcutaneous (s.c.) administrations. The effect of treatment-induced anti-PEG IgM on serum concentrations of Pegasys, following repeated administrations, was evaluated. In addition, the effect of pre-existing anti-PEG IgM elicited by a different PEGylated protein, PEG-OVA, on the systemic clearance of Pegasys, was investigated. At a s.c. dose of 3 μg/kg, single injections of Pegasys barely elicited anti-PEG immune responses. Four repeated doses of 150 μg/kg Pegasys elicited anti-PEG IgM production, depending on dose frequency, and triggered the rapid clearance of subsequent doses. In addition, anti-PEG-IgM produced in response to prior administration of PEG-OVA caused a rapid blood clearance of Pegasys. Our results, therefore, underscore the importance of screening for both pre-existing and treatment-induced anti-PEG antibodies in patients prior to and during treatment with PEGylated protein drugs.
M Sayed M El, Haruka Takata, Taro Shimizu, Yoshino Kawaguchi, Lila Selim Ahmed Ali Abu Amr, Elhewan Emam Elsadek Emam Ali Nehal, E Alaaeldin, Yu Ishima, Hidenori ANDO, A Kamal, A H Sarhan and Tatsuhiro Ishida : Hepatosplenic phagocytic cells indirectly contribute to anti-PEG IgM production in the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes: Appearance of an unexplained mechanism in the ABC phenomenon, Journal of Controlled Release, Vol.323, 102-109, 2020.
(要約)
The accelerated blood clearance (ABC) phenomenon, caused in large degree via in vivo anti-PEG IgM production, is one of obstacles for development of PEGylated liposome and protein formulations, due to decreased efficiency and/or side effects such as anaphylaxis upon repeat administrations. We have shown in murine ABC models that splenectomy suppressed the level of anti-PEG IgM production induced by PEGylated liposomes, indicating that murine splenic B cells play an important role in its production. However, splenectomy did not completely inhibit production of anti-PEG IgM, suggesting that other cells may contribute to its production in the ABC phenomenon. In this study, we examined the contribution of hepatosplenic phagocytic cells to anti-PEG IgM production and clearance of PEGylated liposomes during the ABC phenomenon. Depletion of hepatosplenic phagocytic cells by pretreatment of mice with clodronate-containing non-PEGylated liposomes suppressed anti-PEG IgM production to a considerable degree, without a change in the number of splenic B cells, and attenuated the enhanced clearance of second dose of PEGylated liposomes. These results suggest that hepatosplenic phagocytic cells, in addition to splenic B cells, contribute to the production of anti-PEG IgM and the ABC phenomenon against PEGylated liposomes. The mechanism whereby splenic B cells interact with hepatosplenic phagocytic cells to produce anti-PEG IgM, upon administration of an initial dose of PEGylated liposomes remains to be elucidated.
Ryota Tanaka, Tadashi Imafuku, Yosuke Suzuki, Kento Nishida, Kotaro Matsusaka, Toshitaka Shin, Yuhki Sato, Yu Ishima, Hiroshi Watanabe, Hiromitsu Mimata, Toru Maruyama and Hiroki Itoh : Changes in redox state of albumin before and after kidney transplantation in patients with end-stage renal disease, Clinical Biochemistry, Vol.81, 20-26, 2020.
(要約)
Cardiovascular disease is one of the major causes of death in patients with end-stage kidney disease who have undergone kidney transplantation. Since the complication of cardiovascular disease in patients with chronic kidney disease is strongly linked to oxidative stress, understanding the oxidative stress condition after kidney transplantation would be of great importance for the prevention of cardiovascular disease. This study examined whether improvement of renal function after kidney transplantation has an impact on the redox state of the Cys34 residue of albumin that reflects the level of oxidative stress in blood. We enrolled 23 patients with end-stage renal failure who received kidney transplantation. All patients were followed for 180 days after transplantation. The fractions of albumin isoforms were determined by the electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) method. Serum creatinine decreased significantly immediately after kidney transplantation, suggesting successful transplantations. The ESI-TOFMS method identified three albumin isoforms cysteinylated at the Cys34 residue (Cys-Cys34-albumin) and the three corresponding albumin isoforms without Cys34 cysteinylation. The fraction of total Cys-Cys34-albumin decreased transiently after kidney transplantation, and was followed by an elevation at day 7 and gradual decrease thereafter until day 180. Meanwhile, reduced albumin concentration did not change until day 14 after kidney transplantation, then showed a significant increase compared to pre-transplant level at day 30 and remained stably elevated until day 180. Actual reduced albumin levels were found to exceed pre-transplant levels on or after day 30 following kidney transplantation unlike immediate restoration of renal function. Renal function was recovered immediately following kidney transplantation, but reduced albumen concentration increased above the pre-transplant levels only from day 30 after transplantation.
Nehal Ali Emam Elsadek Emam Elhewan, Amr Abu Ali Ahmed Selim Lila, Sherif Abdallah Emam Emam, Taro Shimizu, Haruka Takata, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM in a dose dependent manner and causes the accelerated blood clearance (ABC) phenomenon upon repeated administration in mice, European Journal of Pharmaceutics and Biopharmaceutics, Vol.152, 56-62, 2020.
(要約)
Pegfilgrastimis a recombinant PEGylated human granulocyte colony-stimulating factor (G-CSF) analog filgrastim (trade names Neulasta® or G-Lasta®) that stimulates the production of white blood cells (neutrophils). It is employed as an alternative to filgrastim (G-CSF) for chemotherapy-induced neutropenia in patients due to its longer half-life. In clinical settings, PEG-G-CSF is administered to cancer patients via both the s.c. and i.v. routes. In a murine study, we showed that, regardless of administration route, initial doses of PEG-G-CSF above 0.06 mg/kg elicited anti-PEG immune response in a dose-dependent manner. I.v. administration elicited higher levels of anti-PEG IgM than the s.c. route. Initial doses of PEG-G-CSF (6 mg/kg) that were high enough to trigger production of anti-PEG IgM, did not trigger the accelerated clearance of a lower subsequent dose (0.06 mg/kg) that was similar to i.v. clinical doses of PEG-G-CSF, but when the subsequent dose of PEG-G-CSF was raised to (6 mg/kg), the initial dose triggered the accelerated clearance of the second dose via an anti-PEG IgM-mediated complement activation. Similar observations were noted when an increased PEG-OVA dose was given as the second dose, indicating that pre-existing and/or treatment-induced anti-PEG antibodies might compromise the therapeutic activity and/or reduce tolerance of other PEGylated formulations. To the best of our knowledge, this is the first report to suggest the induction of the ABC phenomenon upon repeated injections of pegfilgrastim. In the clinic, cancer patients, receiving multiple cycles of chemotherapy, receive multiple cycles of pegfilgrastim to avoid infections and substantial morbidity. The ABC phenomenon to pegfilgrastim appears to be the cause of loss of clinical benefit of sequential treatments with pegfilgrastim in patients.
Kentaro Oniki, Hirofumi Nohara, Ryunosuke Nakashima, Yui Obata, Narumi Muto, Yuki Sakamoto, Keiko Ueno-Shuto, Tadashi Imafuku, Yu Ishima, Hiroshi Watanabe, Toru Maruyama, Koji Otake, Yasuhiro Ogata, Mary Ann Suico, Hirofumi Kai, Tsuyoshi Shuto and Junji Saruwatari : The DsbA-L gene is associated with respiratory function of the elderly via its adiponectin multimeric or antioxidant properties, Scientific Reports, Vol.10, 5973, 2020.
Hidenori ANDO, Noriko Saito-Tarashima, Lila Selim Ahmed Ali Abu Amr, Nozomi Kinjoh, Taro Shimizu, Yu Ishima, Noriaki Minakawa and Tatsuhiro Ishida : A unique gene-silencing approach, using an intelligent RNA expression device (iRed), results in minimal immune stimulation when given by local intrapleural injection in malignant pleural mesothelioma, Molecules, Vol.25, No.7, 1725, 2020.
(要約)
We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers.
Yu Ishima, Ai Minomo, Chuang Tuan Giam Victor, Tetsuya Fukuda, Kohshi Kusumoto, Keiichiro Okuhira, Yoshiaki Suwa, Hiroshi Watanabe, Tatsuhiro Ishida, Hiroshi Morioka, Toru Maruyama and Masaki Otagiri : Albumin domain mutants with enhanced Aβ binding capacity identified by phage display analysis for application in various peripheral Aβ elimination approaches of Alzheimer's disease treatment, IUBMB life, Vol.72, No.4, 641-651, 2020.
(要約)
Deposition of amyloid protein, particularly Aβ , is a major contributor to the onset of Alzheimer's disease (AD). However, almost no deposition of Aβ in the peripheral tissues could be found. Human serum albumin (HSA), the most abundant protein in the blood, has been reported to inhibit amyloid formation through binding Aβ, which is believed to play an important role in the peripheral clearance of Aβ. We identified the Aβ binding site on HSA and developed HSA mutants with high binding capacities for Aβ using a phage display method. HSA fragment 187-385 (Domain II) was found to exhibit the highest binding capacity for Aβ compared with the other two HSA fragments. To elucidate the sequence that forms the binding site for Aβ on Domain II, a random screening of Domain II display phage biopanning was constructed. A number of mutants with higher Aβ binding capacities than the wild type were identified. These mutants exhibited stronger scavenging abilities than the wild type, as revealed via in vitro equilibrium dialysis of Aβ experiments. These findings provide useful basic data for developing a safer alternative therapy than Aβ vaccines and for application in plasma exchange as well as extracorporeal dialysis.
Kazuaki Taguchi, Shigeru Ogaki, Taisei Nagasaki, Hiroki Yanagisawa, Kento Nishida, Hitoshi Maeda, Yuki Enoki, Kazuaki Matsumoto, Hidehisa Sekijima, Kazuya Ooi, Yu Ishima, Hiroshi Watanabe, Masafumi Fukagawa, Masaki Otagiri and Toru Maruyama : Carbon Monoxide Rescues the Developmental Lethality of Experimental Rat Models of Rhabdomyolysis-Induced Acute Kidney Injury, The Journal of Pharmacology and Experimental Therapeutics, Vol.372, No.3, 355-365, 2020.
(要約)
Many victims, after being extricated from a collapsed building as the result of a disaster, suffer from disaster nephrology, a term that is referred to as the crush syndrome (CS). Recommended treatments, which include dialysis or the continuous administration of massive amounts of fluid are not usually easy in cases of such mass natural disasters. In the present study, we examined the therapeutic performance of a biomimetic carbon monoxide (CO) delivery system, CO-enriched red blood cells (CO-RBCs), on experimental animal models of an acute kidney injury (AKI) induced by traumatic and nontraumatic rhabdomyolysis, including CS and rhabdomyolysis with massive hemorrhage shock. A single CO-RBC treatment was found to effectively suppress the pathogenesis of AKI with the mortality in these model rats being improved. In addition, in further studies using glycerol-induced rhabdomyolysis model rats, the pathogenesis of which is similar to that for the CS, AKI and mortality were also reduced as the result of a CO-RBC treatment. Furthermore, CO-RBCs were found to have renoprotective effects via the suppression of subsequent heme protein-associated renal oxidative injury; the oxidation of myoglobin in the kidneys, the generation of reactive oxygen species by free heme produced from degraded-cytochrome P450 and hemoglobin-associated renal injury. Because CO-RBCs can be prepared and used at both hospitals and at a disaster site, these findings suggest that CO-RBCs have the potential for use as a novel cell therapy against both nontraumatic and traumatic rhabdomyolysis including CS-induced AKI. SIGNIFICANCE STATEMENT: After mass natural and man-made disasters, people who are trapped in collapsed buildings are in danger of acute kidney injury (AKI), including crush syndrome (CS)-related AKI. This paper reports that carbon monoxide-enriched red blood cells (CO-RBCs), which can be prepared at both hospitals and disaster sites, dramatically suppressed the pathogenesis of CS-related AKI, thus improving mortality via suppressing heme protein-associated renal injuries. CO-RBCs have the potential for serving as a practical therapeutic agent against disaster nephrology associated with the CS.
Emam Emam Abdallah Sherif, Lila Selim Ahmed Ali Abu Amr, Elhewan Emam Elsadek Emam Ali Nehal, Hidenori ANDO, Taro Shimizu, Keiichiro Okuhira, Yu Ishima, M Mahdy, E Ghazy and Tatsuhiro Ishida : Cancer cell-type tropism is one of crucial determinants for the efficient systemic delivery of cancer cell-derived exosomes to tumor tissues, European Journal of Pharmaceutics and Biopharmaceutics, Vol.145, 27-34, 2019.
(要約)
Exosomes are gaining increasing attention as drug delivery vehicles due to their low toxicity and ability to functionally transfer biological cargos between cells. However, the therapeutic applicability of exosomes is partially hampered by a lack of cell-type specificity. In this study, therefore, we investigated the impact of cell-type tropism on the in vivo systemic delivery of exosomes to tumor tissues. Exosomes derived from murine colorectal cancer cells (C26) (C26-Exos) and murine melanoma cells (B16BL6) (B16BL6-Exos) were collected. In vitro cellular uptake of either autologous (C26) or allogeneic (B16BL6) exosomes by C26 tumor cells was determined. In vivo tumor accumulation of each type of exosomes in mice bearing C26 tumors was monitored with an in vivo imaging system (IVIS). In in vitro studies, autologous C26-Exos were more efficiently taken up by C26 cancer cells, compared to allogeneic B16BL6-Exos. For in vivo studies, exosomes were modified with surface polyethylene glycol (PEG) to improve their circulation lifetimes. Although both types of PEGylated exosomes accumulated in C26-tumor tissue, autologous exosomes were preferentially accumulated within C26-tumor tissue compared to allogeneic exosomes. The increased tumor accumulation of autologous PEGylated exosomes was accompanied by the preferential uptake of exosomes by not only C26-tumor cells but also tumor-associated immune cells. This study implies that cancer cell-type tropism is an important factor in the achievement of tumor cell targeting with cancer cell-derived exosomes.
(キーワード)
Animals / Cell Line, Tumor / Colorectal Neoplasms / Drug Delivery Systems / Exosomes / Male / Melanoma / Mice / Mice, Inbred BALB C / Polyethylene Glycols / Tropism
Hidenori ANDO, M Fukushima, K Eshima, Taichi Hasui, Taro Shimizu, Yu Ishima, C Huang, H Wada and Tatsuhiro Ishida : A novel intraperitoneal therapy for gastric cancer with DFP-10825, a unique RNAi therapeutic targeting thymidylate synthase, in peritoneally disseminated xenograft model, Cancer Medicine, Vol.8, No.17, 7313-7321, 2019.
(要約)
In advanced gastric cancer, peritoneal dissemination is a life-threatening mode of metastasis. Since the treatment options with conventional chemotherapy remain limited, any novel therapeutic strategy that could control such metastasis would improve the outcome of treatment. We recently developed a unique RNA interference therapeutic regimen (DFP-10825) consisting of short hairpin RNA against thymidylate synthase (TS shRNA) and cationic liposomes. The treatment with DFP-10825 has shown remarkable antitumor activity in peritoneally disseminated human ovarian cancer-bearing mice via intraperitoneal administration. In this study, we expanded DFP-10825 to the treatment of peritoneally disseminated gastric cancer. DFP-10825 was administered intraperitoneally into mice with intraperitoneally implanted human gastric cancer cells (MKN45 or NCI-N87). Antitumor activity and host survival benefits were monitored. Intraperitoneal distribution of fluorescence-labeled DFP-10825 was monitored in this MKN45 peritoneally disseminated mouse model. Intraperitoneal injection of DFP-10825 suppressed tumor growth in two peritoneally disseminated cancer models (MKN45 and NCI-N87) and increased the survival time of the MKN45 model without severe side effects. Throughout the treatment regimen, no significant body weight loss was associated with the administration of DFP-10825. Interestingly, after intraperitoneal injection, fluorescence-labeled DFP-10825 retained for more than 72 hours in the peritoneal cavity and selectively accumulated in disseminated tumors. Intraperitoneal injection of DFP-10825 demonstrated effective antitumor activity without systemic severe adverse effects via the selective delivery of RNAi molecules into disseminated tumors in the peritoneal cavity. Our current study indicates that DFP-10825 could become an alternative option to improve the outcomes of patients with peritoneally disseminated gastric cancer.
Kotaro Matsusaka, Yu Ishima, Hitoshi Maeda, Ryo Kinoshita, Shota Ichimizu, Kazuaki Taguchi, Victor Chuang Tuan Giam, Koji Nishi, Keishi Yamasaki, Masaki Otagiri, Hiroshi Watanabe and Toru Maruyama : α1-Acid Glycoprotein Has the Potential to Serve as a Biomimetic Drug Delivery Carrier for Anticancer Agents, Journal of Pharmaceutical Sciences, Vol.108, No.11, 3592-3598, 2019.
(要約)
Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an α-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents.
S Ichimizu, H Watanabe, H Maeda, K Hamasaki, K Ikegami, V Chuang, Ryo Kinoshita, K Nishida, Taro Shimizu, Yu Ishima, Tatsuhiro Ishida, T Seki, H Katsuki, S Futaki, M Otagiri and T Maruyama : Cell-penetrating mechanism of intracellular targeting albumin: Contribution of macropinocytosis induction and endosomal escape, Journal of Controlled Release, Vol.304, 156-163, 2019.
(要約)
We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg). While it is possible that the palmitoyl-cyclic-(D-Arg)/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.
Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, M Fukushima, Rie Matsuoka, Taro Shimizu, Keiichiro Okuhira, Yu Ishima, C Huang, H Wada and Tatsuhiro Ishida : A simplified method for manufacturing RNAi therapeutics for local administration, International Journal of Pharmaceutics, Vol.564, 256-262, 2019.
(要約)
RNA interference (RNAi) is one of the most promising strategies for cancer therapeutics. The successful translation of RNAi therapeutics to a clinic setting requires a delivery system that is efficient and simple to upscale. In this study, we devised a simple industrial method to manufacture lipoplex, which includes short hairpin RNA against the expression of thymidylate synthase (TS shRNA) - a key molecule for DNA biosynthesis. An aqueous solution of TS shRNA was gently mixed with either a precursor of cationic liposome (Presome DF-1) or a cationic lipid mixture in an o/w emulsion. This solution was subsequently lyophilized under optimal conditions to obtain either FD-lipoplex-1 or FD-lipoplex-2, respectively. With this method, a lipoplex in activated form was obtained via a simple "one-step" hydration with saline. Both forms of FD-lipoplex showed physicochemical properties comparable to those of conventional lipoplex. FD-lipoplexes stably retained TS shRNA within their formulations in the presence of tumor ascites fluid. Intraperitoneal treatment with either FD-lipoplex-1 or FD-lipoplex-2 provided a therapeutic level of efficacy comparable to that of conventional lipoplex in the treatment of a peritoneal disseminated gastric cancer mouse model. Collectively, established freeze-drying-based methods for RNAi-therapeutic preparation could realistically be used in a clinical setting for the treatment of patients with peritoneal disseminated cancer.
Yusuke Doi, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Long-term storage of PEGylated liposomal oxaliplatin with improved stability and long circulation times in vivo, International Journal of Pharmaceutics, Vol.564, 237-243, 2019.
(要約)
Liposomal anticancer drugs have been developed with improved clinical effects and reduced side effects. We have developed a PEGylated liposomal formulation of oxaliplatin that has anticancer effects in animal models of colorectal cancer with a favorable toxicity profile. To move this formulation into clinical development, a formulation that is stable during long term storage is needed, which has similar pharmacokinetics and therapeutic activity against solid tumors to the original formulation. In this study, we found that PEGylated liposomal oxaliplatin showed no changes in particle size after long term storage (12 months at 2-8 °C), but phospholipid degradation had occurred. Hence, the stored formulation had compromised membrane integrity, resulting in decreased in vivo circulation times of the liposomes. To improve the stability during long-term storage, a screening study to obtain an appropriate stabilizer was carried out. The buffer 2-morpholinoethansulfonic acid (MES) attenuated not only phospholipid degradation but also oxaliplatin degradation, unlike most other excipients. After 12 months storage at 2-8 °C in the presence of MES only slight degradation of phospholipids in PEGylated liposomal oxaliplatin occurred, resulting in similar in vivo pharmacokinetic profiles of the encapsulated oxaliplatin to the original formulation. Long term stability of PEGylated liposomal oxaliplatin was achieved by addition of MES, resulting in long circulation half-lives in vivo, a property which would be expected to lead to increased suppression of tumor growth and reduced side effects. Our formulation may now be suitable for clinical development.
Zinc (Zn) plays an important role in many organisms in various physiological functions such as cell division, immune mechanisms and protein synthesis. However, excessive Zn release is induced in pathological situations and causes neuronal cell death. Previously, we reported that Cu ions (Cu2+) markedly exacerbates Zn2+-induced neuronal cell death by potentiating oxidative stress and the endoplasmic reticulum stress response. In contrast, the stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK) signaling pathway is important in neuronal cell death. Thus, in this study, we focused on the SAPK/JNK signaling pathway and examined its involvement in Cu2+/Zn2+-induced neurotoxicity. Initially, we examined expression of factors involved in the SAPK/JNK signaling pathway. Accordingly, we found that phosphorylated (ie, active) forms of SAPK/JNK (p46 and p54) are increased by CuCl2 and ZnCl2 co-treatment in hypothalamic neuronal mouse cells (GT1-7 cells). Downstream factors of SAPK/JNK, phospho-c-Jun, and phospho-activating transcription factor 2 are also induced by CuCl2 and ZnCl2 co-treatment. Moreover, an inhibitor of the SAPK/JNK signaling pathway, SP600125, significantly suppressed neuronal cell death and activation of the SAPK/JNK signaling pathway induced by CuCl2 and ZnCl2 cotreatment. Finally, we examined involvement of oxidative stress in activation of the SAPK/JNK signaling pathway, and found that human serum albumin-thioredoxin fusion protein, an antioxidative protein, suppresses activation of the SAPK/JNK signaling pathway. On the basis of these results, our findings suggest that activation of ZnCl2-dependent SAPK/JNK signaling pathway is important in neuronal cell death, and CuCl2-induced oxidative stress triggers the activation of this pathway.
Mayumi Ikeda, Yu Ishima, VTG Chuang, Maki Sakai, Hiroki Osafune, Hidenori ANDO, Taro Shimizu, Keiichiro Okuhira, H Watanabe, T Maruyama, M Otagiri, T Akaike and Tatsuhiro Ishida : Distribution of Polysulfide in Human Biological Fluids and Their. Association with Amylase and Sperm Activities, Molecules, Vol.24, No.9, 1689, 2019.
(要約)
Intracellular polysulfide could regulate the redox balance via its anti-oxidant activity. However, the existence of polysulfide in biological fluids still remains unknown. Recently, we developed a quantitative analytical method for polysulfide and discovered that polysulfide exists in plasma and responds to oxidative stress. In this study, we confirmed the presence of polysulfide in other biological fluids, such as semen and nasal discharge. The levels of polysulfide in these biological fluids from healthy volunteers ( = 9) with identical characteristics were compared. Additionally, the circadian rhythm of plasma polysulfide was also investigated. The polysulfide levels detected from nasal discharge and seminal fluid were approximately 400 and 600 μM, respectively. No correlation could be found between plasma polysulfide and the polysulfide levels of tear, saliva, and nasal discharge. On the other hand, seminal polysulfide was positively correlated with plasma polysulfide, and almost all polysulfide contained in semen was found in seminal fluid. Intriguingly, saliva and seminal polysulfide strongly correlated with salivary amylase and sperm activities, respectively. These results provide a foundation for scientific breakthroughs in various research areas like infertility and the digestive system process.
Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Mizuki Awata, Yukiyo Kubo, Yu Mima, Yosuke Hashimoto, Hidenori ANDO, Keiichiro Okuhira, Yu Ishima and Tatsuhiro Ishida : A cell assay for detecting anti-PEG immune response against PEG-modified therapeutics, Pharmaceutical Research, Vol.35, No.11, 223, 2018.
(要約)
Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry. The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics. The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA. PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.
Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Yoshino Kawaguchi, Yuna Shimazaki, Yu Mima, Yosuke Hashimoto, Keiichiro Okuhira, G Storm, Yu Ishima and Tatsuhiro Ishida : A novel platform for cancer vaccines: Antigen-selective delivery to splenic marginal zone B cells via repeated injections of PEGylated liposomes, The Journal of Immunology, Vol.201, No.10, 2969-2976, 2018.
(要約)
Treating cancer with vaccines has been a challenge. In this study, we introduce a novel Ag delivery platform for cancer vaccines that delivers an encapsulated Ag to splenic marginal zone B (MZ-B) cells via the aid of a PEGylated liposome (PL) system. Splenic MZ-B cells have recently attracted interest as alternative APCs. In mice, preimmunization with empty (no Ag encapsulation) PLs triggered the efficient delivery of a subsequent dose of Ag-containing PLs, injected 3 d later, to the spleen compared with a single dose of Ag-containing PLs. In addition, immunization with empty PLs allowed three subsequent sequential injections of OVA-PLs to efficiently induce a CTL response against OVA-expressing murine thymoma (EG7-OVA) cells and resulted in in vivo growth inhibition of subsequently inoculated EG7-OVA cells. However, these sequential treatments require repeated immunizations to achieve their antitumor effect. Therefore, to improve the antitumor effect of our novel vaccine system, an adjuvant, -galactosylceramide (GC), was incorporated into the OVA-PLs (GC/OVA-PLs). As expected, the incorporation of GC reduced the required number of immunizations with OVA-PLs to the point that a single immunization treatment with empty PLs and an injection of GC/OVA-PL efficiently triggered a potent CTL induction, resulting in a rejection of the development and a suppression of the growth of tumors that had already developed s.c. Results of this study indicate that a novel Ag delivery platform that grants efficient Ag delivery to splenic MZ-B cells shows promise as a therapeutic modality for conquering tumor growth and/or progression.
Emam Emam Abdallah Sherif, Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Taro Shimizu, Keiichiro Okuhira, Yu Ishima, M Mahdy, F Ghazy, I Sagawa and Tatsuhiro Ishida : Liposome co-incubation with cancer cells secreted exosomes (extracellular vesicles) with different proteins expressions and different uptake pathways, Scientific Reports, Vol.8, No.1, 14493, 2018.
(要約)
We recently showed that in vitro incubation of cells with liposomes of varying compositions can increase exosome secretion and increase the yield of harvested exosomes (extracellular vesicles, EVs). This might foster their potential therapeutic implementations. In the current study, we investigated the surface proteins and the uptake of the harvested exosomes (EVs) to see if the incubation of cells with liposomes would change the biological properties of these exosomes (EVs). Interestingly, exosomes (EVs) induced by solid cationic liposomes lacked some major exosome marker proteins such as CD9, flotillin-1, annexin-A2 and EGF, and subsequently had lower levels of cellular uptake upon re-incubation with donor cancer cells. However, exosomes (EVs) induced under normal condition and by fluid cationic liposomes, displayed the entire spectrum of proteins, and exhibited higher uptake by the donor cancer cells. Although endocytosis was the major uptake pathway of exosomes (EVs) by tumor cells, endocytosis could occur via more than one mechanism. Higher exosome uptake was observed in donor B16BL6 cells than in allogeneic C26 cells, indicating that donor cells might interact specifically with their exosomes (EVs) and avidly internalize them. Taken together, these results suggest a technique for controlling the characteristics of secreted exosomes (EVs) by incubating donor cancer cells with liposomes of varying physiochemical properties.
Emam Emam Abdallah Sherif, Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Shinya Kobayashi, Taro Shimizu, Keiichiro Okuhira, Yu Ishima and Tatsuhiro Ishida : Doxorubicin expands in vivo secretion of circulating exosome in mice, Biological & Pharmaceutical Bulletin, Vol.41, No.7, 1078-1083, 2018.
(要約)
Modulation of tumor immunity is a known factor in the antitumor activity of many chemotherapeutic agents. Exosomes are extracellular nanometric vesicles that are released by almost all types of cells, which includes cancer cells. These vesicles play a crucial role in tumor immunity. Many in vitro studies have reproduced the aggressive secretion of exosomes following treatment with conventional anticancer drugs. Nevertheless, how chemotherapeutic agents including nanomedicines such as Doxil affect the in vivo secretion of exosomes is yet to be elucidated. In this study, the effect of intravenous injection of either free doxorubicin (DXR) or liposomal DXR formulation (Doxil) on exosome secretion was evaluated in BALB/c mice. Exosomes were isolated from serum by using an ExoQuick™ kit. Free DXR treatment markedly increased serum exosome levels in a post-injection time-dependent manner, while Doxil treatment did not. Exosomal size distribution and marker protein expressions (CD9, CD63, and TSG101) were studied. The physical/biological characteristics of treatment-induced exosomes were comparable to those of control mice. Interestingly, splenectomy significantly suppressed the copious exosomal secretions induced by free DXR. Collectively, our results indicate that conventional anticancer agents induce the secretion of circulating exosomes, presumably via stimulating immune cells of the spleen. As far as we know, this study represents the first report indicating that conventional chemotherapeutics may induce exosome secretion which might, in turn, contribute partly to the antitumor effect of chemotherapeutic agents.
K Oniki, T Watanabe, M Kudo, T Izuka, T Ono, K Matsuda, Y Sakamoto, K Nagaoka, T Imafuku, Yu Ishima, H Watanabe, T Maruyama, K Otake, Y Ogata and J Saruwatari : Modeling of the Weight Status and Risk of Nonalcoholic Fatty Liver Disease in Elderly Individuals: The Potential Impact of the Disulfide Bond-Forming Oxidoreductase A-Like Protein (DsbA-L) Polymorphism on the Weight Status, CPT: Pharmacometrics & Systems Pharmacology, Vol.7, No.6, 384-393, 2018.
(要約)
Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity. Disulfide bond-forming oxidoreductase A-like protein (DsbA-L) is known to be a key molecule in protection against obesity and obesity-induced inflammation. In the present study, we used a modeling and simulation approach in an attempt to develop body mass index (BMI) and BMI-based NAFLD prediction models incorporating the DsbA-L polymorphism to predict the BMI and NAFLD in 341 elderly subjects. A nonlinear mixed-effect model best represented the sigmoidal relationship between the BMI and the logit function of the probability of NAFLD prevalence. The final models for BMI and NAFLD showed that DsbA-L rs1917760 polymorphism, age, and gender were associated with the BMI, whereas gender, patatin-like phospholipase 3 rs738409 polymorphism, HbA1c, and high-density and low-density lipoprotein cholesterol levels were associated with the risk of NAFLD. This information may aid in the genetic-based prevention of obesity and NAFLD in the general elderly population.
Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Risako Fujita, Mizuki Awata, Munehira Kawanishi, Yosuke Hashimoto, Keiichiro Okuhira, Yu Ishima and Tatsuhiro Ishida : A hydroxyl PEG version of PEGylated liposomes and its impact on anti-PEG IgM induction and on the accelerated clearance of PEGylated liposomes, European Journal of Pharmaceutics and Biopharmaceutics, Vol.127, 142-149, 2018.
(要約)
Surface decoration of liposomes with polyethylene glycol (PEG), PEGylation, is recognized as a method to bestow liposomes with a prolonged circulation time following intravenous administration. However, many reports have emphasized that a first dose of PEGylated liposomes (PL) elicits an anti-PEG IgM antibody response that can trigger a rapid systemic clearance of a second dose of PL via a phenomenon that is referred to as "accelerated blood clearance (ABC)." Such a phenomenon is usually observed with PL that has been modified with methoxy-PEG. In the current study, we introduced various functional groups, methoxy (OCH), amino (NH), carboxyl (COOH), and hydroxyl (OH), at the chain ends of PEG to investigate the effect on anti-PEG IgM induction. Among different PEG-modified liposomes, hydroxyl PEG-modified liposomes (PL-OH) efficiently attenuated the anti-PEG IgM response in vitro. In addition, PL-OH was less recognizable by anti-PEG IgM compared with other PLs. These findings raised the possibility that PL-OH could attenuate/abrogate elicitation of the ABC phenomenon. Nonetheless, upon repeated intravenous injection, PL-OH triggered the enhanced clearance of a subsequently injected second dose. Furthermore, in vitro studies have demonstrated that, as a complement activator, PL-OH is stronger than PL-OCH and induces further complement activation in the presence of anti-PEG IgM, which was the predominant contributor to the rapid clearance of a second dose of PL-OH. Our results suggest that the screening of complement activation by polymer-modified products in tandem with anti-polymer antibody production should be a prerequisite in the development of polymers that might enhance the therapeutic efficacy of nanocarriers.
S Ichimizu, H Watanabe, H Maeda, K Hamasaki, Y Nakamura, VTG Chuang, R Kinoshita, K Nishida, R Tanaka, Y Enoki, Yu Ishima, A Kuniyasu, Y Kobashigawa, H Morioka, S Futaki, M Otagiri and T Maruyama : Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery, Journal of Controlled Release, Vol.277, 23-34, 2018.
(要約)
Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg) was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.
(キーワード)
Cell Membrane Permeability / Cell-Penetrating Peptides / Dose-Response Relationship, Drug / Drug Delivery Systems / Drug Design / HeLa Cells / Humans / Intracellular Fluid / Nanoparticles / Serum Albumin, Human / Structure-Activity Relationship
Emam Emam Abdallah Sherif, Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Taro Shimizu, Masami Ukawa, Keiichiro Okuhira, Yu Ishima, A M Mahdy, S F Ghazy and Tatsuhiro Ishida : A novel strategy to increase the yield of exosomes (extracellular vesicles) for an expansion of basic research, Biological & Pharmaceutical Bulletin, Vol.41, No.5, 733-742, 2018.
(要約)
Exosomes are tiny extracellular vesicles that are usually harvested in small quantities. Such small yield has been an obstacle for the expansion of the basic research regarding exosome analysis and applications in drug delivery. To increase exosome yield, we attempted to stimulate tumor cells via the addition of liposomes in vitro. Neutral, cationic-bare or PEGylated liposomes were incubated with four different tumor cell lines. The stimulatory effect of liposomal formulations on exosome secretion and cellular uptake propensity of the collected exosome by mother cells or different cells was evaluated. Both neutral and cationic-bare liposomes enhanced exosome secretion in a dose-dependent manner. Fluid cationic liposomes provided the strongest stimulation. Surprisingly, the PEGylation of bare liposomes diminished exosome secretion. Exosomes harvested in the presence of fluid cationic liposomes showed increased cellular uptake, but solid cationic liposomes did not. Our findings indicate that the physicochemical properties of liposomes determine whether they will act as a stimulant or as a depressant on exosome secretion from tumor cells. Liposomal stimulation may be a useful strategy to increase exosome yield, although further preparation to increase the purity of exosomes may be needed. In addition, fine-tuning of the biological properties of induced exosomes could be achieved via controlling the physicochemical properties of the stimulant liposomes.
Yuki Minayoshi, Hitoshi Maeda, Hiroki Yanagisawa, Keisuke Hamasaki, Yuki Mizuta, Kento Nishida, Ryo Kinoshita, Yuki Enoki, Tadasi Imafuku, Giam Victor Tuan Chuang, Tomoaki Koga, Yukiko Fujiwara, Motohiro Takeya, Kayoko Sonoda, Tomohiro Wakayama, Kazuaki Taguchi, Yu Ishima, Tatsuhiro Ishida, Yasuko Iwakiri, Motohiko Tanaka, Yutaka Sasaki, Hiroshi Watanabe, Masako Otagiri and Toru Maruyama : Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions, Drug Delivery, Vol.25, No.1, 1067-1077, 2018.
(要約)
Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.
(キーワード)
Animals / Anti-Inflammatory Agents / B7-H1 Antigen / Cell Line / Hepatitis / Humans / Immunologic Factors / Interferon Type I / Interferon-alpha / Interleukin 1 Receptor Antagonist Protein / Interleukin-10 / Kupffer Cells / Liver / Male / Mannose / Mice / Mice, Inbred C57BL / Mice, Inbred ICR / RAW 264.7 Cells / Recombinant Proteins / Serum Albumin
M Ikeda, Yu Ishima, R Kinoshita, V Chuang, N Tasaka, N Matsuo, H Watanabe, Taro Shimizu, Tatsuhiro Ishida, M Otagiri and T Maruyama : A Novel S-Sulfhydrated Human Serum Albumin Preparation Suppresses Melanin Synthesis, Redox Biology, Vol.14, 354-360, 2018.
S Oshiro, Yu Ishima, M Hitoshi, N Honda, B Jing, Ryo Kinoshita, Mayumi Ikeda, Y Iwao, T Imafuku, K Nishida, S Miyamura, H Watanabe, M Otagiri and T Maruyama : Dual Therapeutic Effects of an Albumin-based Nitric Oxide Donor on Two Experimental Models of Chronic Kidney Disease, Journal of Pharmaceutical Sciences, Vol.107, No.3, 848-855, 2018.
Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Masao Tanaka, Yusuke Doi, Yasuko Terada, Naoto Yagi, Taro Shimizu, Keiichiro Okuhira, Yu Ishima and Tatsuhiro Ishida : Intratumoral visualization of oxaliplatin within a liposomal formulation using X-ray fluorescence spectrometry, Molecular Pharmaceutics, Vol.15, No.2, 403-409, 2018.
(要約)
Microsynchrotron radiation X-ray fluorescence spectrometry (-SR-XRF) is an X-ray procedure that utilizes synchrotron radiation as an excitation source. -SR-XRF is a rapid, nondestructive technique that allows mapping and quantification of metals and biologically important elements in cell or tissue samples. Generally, the intratumor distribution of nanocarrier-based therapeutics is assessed by tracing the distribution of a labeled nanocarrier within tumor tissue, rather than by tracing the encapsulated drug. Instead of targeting the delivery vehicle, we employed -SR-XRF to visualize the intratumoral microdistribution of oxaliplatin (l-OHP) encapsulated within PEGylated liposomes. Tumor-bearing mice were intravenously injected with either l-OHP-containing PEGylated liposomes (l-OHP liposomes) or free l-OHP. The intratumor distribution of l-OHP within tumor sections was determined by detecting the fluorescence of platinum atoms, which are the main elemental components of l-OHP. The l-OHP in the liposomal formulation was localized near the tumor vessels and accumulated in tumors at concentrations greater than those seen with the free form, which is consistent with the results of our previous study that focused on fluorescent labeling of PEGylated liposomes. In addition, repeated administration of l-OHP liposomes substantially enhanced the tumor accumulation and/or intratumor distribution of a subsequent dose of l-OHP liposomes, presumably via improvements in tumor vascular permeability, which is also consistent with our previous results. In conclusion, -SR-XRF imaging efficiently and directly traced the intratumor distribution of the active pharmaceutical ingredient l-OHP encapsulated in liposomes within tumor tissue. -SR-XRF imaging could be a powerful means for estimating tissue distribution and even predicting the pharmacological effect of nanocarrier-based anticancer metal compounds.
Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Munehira Kawanishi, Taro Shimizu, Keiichiro Okuhira, Yu Ishima and Tatsuhiro Ishida : Reactivity of IgM antibodies elicited by PEGylated liposomes or PEGylated lipoplexes against auto and foreign antigens, Journal of Controlled Release, Vol.270, 114-119, 2018.
(要約)
Polyethylene glycol (PEG) is an attractive tool for the development of nanoparticle-based cancer therapy since it endows nanoparticles with extended-circulation properties. Nevertheless, recent reports have revealed that intravenous injection of either PEGylated liposomes (SLs) or PEGylated lipoplex (PLpx) could elicit an anti-PEG immunoglobulin (IgM) response in a T cell-independent (TI) manner that would substantially compromise the in vivo fate of PEGylated products upon repeated administration. In the same context, viral or bacterial infections trigger the production of polyreactive IgM that binds both self and foreign antigens. The polyreactivity of IgM elicited by SLs or PLpx, to bacteria and other polymers, however, is yet to be elucidated. In this study, the polyreactivity of IgM elicited by SLs or PLpx was challenged against different bacteria (TI antigens) and against synthetic polymer composed of repetitive structures (PVP-360 or FITC-dextran). Results demonstrated that anti-PEG IgM elicited by either SLs or PLpx showed no reactivity to various bacteria examined, while the IgM showed remarkable reactivity to both PVP-360 and FITC-dextran. In addition, interestingly, anti-PEG IgM elicited by either SLs or PLpx showed no antinuclear antibody-like immune reactivity, and, therefore, treatment with either SLs or PLpx was not expected to exacerbate autoimmune diseases such as systemic lupus erythematosus. Collectively, our findings could provide information supporting the safety of PEGylated nanoparticle-based pharmaceutics, particularly in patients with autoimmune diseases.
K Tanaka, M Shimoda, V Chuang, K Nishida, M Kawahara, Tatsuhiro Ishida, M Otagiri, T Maruyama and Yu Ishima : Thioredoxin-Albumin Fusion Protein Prevents Copper enhanced Zinc-induced Neurotoxicity via Its Antioxidative activity, International Journal of Pharmaceutics, Vol.535, No.1-2, 140-147, 2018.
(要約)
Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer's disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu/Zn-induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu/Zn-induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu/Zn-induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu and Zn after Cu/Zn treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu/Zn treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu/Zn-induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases.
Hiroshi Watanabe, Ryusei Sugimoto, Komei Ikegami, Yuki Enoki, Tadashi Imafuku, Rui Fujimura, Jing Bi, Kento Nishida, Yoshiaki Sakaguchi, Michiya Murata, Hitoshi Maeda, Kenshiro Hirata, Sachiko Jingami, Yu Ishima, Motoko Tanaka, Kazutaka Matsushita, Hirotaka Komaba, Masafumi Fukagawa, Masaki Otagiri and Toru Maruyama : Parathyroid hormone contributes to the down-regulation of cytochrome P450 3A through the cAMP/PI3K/PKC/PKA/NF-B signaling pathway in secondary hyperparathyroidism, Biochemical Pharmacology, Vol.145, 192-201, 2017.
(要約)
Chronic kidney disease (CKD), which affects, not only renal clearance, but also non-renal clearance, is accompanied by a decline in renal function. Although it has been suggested that humoral factors, such as uremic toxins that accumulate in the body under CKD conditions, could be involved in the changes associated with non-renal drug clearance, the overall process is not completely understood. In this study, we report on the role of parathyroid hormone (PTH), a middle molecule uremic toxin, on the expression of drug metabolizing or transporting proteins using rats with secondary hyperparathyroidism (SHPT) as models. In SHPT rats, hepatic and intestinal CYP3A expression was suppressed, but the changes were recovered by the administration of the calcimimetic cinacalcet, a PTH suppressor. Under the same experimental conditions, a pharmacokinetic study using orally administered midazolam, a substrate for CYP3A, showed that the AUC was increased by 5 times in SHPT rats, but that was partially recovered by a cinacalcet treatment. This was directly tested in rat primary hepatocytes and intestinal Caco-2 cells where the expression of the CYP3A protein was down-regulated by PTH (1-34). In Caco-2 cells, PTH (1-34) down-regulated the expression of CYP3A mRNA, but an inactive PTH derivative (13-34) had no effect. 8-Bromo-cyclic adenosine monophosphate, a membrane-permeable cAMP analog, reduced mRNA expression of CYP3A whereas the inhibitors of PI3K, NF-κB, PKC and PKA reversed the PTH-induced CYP3A down-regulation. These results suggest that PTH down-regulates CYP3A through multiple signaling pathways, including the PI3K/PKC/PKA/NF-κB pathway after the elevation of intracellular cAMP, and the effect of PTH can be prevented by cinacalcet treatment.
Sadaharu Matsushita, Koji Nishi, Yasunori Iwao, Yu Ishima, Hiroshi Watanabe, Kazuaki Taguchi, Keishi Yamasaki, Toru Maruyama and Masaki Otagiri : Recombinant Human Serum Albumin Containing 3 Copies of Domain I, Has Significant in Vitro Antioxidative Capacity Compared to the Wild-Type, Biological & Pharmaceutical Bulletin, Vol.40, No.10, 1813-1817, 2017.
(要約)
Human serum albumin (HSA), the most abundant protein in serum, functions as carrier of drugs and contributes to maintaining serum colloid osmotic pressure. We report herein on the preparation of a genetic recombinant HSA, in which domains II and III were changed to domain I (triple domain I; TDI). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that the purity of the TDI was equivalent to that of the wild type (WT). Both far- and near-UV circular dichroism (CD) spectra of the TDI showed that its structural characteristics were similar to the WT. Ligand binding capacity was examined by an ultrafiltration method using 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and ketoprofen as markers for site I and site II, respectively. The binding capacity of TDI for both ligands was lower than that for the wild type. TDI significantly suppressed the oxidation of dihydrorhodamine 123 (DRD) by HO compared to the WT. Our current results suggest that TDI has great potential for further development as HSA a product having antioxidative functions.
(キーワード)
Antioxidants / Furans / 過酸化水素水 (hydrogen peroxide) / Ketoprofen / Ligands / 酸化と還元 (oxidation and reduction) / Propionates / Protein Binding / Protein Domains / Recombinant Proteins / Rhodamines / Serum Albumin, Human
Yuki Enoki, Hiroshi Watanabe, Riho Arake, Rui Fujimura, Kana Ishiodori, Tadashi Imafuku, Kento Nishida, Ryusei Sugimoto, Saori Nagao, Shigeyuki Miyamura, Yu Ishima, Motoko Tanaka, Kazutaka Matsushita, Hirotaka Komaba, Masafumi Fukagawa, Masaki Otagiri and Toru Maruyama : Potential therapeutic interventions for chronic kidney disease-associated sarcopenia via indoxyl sulfate-induced mitochondrial dysfunction, Journal of Cachexia, Sarcopenia and Muscle, Vol.8, No.5, 735-747, 2017.
(要約)
Chronic kidney disease (CKD) patients experience skeletal muscle wasting and decreased exercise endurance. Our previous study demonstrated that indoxyl sulfate (IS), a uremic toxin, accelerates skeletal muscle atrophy. The purpose of this study was to examine the issue of whether IS causes mitochondria dysfunction and IS-targeted intervention using AST-120, which inhibits IS accumulation, or mitochondria-targeted intervention using L-carnitine or teneligliptin, a dipeptidyl peptidase-4 inhibitor which retains mitochondria function and alleviates skeletal muscle atrophy and muscle endurance in chronic kidney disease mice. The in vitro effect of IS on mitochondrial status was evaluated using mouse myofibroblast cells (C2C12 cell). The mice were divided into sham or 5/6-nephrectomized (CKD) mice group. Chronic kidney disease mice were also randomly assigned to non-treatment group and AST-120, L-carnitine, or teneligliptin treatment groups. In C2C12 cells, IS induced mitochondrial dysfunction by decreasing the expression of PGC-1α and inducing autophagy in addition to decreasing mitochondrial membrane potential. Co-incubation with an anti-oxidant, ascorbic acid, L-carnitine, or teneligliptine restored the values to their original state. In CKD mice, the body and skeletal muscle weights were decreased compared with sham mice. Compared with sham mice, the expression of interleukin-6 and atrophy-related factors such as myostatin and atrogin-1 was increased in the skeletal muscle of CKD mice, whereas muscular Akt phosphorylation was decreased. In addition, a reduced exercise capacity was observed for the CKD mice, which was accompanied by a decreased expression of muscular PCG-1α and increased muscular autophagy, as reflected by decreased mitochondria-rich type I fibres. An AST-120 treatment significantly restored these changes including skeletal muscle weight observed in CKD mice to the sham levels accompanied by a reduction in IS levels. An L-carnitine or teneligliptin treatment also restored them to the sham levels without changing IS level. Our results indicate that IS induces mitochondrial dysfunction in skeletal muscle cells and provides a potential therapeutic strategy such as IS-targeted and mitochondria-targeted interventions for treating CKD-induced muscle atrophy and decreased exercise endurance.
Mayumi Ikeda, Yu Ishima, Victor Chuang, Tsuyoshi Ikeda, Ryo Kinoshita, Hiroshi Watanabe, Tatsuhiro Ishida, Masaki Otagiri and Toru Maruyama : Apoptosis induction of Poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition, Nitric Oxide: Biology and Chemistry, Vol.69, 28-34, 2017.
(要約)
Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.
Ryo Kinoshita, Yu Ishima, Victor T.G. Chuang, Hideaki Nakamura, Jun Fang, Hiroshi Watanabe, Taro Shimizu, Keiichiro Okuhira, Tatsuhiro Ishida, Hiroshi Maeda, Masaki Otagiri and Toru Maruyama : Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-Nitrosated Human Serum Albumin Dimer, Biomaterials, Vol.140, 162-169, 2017.
(要約)
In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed.
Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Miho Nishio, Yusuke Doi, Hidenori ANDO, Masami Ukawa, Yu Ishima and Tatsuhiro Ishida : Modulation of antitumor immunity contributes to the enhanced therapeutic efficacy of liposomal oxaliplatin in mouse model, Cancer Science, Vol.108, No.9, 1864-1869, 2017.
(要約)
Immune modulation of the tumor microenvironment has been reported to participate in the therapeutic efficacy of many chemotherapeutic agents. Recently, we reported that liposomal encapsulation of oxaliplatin (l-OHP) within PEGylated liposomes conferred a superior antitumor efficacy to free l-OHP in murine colorectal carcinoma-bearing mice through permitting preferential accumulation of the encapsulated drug within tumor tissue. However, the contribution of the immune-modulatory properties of liposomal l-OHP and/or free l-OHP to the overall antitumor efficacy was not elucidated. In the present study, therefore, we investigated the effect of liposomal encapsulation of l-OHP within PEGylated liposomes on the antitumor immunity in both immunocompetent and immunodeficient mice. Liposomal l-OHP significantly suppressed the growth of tumors implanted in immunocompetent mice, but not in immunodeficient mice. In immunocompetent mice, liposomal l-OHP increased the tumor MHC-1 level and preserved antitumor immunity through decreasing the number of immune suppressor cells, including regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages, which collectively suppress CD8(+) T cell-mediated tumor cells killing. In contrast, free l-OHP ruined antitumor immunity. These results suggest that the antitumor efficacy of liposomal l-OHP is attributed, on the one hand, to its immunomodulatory effect on tumor immune microenvironment that is superior to that of free l-OHP, and on the other hand, to its direct cytotoxic effect on tumor cells.
Mayumi Ikeda, Yu Ishima, Akitomo Shibata, Victor T.G. Chuang, Tomohiro Sawa, Hideshi Ihara, Hiroshi Watanabe, Ming Xian, Yuya Ouchi, Taro Shimizu, Hidenori ANDO, Masami Ukawa, Tatsuhiro Ishida, T Akaike, M Otagiri and T Maruyama : Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach, Analytica Chimica Acta, Vol.969, 18-25, 2017.
(要約)
Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, 1-anti-trypsin, 1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 M of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.
Hyperuricemia occurs with increasing frequency among patients with hyperparathyroidism. However, the molecular mechanism by which the serum parathyroid hormone (PTH) affects serum urate levels remains unknown. This was studied in uremic rats with secondary hyperparathyroidism where serum urate levels were found to be increased and urate excretion in the intestine and kidney decreased, presumably due to down-regulation of the expression of the urate exporter ABCG2 in intestinal and renal epithelial membranes. These effects were prevented by administration of the calcimimetic cinacalcet, a PTH suppressor, suggesting that PTH may down-regulate ABCG2 expression. This was directly tested in intestinal Caco-2 cells where the expression of ABCG2 on the plasma membrane was down-regulated by PTH (1-34) while its mRNA level remained unchanged. Interestingly, an inactive PTH derivative (13-34) had no effect, suggesting that a posttranscriptional regulatory system acts through the PTH receptor to regulate ABCG2 plasma membrane expression. As found in an animal study, additional clinical investigations showed that treatment with cinacalcet resulted in significant reductions in serum urate levels together with decreases in PTH levels in patients with secondary hyperparathyroidism undergoing dialysis. Thus, PTH down-regulates ABCG2 expression on the plasma membrane to suppress intestinal and renal urate excretion, and the effects of PTH can be prevented by cinacalcet treatment.
Yu Ishima : Albumin-Based Nitric Oxide Traffic System for the Treatment of Intractable Cancers, Biological & Pharmaceutical Bulletin, Vol.40, No.2, 128-134, 2017.
(要約)
Biomacromolecules (>40 kDa) have been developed as drug delivery system (DDS) carriers of low-molecular weight drugs to promote these drugs' uptake by cancer tissues via enhanced permeability and retention (EPR) effects. Human serum albumin (HSA) has been found to accumulate in cancer tissues via this EPR effect. HSA is the most abundant protein in serum, which performs essential physiological functions such as the transportation of many endogenous and exogenous ligands. Nitric oxide (NO) is a very small ligand of HSA; it is a unique and diffusible molecular messenger that plays a central role in mammalian physiology. Although the in vivo half-life of NO is extremely short, HSA could prolong the half-life of NO via S-nitrosation at the position of Cys-34. S-Nitrosated HSA (mono-SNO-HSA) is called an 'Endogenous NO traffic protein,' due to the highly stable S-nitroso form in circulating blood, and to the efficiency of S-transnitrosation in cells that require NO. Mono-SNO-HSA possesses a very strong cytoprotective action via the induction of heme oxygenase-1. On the other hand, HSA reinforced with approximately seven NO molecules (poly-SNO-HSA), which we developed by means of chemical modification, possesses multiple anticancer activities. Our previous data clarified that the high expression of protein disulfide isomerase on the surface of cancer cells plays a very important role in the anticancer action of poly-SNO-HSA. In this review, we focus on the advantage of poly-SNO-HSA in treating intractable cancers from the viewpoint of drug delivery systems and drug resistance.
(キーワード)
Animals / Antineoplastic Agents / Drug Delivery Systems / Humans / Neoplasms / Nitric Oxide / Protein Structure, Secondary / Protein Transport / Serum Albumin / Treatment Outcome
Akitomo Shibata, Yu Ishima, Mayumi Ikeda, Hirokazu Sato, Tadashi Imafuku, Victor T. G. Chuang, Yuya Ouchi, Takaya Abe, Hiroshi Watanabe, Tatsuhiro Ishida, Masaki Otagiri and Toru Maruyama : Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease, Biochemical and Biophysical Research Communications, Vol.479, No.3, 578-583, 2016.
(要約)
Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na2S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximately 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases.
U Kragh-Hansen, L Minchiotti, A Coletta, K Bienk, M Galliano, B Schiøtt, Y Iwao, Yu Ishima and M Otagiri : Mutants and molecular dockings reveal that the primary L-thyroxine binding site in human serum albumin is not the one which can cause familial dysalbuminemic hyperthyroxinemia, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1860, No.4, 648-660, 2016.
(要約)
Natural mutations of R218 in human serum albumin (HSA) result in an increased affinity for L-thyroxine and lead to the autosomal dominant condition of familial dysalbuminemic hyperthyroxinemia. Binding was studied by equilibrium dialysis and computer modeling. Ten of 32 other isoforms tested had modified high-affinity hormone binding. L-thyroxine has been reported to bind to four sites (Tr) in HSA; Tr1 and Tr4 are placed in the N-terminal and C-terminal part of the protein, respectively. Site-directed mutagenesis gave new information about all the sites. It is widely assumed that Tr1 is the primary hormone site, and that this site, on a modified form, is responsible for the above syndrome, but the binding experiments with the genetic variants and displacement studies with marker ligands indicated that the primary site is Tr4. This new assignment of the high-affinity site was strongly supported by results of MM-PBSA analyses and by molecular docking performed on relaxed protein structure. However, dockings also revealed that mutating R218 for a smaller amino acid increases the affinity of Tr1 to such an extent that it can become the high-affinity site. Placing the high-affinity binding site (Tr4) and the one which can result in familial dysalbuminemic hyperthyroxinemia (Tr1) in two very different parts of HSA is not trivial, because in this way persons with and without the syndrome can have different types of interactions, and thereby complications, when given albumin-bound drugs. The molecular information is also useful when designing drugs based on L-thyroxine analogues.
S Miyamura, T Imafuku, M Anraku, K Taguchi, K Yamasaki, Y Tominaga, H Maeda, Yu Ishima, H Watanabe, M Otagiri and T Maruyama : Comparison of Posttranslational Modification and the Functional Impairment of Human Serum Albumin in Commercial Preparations, Journal of Pharmaceutical Sciences, Vol.105, No.3, 1043-1049, 2016.
(要約)
On account of its long circulating half-life, human serum albumin (HSA) is susceptible to posttranslational modifications that can alter its functions. Here, we comprehensively compared the degree of posttranslational modifications with the functional impairment of HSA derived from 5 different commercially available albumin preparations and clarified their relationships. We used electrospray ionization-time of flight mass spectrometry to evaluate the degree of posttranslational modification of the entire HSA molecule that was associated with disease development and found that the fraction of Cys34-cysteinylated HSA (Cys-Cys34-HSA), a major form of oxidative modification, varied substantially among the albumin preparations. Meanwhile, no remarkable difference was found in the degree of glycated or N-terminal truncated HSA among the preparations tested. The nonosmotic pressure maintenance functions of HSA, such as its antioxidative and ligand-binding activities significantly differed among the preparations. Interestingly, the alternations of these functions showed a significantly negative correlation only with the Cys-Cys34-HSA fraction. These findings suggest that the Cys-Cys34-HSA fraction, as estimated by electrospray ionization-time of flight mass spectrometry can be used as a predictive marker for the functional impairment of albumin preparations and that it would be preferable to use albumin preparations with higher contents of functionally effective albumin that correspond to a lower degree of cysteinylation of Cys34 in clinical practice.
Yu Ishima and T Maruyama : Human Serum Albumin as Carrier in Drug Delivery Systems, Journal of the Pharmaceutical Society of Japan, Vol.136, No.1, 39-47, 2016.
(要約)
Recently, human serum albumin (HSA) has emerged as a versatile carrier for therapeutic agents against diabetes, cancer, and infectious diseases. Market-approved products include fatty acid derivatives of human insulin for diabetes and the paclitaxel-HSA nanoparticle for various cancers such as metastatic breast cancer and advanced pancreatic cancer. In this review, we focus on the next-generation approach including HSA-binding bioactive gas such as nitric oxide (NO) for treating ischemic/reperfusion injury, cancer, and bacterial infection. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated HSA (SNO-HSA), which is why we are investigating whether this albumin form can be therapeutically useful. Recently, we have developed SNO-HSA analogues such as SNO-HSA with many conjugated SNO groups (poly-SNO-HSA) prepared using chemical modification. Unexpectedly, we found striking inverse effects between poly-SNO-HSA and SNO-HSA. Despite the fact that SNO-HSA inhibits apoptosis, poly-SNO-HSA possesses very strong pro-apoptotic effects against tumor cells. Furthermore, poly-SNO-HSA can reduce or even completely eliminate the multidrug resistance often developed by cancer cells. In this review, we put forward the possibility that poly-SNO-HSA can be used as a safe, effective multifunctional antitumor agent.
(キーワード)
Apoptosis / Drug Carriers / Drug Delivery Systems / Drug Resistance, Multiple / Drug Resistance, Neoplasm / Gases / Humans / Neoplasms / Nitric Oxide / Nitric Oxide Donors / Nitroso Compounds / Protein Binding / Serum Albumin
Yu Ishima, Ryo Kinoshita, Mayumi Ikeda, U Kragh-Hansen, J Fang, H Nakamura, VT Chuang, R Tanaka, H Maeda, A Kodama, H Watanabe, H Maeda, M Otagiri and T Maruyama : S-Nitrosated human serum albumin dimer as novel nano-EPR enhancer applied to macromolecular anti-tumor drugs such as micelles and liposomes, Journal of Controlled Release, Vol.217, 1-9, 2015.
(要約)
The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors, and it can serve as a basis for the development of macromolecular anticancer therapy. We have previously found that recombinant human serum albumin dimer, and especially its S-nitrosated form (SNO-HSA-Dimer), is an enhancer of the EPR effect. In this study, we investigated the influence of SNO-HSA-Dimer on the anti-tumor effect of two types of macromolecular anti-tumor drugs, namely N-(2-hydroxypropyl)methacrylamide polymer conjugated with zinc protoporphyrin, which forms micelles and can be used for fluorescence studies. The other was PEGylated liposomal doxorubicin (Doxil), a typical example of a stealth liposome approved for medical usage. In mice having C26 tumors with highly permeable vasculature, SNO-HSA-Dimer increases tumor accumulation of the drugs by a factor 3-4 and thereby their anti-tumor effects. Experiments with Evans blue revealed increased EPR effect in all parts of the tumor. Furthermore, SNO-HSA-Dimer improves the anti-metastatic effects of Doxil and reduces its minor uptake in non-tumorous organs such as liver and kidney. Tumor accumulation of Doxil in B16 tumors, which are characterized by a low permeable vasculature, increased even more (6-fold) in the presence of SNO-HSA-Dimer, and the improved accumulation lead to decreased tumor volume and increased survival of the animals. The administration of SNO-HSA-Dimer itself is safe, because it has no effect on blood pressure, heart rate or on several biochemical parameters. The present findings indicate that SNO-HSA-Dimer is promising for enhancing the EPR effect and consequently the specific, therapeutic effects of macromolecular anticancer drugs.
S Ogaki, K Taguchi, H Maeda, H Watanabe, Yu Ishima, M Otagiri and T Maruyama : Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock, Biochemical Pharmacology, Vol.97, No.3, 310-319, 2015.
(要約)
Red blood cell (RBC) transfusions for controlling hemorrhaging induce systemic ischemia reperfusion, resulting in a decrease in hepatic cytochrome P450 (CYP) levels. Carbon monoxide (CO), when bound to red blood cells (CO-RBC) has the potential to protect the hepatic CYP protein to produce a resuscitative effect in a hemorrhagic shock rat model. The aim of this study was to investigate the mechanism by which CO-RBC resuscitation from a massive hemorrhage protects against a decrease in hepatic CYP. In the early phase (∼1h) after a hemorrhage and RBC resuscitation, hepatic CYP protein levels were significantly decreased with increasing hepatic free heme levels, but were maintained by a pre-treatment of gadolinium chloride (GdCl3), a Kupffer cell inhibitor, and Trolox, an anti-oxidant agent, as well as CO-RBC resuscitation. Under these conditions, the production of reactive oxygen species (ROS) derived from activated Kupffer cells was increased, but this increase was suppressed by CO-RBC resuscitation. At a late phase (6∼24h), CYP mRNA levels decreased after hemorrhage and RBC resuscitation, but not in the case of CO-RBC resuscitation. The increases in plasma IL-6 and TNF-α levels were decreased by CO-RBC resuscitation via the suppression of the toll-like receptor-4 (TLR-4) and the expression of the high mobility group box-1 (HMGB-1). Hepatic CYP protection after a hemorrhage and CO-RBC resuscitation can be attributed to the inactivation of Kupffer cells, resulting in the suppression of ROS production in the early phase and the suppression of inflammatory cytokine production via the TLR-4/HMGB-1signal pathway in the late phase.
K Nishida, H Watanabe, S Ogaki, A Kodama, R Tanaka, T Imafuku, Yu Ishima, VT Chuang, M Toyoda, M Kondoh, Q Wu, M Fukagawa, M Otagiri and T Maruyama : Renoprotective effect of long acting thioredoxin by modulating oxidative stress and macrophage migration inhibitory factor against rhabdomyolysis-associated acute kidney injury, Scientific Reports, Vol.5, 14471, 2015.
(要約)
Rhabdomyolysis-associated acute kidney injury (AKI) is a serious life-threatening condition. As such, more effective strategies are needed for its prevention. Thioredoxin-1 (Trx), a redox-active and macrophage migration inhibitory factor (MIF) modulating protein, has a short retention time in the blood. We examined the renoprotective effect of long acting Trx that was genetically fused with human serum albumin (HSA-Trx) against glycerol-induced AKI. An intravenous HSA-Trx pre-treatment attenuated the glycerol-induced decline in renal function, compared to a PBS, HSA or Trx alone. HSA-Trx caused a reduction in the tubular injuries and in the number of apoptosis-positive tubular cells. Renal superoxide, 8-hydroxy deoxyguanosine, nitrotyrosine and the plasma Cys34-cysteinylated albumin were clearly suppressed by the HSA-Trx treatment. Prior to decreasing TNF-α and IL-6, HSA-Trx suppressed an increase of plasma MIF level. In LLC-PK1 cells, HSA-Trx decreased the level of reactive oxygen species and lactate dehydrogenase release induced by myoglobin. HSA-Trx treatment resulted in a threefold increase in the survival of lethal glycerol-treated mice. The post-administration of HSA-Trx at 1 and 3 hr after glycerol injection exerted a significant renoprotective effect. These results suggest HSA-Trx has potential for use in the treatment of rhabdomyolysis-associated AKI via its extended effects of modulating oxidative stress and MIF.
Yu Ishima, T Narisoko, U Kragh-Hansen, S Kotani, M Nakajima, M Otagiri and T Maruyama : Nitration of indoxyl sulfate facilitates its cytotoxicity in human renal proximal tubular cells via expression of heme oxygenase-1, Biochemical and Biophysical Research Communications, Vol.465, No.3, 481-487, 2015.
(要約)
Peroxynitrite, the reaction product of superoxide [Formula: see text] and nitric oxide (NO), nitrates tyrosine residues, unsaturated fatty acids, cyclic guanosine monophosphate and other phenolics. We report herein that indoxyl sulfate (IS) is also nitrated by peroxynitrite in vitro and forms 2-nitro-IS, as determined from spectral characteristics and (1)H-NMR. IS is one of the very important uremic toxins that accelerate the progression of chronic kidney disease via various mechanisms. However, cell viability experiments with human proximal tubular cells show that the cytotoxicity of 2-nitro-IS is several-fold higher than that of IS. The explanation for this finding seems to be that 2-nitro-IS induces a much more pronounced generation of intracellular reactive oxygen species (ROS) than IS. Results with inhibitors revealed that an organic anion transporter, several intracellular enzymes and nonprotein-bound iron ions are reasons for this finding. Most importantly, however, as detected by immunofluorescence and Western blotting, 2-nitro-IS induces the expression of heme oxygenase-1 and thereby the formation of ROS; most probably through the Fenton reaction. The final result of the increased amounts of ROS is death of the kidney cells. Thus, nitration of uremic toxins by peroxynitrite may help us to understand the initiation and progress of chronic kidney diseases.
Y Enoki, Yu Ishima, R Tanaka, K Sato, K Kimachi, T Shirai, H Watanabe, VT Chuang, Y Fujiwara, M Takeya, M Otagiri and T Maruyama : Pleiotropic Effects of Levofloxacin, Fluoroquinolone Antibiotics, against Influenza Virus-Induced Lung Injury, PLoS ONE, Vol.10, No.6, e0130248, 2015.
(要約)
Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs.
H Maeda, K Hirata, H Watanabe, Yu Ishima, VT Chuang, K Taguchi, A Inatsu, M Kinoshita, M Tanaka, Y Sasaki, M Otagiri and T Maruyama : Polythiol-Containing, Recombinant Mannosylated-Albumin Is a Superior CD68+/CD206+ Kupffer Cell-Targeted Nanoantioxidant for Treatment of Two Acute Hepatitis Models, The Journal of Pharmacology and Experimental Therapeutics, Vol.352, No.2, 244-257, 2015.
(要約)
Since reactive oxygen species (ROS) derived from Kupffer cells (KC), especially CD68(+) KC, play a key role in the induction of hepatic oxidative stress and injuries, we developed a polythiolated- and mannosylated human serum albumin (SH-Man-HSA), which functions as a novel nanoantioxidant for delivering thiol to CD68(+) KC. In vitro electron paramagnetic resonance coupled with pharmacokinetics and immunohistochemical studies showed that SH-Man-HSA possessed powerful radical-scavenging activity and rapidly and selectively delivered thiols to the liver via mannose receptor (CD206) on CD68(+) cells. SH-Man-HSA significantly improved the survival rate of concanavalin-A (Con-A)-treated mice. Moreover, SH-Man-HSA exhibited excellent hepatoprotective functions, not by decreasing tumor necrosis factor or interferon-γ production that is closely associated with Con-A-induced hepatitis, but by suppressing ROS production. Interestingly, the protective effect of SH-Man-HSA was superior to N-acetyl cysteine (NAC). This could be attributed to the difference in the inhibition of hepatic oxidative stress between the two antioxidants depending on their potential for thiol delivery to the liver. Similar results were also observed for acetaminophen (APAP)-induced hepatopathy models. Flow cytometric data further confirmed that an increase in F4/80(+)/ROS(+) cells was dramatically decreased by SH-Man-HSA. The administration of SH-Man-HSA at 4 hours following a Con-A or APAP injection also exhibited a profound hepatoprotective action against these hepatitis models, whereas this was not observed for NAC. It can be concluded therefore that SH-Man-HSA has great potential for use in a rescue therapy for hepatopathy as a nanoantioxidant because of its ability to efficiently and rapidly deliver thiols to CD68(+)/CD206(+) KC.
(キーワード)
Acetaminophen / Acute Disease / Albumins / Animals / Antigens, CD / Antigens, Differentiation, Myelomonocytic / Antioxidants / Chemical and Drug Induced Liver Injury / Concanavalin A / Disease Models, Animal / Flow Cytometry / Glycoproteins / Kupffer Cells / Lectins, C-Type / Male / Mannose-Binding Lectins / Mice, Inbred C57BL / Nanoparticles / Receptors, Cell Surface
Yu Ishima, A Inoue, J Fang, R Kinoshita, M Ikeda, H Watanabe, H Maeda, M Otagiri and T Maruyama : Poly-S-nitrosated human albumin enhances the antitumor and antimetastasis effect of bevacizumab, partly by inhibiting autophagy via the generation of nitric oxide, Cancer Science, Vol.106, No.2, 194-200, 2015.
(要約)
Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short-term therapeutic effects, whereas long-term therapeutic benefits are rarely observed, probably due to hypoxia-induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S-nitrosated human serum albumin (SNO-HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO-HSA significantly suppressed hypoxia-induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO-HSA was also confirmed in vivo by combining it with Bev. In C26-bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO-HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO-HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO-HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO-HSA to suppress autophagy-mediated drug resistance and enhance the efficacy of chemotherapy.
H Watanabe, Y Miyamoto, Y Enoki, Yu Ishima, D Kadowaki, S Kotani, M Nakajima, M Tanaka, K Matsushita, Y Mori, T Kakuta, M Fukagawa, M Otagiri and T Maruyama : p-Cresyl sulfate, a uremic toxin, causes vascular endothelial and smooth muscle cell damages by inducing oxidative stress, Pharmacology Research & Perspectives, Vol.3, No.1, e00092, 2015.
(要約)
The major cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease. Here, p-Cresyl sulfate (PCS), a uremic toxin, is considered to be a risk factor for cardiovascular disease in CKD. However, our understanding of the vascular toxicity induced by PCS and its mechanism is incomplete. The purpose of this study was to determine whether PCS enhances the production of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (HASMC) by enhancing NADPH oxidase expression. PCS also up-regulates the mRNA levels and the protein secretion of monocyte chemotactic protein-1 (MCP-1) in HUVEC. In HASMC, PCS increased the mRNA levels of alkaline phosphatase (ALP), osteopontin (OPN), core-binding factor alpha 1, and ALP activity. The knockdown of Nox4, a subunit of NADPH oxidase, suppressed the cell toxicity induced by PCS. The vascular damage induced by PCS was largely suppressed in the presence of probenecid, an inhibitor of organic anion transporters (OAT). In PCS-overloaded 5/6-nephrectomized rats, plasma MCP-1 levels, OPN expression, and ALP activity of the aortic arch were increased, accompanied by the induction of Nox4 expression. Collectively, the vascular toxicity of PCS can be attributed to its intracellular accumulation via OAT, which results in an enhanced NADPH oxidase expression and increased ROS production. In conclusion, we found for the first time that PCS could play an important role in the development of cardiovascular disease by inducing vascular toxicity in the CKD condition.
R Tanaka, Yu Ishima, Y Enoki, K Kimachi, T Shirai, H Watanabe, VT Chuang, T Maruyama and M Otagiri : Therapeutic impact of human serum albumin-thioredoxin fusion protein on influenza virus-induced lung injury mice, Frontiers in Immunology, Vol.5, 561, 2014.
(要約)
Reactive oxygen species (ROS) are the primary pathogenic molecules produced in viral lung infections. We previously reported on the use of a recombinant human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) for extending the half-life Trx, an endogenous protein with anti-oxidant properties. As a result, it was possible to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx. We hypothesized that HSA-Trx would attenuate the enhanced ROS production of species such as hydroxyl radicals by neutrophils during an influenza viral infection. The levels of 8-hydroxy-2'-deoxyguanosine and 3-nitrotyrosine were used as indices of the anti-oxidant activity of HSA-Trx. In addition, the cytoprotective effects of HSA-Trx were examined in PR8 (H1N1) influenza virus-induced lung injured mice. The findings show that HSA-Trx reduced the number of total cells, neutrophils, and total protein in BALF of influenza virus-induced lung injured mice. The HSA-Trx treatment significantly decreased the level of 8-hydroxy-2'-deoxyguanosine and 3-nitrotyrosine, but failed to inhibit inducible nitric oxide synthase expression, in the lungs of the virus-infected mice. On the other hand, Tamiflu(®) treatment also significantly suppressed the production of inflammatory cells and neutrophil infiltration, as well as the protein level in BALF and lung histopathological alterations caused by the influenza virus. The suppressive effect of Tamiflu(®) was slightly stronger than that of HSA-Trx. Interestingly, Tamiflu(®) significantly decreased virus proliferation, while HSA-Trx had no effect. These results indicate that HSA-Trx may be of therapeutic value for the treatment of various acute inflammatory disorders such as influenza-virus-induced pneumonia, by inhibiting inflammatory-cell responses and suppressing the overproduction of NO in the lung.
Y Miyazaki, K Taguchi, K Sou, H Watanabe, Yu Ishima, T Miyakawa, H Mitsuya, M Fukagawa, M Otagiri and T Maruyama : Therapeutic impact of erythropoietin-encapsulated liposomes targeted to bone marrow on renal anemia, Molecular Pharmaceutics, Vol.11, No.11, 4238-4248, 2014.
(要約)
Bone marrow is a key element in the diagnosis of disorders of erythropoiesis, including anemia, and a potential target in their treatment. However, because efficient delivery of diagnostic and therapeutic agents to bone marrow is difficult, such delivery is achieved by administering drugs in large quantities that often have adverse effects. Here, we achieved selective delivery of recombinant human erythropoietin (rHuEPO) to bone marrow, via its encapsulation in liposomes with l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) (liposome-EPO). The result, in a rabbit model of renal anemia, was a beneficial effect on hematopoiesis, better than with rHuEPO alone. Also, we determined that liposome-EPO delivery to bone marrow depended on specific uptake by bone marrow macrophages because of the presence of SA. These results indicate both that liposome-EPO is a new, promising erythropoietin-stimulating agent and that liposomes with SA have potential for diagnostic and therapeutic applications in diseases originating from bone marrow.
(キーワード)
Anemia / Animals / Bone Marrow / Cells, Cultured / Drug Carriers / Drug Delivery Systems / Epoetin Alfa / Erythropoiesis / Erythropoietin / Flow Cytometry / Humans / Kidney Diseases / Liposomes / Macrophages / Male / Rabbits / Recombinant Proteins
H Watanabe, Y Sakaguchi, R Sugimoto, KI Kaneko, H Iwata, S Kotani, M Nakajima, Yu Ishima, M Otagiri and T Maruyama : Human organic anion transporters function as a high-capacity transporter for p-cresyl sulfate, a uremic toxin, Clinical and Experimental Nephrology, Vol.18, No.5, 814-820, 2014.
(要約)
Recent clinical studies have shown that increased serum levels of p-cresyl sulfate (PCS), a uremic toxin, are associated with the progression of chronic kidney disease (CKD) and cardiovascular outcomes. Using rat renal cortical slices, we previously reported that the rat organic anion transporter (OAT) could play a key role in the renal tubular secretion of PCS. However, no information is currently available regarding the transport of PCS via human OAT (hOAT) isoforms, hOAT1 and hOAT3. Uptake experiments of PCS were performed using HEK293 cells, which stably express hOAT1 or hOAT3. PCS was taken up by hOAT1/HEK293 and hOAT3/HEK293 cells in a time- and concentration-dependent manner. The apparent K m for the hOAT1-mediated transport of PCS was 128 μM, whereas in hOAT3/HEK293, saturation was not observed at the highest tested PCS concentration of 5 mM. Probenecid, an OAT inhibitor, inhibited PCS transport by hOAT1 and hOAT3. The uptake of p-aminohippurate by hOAT1 and estron-3-sulfate by hOAT3 was decreased with increasing PCS concentration. The apparent 50 % inhibitory concentrations for PCS were 690 and 485 μM for hOAT1 and hOAT3, respectively. PCS is a substrate for hOAT1 and hOAT3, and hOAT1 and hOAT3 appear to play a physiological role as a high-capacity PCS transporter. Since hOATs are expressed not only in the kidneys, but also in blood vessels and osteoblasts, etc., these findings are of great significance in terms of elucidating the renal clearance, tissue disposition of PCS and the mechanism of its toxicity in CKD.
Y Kouno, M Anraku, K Yamasaki, Y Okayama, D Iohara, Yu Ishima, T Maruyama, U Kragh-Hansen, F Hirayama and M Otagiri : N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1840, No.9, 2806-2812, 2014.
(要約)
Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products. Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration. N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present. The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations. N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.
(キーワード)
Free Radical Scavengers / Humans / Methionine / Oxidation-Reduction / Photochemical Processes / Protein Stability / Reactive Oxygen Species / Serum Albumin / Tryptophan
Yu Ishima, J Fang, U Kragh-Hansen, H Yin, L Liao, N Katayama, H Watanabe, T Kai, A Suenaga, H Maeda, M Otagiri and T Maruyama : Tuning of poly-s-nitrosated human serum albumin as superior antitumor nanomedicine, Journal of Pharmaceutical Sciences, Vol.103, No.7, 2184-2188, 2014.
(要約)
Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. We have previously demonstrated that poly-nitric oxide (NO) conjugated human serum albumin (Poly-SNO-HSA) has the potential to be a DDS carrier capable of accumulating NO in tumors. However, the stability of Poly-SNO-HSA in the circulation has to be improved, and its optimal molecular size for using the EPR effects has to be evaluated. In the present study, we performed two tuning methods for refining Poly-SNO-HSA, namely, pegylation and dimerization. We observed that pegylation enhanced the stability of Poly-SNO-HSA both in vitro and in vivo, and that dimerization of Poly-SNO-HSA enhanced the antitumor activity via more efficient delivery of NO in Colon 26 tumor-bearing mice. Intriguingly, dimerization resulted in a 10 times higher antitumor activity. These data suggest that pegylation and dimerization of Poly-SNO-HSA are very important tuners to optimize NO stability and accumulation, and thereby effect, in tumors. Thus, polyethylene glycol-Poly-SNO-HSA dimer seems to be a very appealing and safe NO carrier and thereby a strong candidate as an antitumor drug in future development of cancer therapeutics.
(キーワード)
Animals / Antineoplastic Agents / Cell Line, Tumor / Drug Carriers / Drug Liberation / Drug Stability / Humans / Male / Mice, Inbred Strains / Nanomedicine / Nitric Oxide / Nitroso Compounds / Polyethylene Glycols / Protein Multimerization / Rats, Inbred Strains / Serum Albumin / Serum Albumin, Human / Xenograft Model Antitumor Assays
S Ogaki, K Taguchi, H Watanabe, Yu Ishima, M Otagiri and T Maruyama : Carbon Monoxide-Bound Red Blood Cell Resuscitation Ameliorates Hepatic Injury Induced by Massive Hemorrhage and Red Blood Cell Resuscitation via Hepatic Cytochrome P450 Protection in Hemorrhagic Shock Rats, Journal of Pharmaceutical Sciences, Vol.103, No.7, 2199-2206, 2014.
(要約)
Red blood cell (RBC) transfusions are the gold standard in cases of massive hemorrhage, but induce hepatic ischemia-reperfusion injury, a serious complication associated with hemorrhage and RBC resuscitation. Thus, the development of a novel resuscitable fluid that is not associated with hepatic ischemia-reperfusion injury would be desirable. It was reported that exogenous carbon monoxide (CO) treatment ameliorated hepatic ischemia-reperfusion injury accompanying liver transplantation. This suggests that transfusions with CO-bound RBC (CO-RBC) might protect against hepatic ischemia-reperfusion injury following massive hemorrhage and resuscitation compared with RBC resuscitation. To investigate this, we created a hemorrhagic shock model rat, followed by resuscitation with RBC and CO-RBC. Hepatic ischemia-reperfusion injury and the destruction of hepatic cytochrome P450 (CYP) were significantly ameliorated in the CO-RBC resuscitation group compared with the RBC resuscitation group. The free heme derived from the destruction of hepatic CYP was correlated with hepatic oxidation and injury, suggesting that CO-RBC was a major factor in the amelioration of hepatic ischemia-reperfusion injury induced by hemorrhage and resuscitation via hepatic CYP protection. These results indicate that CO-RBC has potential for use as a resuscitative fluid in blood transfusion and does not suffer from the limitations associated with the RBC transfusions that are currently in use.
R Tanaka, Yu Ishima, H Maeda, A Kodama, S Nagao, H Watanabe, VT Chuang, M Otagiri and T Maruyama : Albumin fusion prolongs the antioxidant and anti-inflammatory activities of thioredoxin in mice with acetaminophen-induced hepatitis, Molecular Pharmaceutics, Vol.11, No.4, 1228-1238, 2014.
(要約)
Overdoses of acetaminophen (APAP) are a major cause of acute liver failure. N-Acetylcysteine (NAC) is the standard therapy for patients with such an overdose because oxidative stress plays an important role in the pathogenesis of APAP-induced hepatitis. However, NAC is not sufficiently efficacious. We previously developed a recombinant human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx), designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an endogenous protein with antioxidative and anti-inflammatory properties. In this study, we investigated the therapeutic impact of HSA-Trx in mice with APAP-induced hepatitis. The systemic administration of HSA-Trx significantly improved the survival rate of mice treated with a lethal dose of APAP compared with saline. HSA-Trx strongly attenuated plasma transaminases in APAP-induced hepatitis mice compared with HSA or Trx, components of the fusion protein. HSA-Trx also markedly caused a diminution in the histopathological features of hepatic injuries and the number of apoptosis-positive hepatic cells. In addition, an evaluation of oxidative stress markers and plasma cytokine and chemokine levels clearly showed that HSA-Trx significantly improved the breakdown of hepatic redox conditions and inflammation caused by the APAP treatment. HSA-Trx also significantly decreased oxidative and nitrosative/nitrative stress induced by SIN-1 in vitro. Finally, HSA-Trx, but not the NAC treatment at 4 h after APAP injection, significantly inhibited the elevation in plasma transaminase levels. In conclusion, the findings suggest that HSA-Trx has considerable potential for use as a novel therapeutic agent for APAP-induced hepatitis, due to its long-lasting antioxidative and anti-inflammatory effects.
(キーワード)
Acetaminophen / Alanine Transaminase / Animals / Anti-Inflammatory Agents / Antioxidants / Aspartate Aminotransferases / Chemical and Drug Induced Liver Injury / Cytokines / Hep G2 Cells / Humans / Male / Mice / Mice, Inbred C57BL / Oxidative Stress / Serum Albumin
A Kodama, H Watanabe, R Tanaka, M Kondo, VT Chuang, Q Wu, M Endo, Yu Ishima, M Fukagawa, M Otagiri and T Maruyama : Albumin fusion renders thioredoxin an effective anti-oxidative and anti-inflammatory agent for preventing cisplatin-induced nephrotoxicity, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1840, No.3, 1152-1162, 2014.
(要約)
A strategy for preventing cisplatin nephrotoxicity due to enhanced oxidative stress and inflammatory response is highly desirable. Thioredoxin-1 (Trx), an endogenous redox-active protein, has a short retention time in the blood. A long acting form of Trx, human serum albumin-Trx (HSA-Trx), was produced by recombinant HSA fusion and its effectiveness in preventing cisplatin nephrotoxicity was examined. HSA-Trx was prepared in Pichia expression system. Cisplatin-induced nephropathy mouse model was established by a single administration of cisplatin. Compared to saline, Trx or N-acetylcysteine, an intravenous administration of HSA-Trx attenuated the cisplatin-induced elevation in serum creatinine, blood urea nitrogen and urinary N-acetyl-β-d-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx caused a substantial reduction in the histological features of renal tubular injuries and the apoptosis-positive tubular cells. Changes in superoxide, 8-OHdG, glutathione and nitrotyrosine levels indicated that HSA-Trx significantly suppressed renal oxidative stress. HSA-Trx also suppressed the elevation of TNF-α, IL-1β and IL-6. Administered fluorescein isothiocyanate-labeled HSA-Trx was found partially localized in the proximal tubular cells whereas majority remained in the blood circulation. Specific cellular uptake and the scavenging of intracellular reactive oxygen species by HSA-Trx were observed in HK-2 cells. HSA-Trx could be a novel and effective approach for preventing cisplatin nephrotoxicity due to its prolonged anti-oxidative and anti-inflammatory action not only in extracellular compartment but also inside the proximal tubular cell. We report the renoprotective effect of HSA-Trx against cisplatin nephrotoxicity. This work would enhance developing therapeutics against acute kidney injuries including cisplatin nephrotoxicity.
H Sato, VT Chuang, K Yamasaki, N Yamaotsu, H Watanabe, K Nagumo, M Anraku, D Kadowaki, Yu Ishima, M Otagiri and T Maruyama : Differential effects of methoxy group on the interaction of curcuminoids with two major ligand binding sites of human serum albumin, PLoS ONE, Vol.9, No.2, e87919, 2014.
(要約)
Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.
K Nagumo, M Tanaka, VT Chuang, H Setoyama, H Watanabe, N Yamada, K Kubota, M Tanaka, K Matsushita, A Yoshida, H Jinnouchi, M Anraku, D Kadowaki, Yu Ishima, Y Sasaki, M Otagiri and T Maruyama : Cys34-cysteinylated human serum albumin is a sensitive plasma marker in oxidative stress-related chronic diseases, PLoS ONE, Vol.9, No.1, e85216, 2014.
(要約)
The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.
R Tanaka, H Watanabe, A Kodama, VT Chuang, Yu Ishima, K Hamasaki, K Tanaka, T Mizushima, M Otagiri and T Maruyama : Long-acting human serum albumin-thioredoxin fusion protein suppresses bleomycin-induced pulmonary fibrosis progression, The Journal of Pharmacology and Experimental Therapeutics, Vol.345, No.2, 271-283, 2013.
(要約)
Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory cells and reactive oxygen species (ROS), such as superoxide anion radical (O2(·-)). There is currently no effective treatment of IPF. We previously developed a human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an antioxidative and anti-inflammatory protein. In this study, we examined the therapeutic effect of HSA-Trx on an IPF animal model of bleomycin (BLM)-induced pulmonary fibrosis. A pharmacokinetic study of HSA-Trx or Trx in BLM mice showed that the plasma retention and lung distribution of Trxc was markedly improved by fusion with HSA. A weekly intravenous administration of HSA-Trx, but not Trx, ameliorated BLM-induced fibrosis, as evidenced by a histopathological analysis and pulmonary hydroxyproline levels. HSA-Trx suppressed active-transforming growth factor (TGF)-β levels in the lung and inhibited the increase of inflammatory cells in bronchoalveolar lavage fluid, pulmonary inflammatory cytokines, and oxidative stress markers. An in vitro EPR experiment using phosphate-buffered saline-stimulated neutrophils confirmed the O2(·-) scavenging ability of HSA-Trx. Furthermore, post-treatment of HSA-Trx had a suppressive effect against BLM-induced fibrosis. These results suggest that HSA-Trx has potential as a novel therapeutic agent for IPF, because of its long-acting antioxidative and anti-inflammatory modulation effects.
Yu Ishima, T Shinagawa, S Yoneshige, U Kragh-Hansen, Y Ohya, Y Inomata, T Kai, M Otagiri and T Maruyama : UW solution improved with high anti-apoptotic activity by S-nitrosated human serum albumin, Nitric Oxide: Biology and Chemistry, Vol.30, 36-42, 2013.
(要約)
S-Nitrosated human serum albumin (SNO-HSA) is useful in preventing liver ischemia/reperfusion injury, and SNO-HSA should thus be able to prevent cell injury during liver transplantation. However, the potential protective effect of SNO-HSA on a combination of cold and warm ischemia, which is obligatory when performing liver transplantation, has not been examined. Therefore, we evaluated the protective effect of SNO-HSA added to University of Wisconsin (UW) solution during cold or/and warm ischemia in situ and in vitro. First, we observed that apoptotic and necrotic cell death were increased during cold and warm ischemia, respectively. SNO-HSA, which possesses anti-apoptosis activity at low NO concentrations, can inhibit cold ischemia injury both in situ and in vitro. In contrast, SNO-HSA had no significant effect on warm liver ischemia injury which, however, can be reduced by UW solution. We also demonstrated that the cellular uptake of NO from SNO-HSA can occur during cold ischemia resulting in induction of heme oxygenase-1 within 3h of cold ischemia. Our results indicate that treatment with SNO-HSA or UW solution alone is not sufficient to inhibit liver injury during a period of both cold and warm ischemia. However, a combination of SNO-HSA and UW solution can be used to prevent the two types of ischemia. SNO-HSA-added UW solution could be very useful in transplantation, because the previously imposed constraints on preservation time can be removed. This is a great advantage in a situation as the present one with increased utilization of scarce donor organs for more recipients.
H Watanabe, Y Miyamoto, D Honda, H Tanaka, Q Wu, M Endo, T Noguchi, D Kadowaki, Yu Ishima, S Kotani, M Nakajima, K Kataoka, S Kim-Mitsuyama, M Tanaka, M Fukagawa, M Otagiri and T Maruyama : p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase, Kidney International, Vol.83, No.4, 582-592, 2013.
Yu Ishima, A Minomo, VT Chuang, Y Suwa, U Kragh-Hansen, T Narisoko, H Morioka, T Maruyama and M Otagiri : Albumin domain II mutant with high bilirubin binding affinity has a great potential as serum bilirubin excretion enhancer for hyperbilirubinemia treatment, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1830, No.4, 2917-2923, 2013.
(要約)
4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function. Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice. After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment. These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia. This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.
A Kodama, H Watanabe, R Tanaka, H Tanaka, VT Chuang, Y Miyamoto, Q Wu, M Endo, K Hamasaki, Yu Ishima, M Fukagawa, M Otagiri and T Maruyama : A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy, Kidney International, Vol.83, No.3, 446-456, 2013.
Yu Ishima, K Watanabe, T Akaike, T Sawa, T Kuroda, W Ogawa, H Watanabe, A Suenaga, T Kai, M Otagiri and T Maruyama : S-nitrosated _-1-acid glycoprotein kills drug-resistant bacteria and aids survival in sepsis, The FASEB journal, Vol.27, No.1, 391-398, 2013.
(要約)
Treating infections with exogenous NO, which shows broad-spectrum antimicrobial activity, appears to be effective. Similar to NO biosynthesis, biosynthesis of α-1-acid glycoprotein variant A (AGPa), with a reduced cysteine (Cys149), increases markedly during inflammation and infection. We hypothesized that AGPa is an S-nitrosation target in acute-phase proteins. This study aimed to determine whether S-nitrosated AGPa (SNO-AGPa) may be the first compound of this novel antibacterial class against multidrug-resistant bacteria. AGPa was incubated with RAW264.7 cells activated by lipopolysaccharide and interferon-γ. The antimicrobial effects of SNO-AGPa were determined by measuring the turbidity of the bacterial suspensions in vitro and survival in a murine sepsis model in vivo, respectively. Results indicated that endogenous NO generated by activated RAW264.7 cells caused S-nitrosation of AGPa at Cys149. SNO-AGPa strongly inhibited growth of gram-positive, gram-negative, and multidrug-resistant bacteria and was an extremely potent bacteriostatic compound (IC(50): 10(-9) to 10(-6) M). The antibacterial mechanism of SNO-AGPa involves S-transnitrosation from SNO-AGPa to bacterial cells. Treatment with SNO-AGPa, but not with AGPa, markedly reduced bacterial counts in blood and liver in a mouse sepsis model. The sialyl residues of AGPa seem to suppress the antibacterial activity, since SNO-asialo AGPa was more potent than SNO-AGPa.
Yu Ishima, M Hara, U Kragh-Hansen, A Inoue, A Suenaga, T Kai, H Watanabe, M Otagiri and T Maruyama : Elucidation of the therapeutic enhancer mechanism of poly-S-nitrosated human serum albumin against multidrug-resistant tumor in animal models, Journal of Controlled Release, Vol.164, No.1, 1-7, 2012.
(要約)
Human serum albumin (HSA) is the most abundant circulating protein and its S-nitrosated form serves as a reservoir of nitric oxide (NO). Previously, we prepared poly-S-nitrosated HSA (Poly-SNO-HSA) by incubation with Traut's Reagent and isopentyl nitrite and evaluated its potential as a novel anticancer agent through apoptosis involving the caspase-3 pathway. Recently, NO donors such as nitroglycerin were reported to revert the resistance to anticancer agents. Therefore, now we have evaluated the effect of the above type of Poly-SNO-HSA on the resistance to doxorubicin (dx) in human myelogenous leukemic cells (K562 cells). P-gp expression and dx accumulation in K562 and dx-resistant K562 cells (K562/dx cells) were quantified using Western blot and FACS analysis, respectively. Compared with parent K562 cells, higher expression of P-gp and lower accumulation of dx were shown in K562/dx cells. Poly-SNO-HSA caused increased dx accumulation in K562/dx cells by decreasing the expressions of P-gp and HIF-1α. Other experiments with the guanylate cyclase inhibitor ODQ and 8-Br-cGMP revealed that also a cGMP signaling pathway is involved in the Poly-SNO-HSA induced increase in dx accumulation. Furthermore, in vivo studies showed that co-treatment with Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx cells-bearing mice. Thus, in addition to its proapoptotic effect Poly-SNO-HSA can in an efficient manner revert drug resistance both in vitro and in vivo, and two pathways for this effect have been identified.
(キーワード)
ATP-Binding Cassette, Sub-Family B, Member 1 / Animals / Antibiotics, Antineoplastic / Blotting, Western / Cell Survival / Cyclic GMP / Doxorubicin / Drug Resistance, Neoplasm / Drug Synergism / Female / Humans / Hypoxia-Inducible Factor 1, alpha Subunit / K562 Cells / Mice / Mice, Inbred BALB C / Mice, Nude / Nitric Oxide Donors / Nitroso Compounds / Serum Albumin / Serum Albumin, Human / Xenograft Model Antitumor Assays
Y Miyamoto, Y Iwao, K Mera, H Watanabe, D Kadowaki, Yu Ishima, VT Chuang, K Sato, M Otagiri and T Maruyama : A uremic toxin, 3-carboxy-4-methyl-5-propyl-2-furanpropionate induces cell damage to proximal tubular cells via the generation of a radical intermediate, Biochemical Pharmacology, Vol.84, No.9, 1207-1214, 2012.
(要約)
3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a furan fatty acid uremic toxin (UT) and a substrate for organic ion transporters, contributes to the accumulation of CMPF in renal tubular cells. Although oxidative stress induced by UTs has been proposed as a mechanism of its toxicity in chronic kidney disease, little information is available regarding the redox property of CMPF and its relation to renal cell damage. The findings herein show that CMPF enhances the production of reactive oxygen species (ROS) in HK-2 cells in the presence of angiotensin II (A-II), an inducer of O(2)(·-). When iron is also present, CMPF and A-II induce the Fenton reaction, resulting in a further increase in ROS production. Such CMPF-induced oxidative stress increases TGF-β1 secretion in HK-2 cells, and a positive correlation between CMPF-induced ROS production and the secretion of active TGF-β1 was observed. CMPF caused a reduction in cell viability which was negatively correlated with intracellular ROS production. These negative effects of CMPF in HK-2 cells were completely suppressed by probenecid, an inhibitor of organic anion transport. Interestingly, in vitro ROS assays indicate that CMPF directly interacts with superoxide anion radicals (O(2)(·-)) and peroxy radicals (LOO) to produce CMPF radicals. The subsequent interaction of CMPF radicals with dissolved oxygen leads to the overproduction of O(2)(·-). Based on these findings, we conclude that CMPF, which accumulates in the renal cells, appears to play a prominent role as a pro-oxidant which subsequently leads to renal cellular damage via the overproduction of O(2)(·-).
Yu Ishima, H Hoshino, T Shinagawa, K Watanabe, T Akaike, T Sawa, U Kragh-Hansen, T Kai, H Watanabe, T Maruyama and M Otagiri : S-guanylation of human serum albumin is a unique posttranslational modification and results in a novel class of antibacterial agents, Journal of Pharmaceutical Sciences, Vol.101, No.9, 3222-3229, 2012.
(要約)
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitric oxide metabolite and an important second messenger. 8-Nitro-cGMP reacts with sulfhydryl groups forming a novel posttranslational modification, namely, S-guanylation. In this work, we found, by using a quantitative competition enzyme-linked immunosorbent assay procedure, that S-guanylated human serum albumin (S-cGMP-HSA) is a component of normal plasma, and that hemodialysis patients decrease its concentration, on an average, from 68 to 34 nM. End-stage renal disease is often accompanied by septicemia, and we found that S-cGMP-HSA possesses an in vitro antibacterial effect with half maximal inhibitory concentration of approximately 2 μM against Escherichia coli American Type Culture Collection. Our findings indicate that S-cGMP-HSA can be regarded as an endogenous antibacterial agent in healthy conditions and as a useful new class of antibacterial agents with a circulation time sufficient for in vivo biological activity. The clinical development of S-cGMP-HSA as a safe and strong antibacterial agent arisen from endogenous posttranslational modification would be expected.
(キーワード)
Adult / Aged / Aged, 80 and over / Anti-Bacterial Agents / Binding, Competitive / Case-Control Studies / Chemistry, Pharmaceutical / Circular Dichroism / Cyclic GMP / Cysteine / Dose-Response Relationship, Drug / Drug Design / Enzyme-Linked Immunosorbent Assay / Escherichia coli / Female / Humans / Japan / Kidney Failure, Chronic / Ligands / Male / Microbial Sensitivity Tests / Middle Aged / Protein Binding / Protein Processing, Post-Translational / Renal Dialysis / Serum Albumin / Serum Albumin, Human / Spectrometry, Fluorescence / Technology, Pharmaceutical
H Watanabe, T Noguchi, Y Miyamoto, D Kadowaki, S Kotani, M Nakajima, S Miyamura, Yu Ishima, M Otagiri and T Maruyama : Interaction between two sulfate-conjugated uremic toxins, p-cresyl sulfate and indoxyl sulfate, during binding with human serum albumin, Drug Metabolism and Disposition, Vol.40, No.7, 1423-1428, 2012.
(要約)
Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 10(5) M(-1) (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.
Yu Ishima, Y Iwao, J Yamada, T Noguchi, U Kragh-Hansen, K Mera, D Honda, A Suenaga, T Maruyama and M Otagiri : Quantitative evaluation of the role of cysteine and methionine residues in the antioxidant activity of human serum albumin using recombinant mutants, IUBMB life, Vol.64, No.5, 450-454, 2012.
(要約)
The importance of cysteine (Cys) and methionine (Met) residues for the antioxidant activity of human serum albumin (HSA) was investigated using recombinant HSA mutants, in which Cys34 and/or the six Met residues had been mutated to Ala. The scavenging activities of the mutants against five reactive oxygen and nitrogen species were evaluated by a chemiluminescence assay, electron paramagnetic resonance spectroscopy, or a HPLC-flow reactor assay. Our results showed that the contributions of Cys34 and the Met residues to the antioxidant activity of HSA were 61% and 29% against O(2)(·-), 68% and 61% against H(2)O(2), 38% and 6% against HO(·), 36% and 13% against HOCl, and 51% and 1% against (·)NO, respectively. Thus, the findings propose in a direct way that Cys34 plays a more important role than the Met residues in the antioxidant activity of HSA.
Yu Ishima, D Chen, J Fang, H Maeda, A Minomo, U Kragh-Hansen, T Kai, T Maruyama and M Otagiri : S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects, Bioconjugate Chemistry, Vol.23, No.2, 264-271, 2012.
(要約)
Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.
Ibrahim Mohamed, Eslam Mostafa Ramadan Abdelhameed, Nehal Ali Emam Elsadek Emam Elhewan, Sherif Abdallah Emam Emam, Taro Shimizu, Hidenori ANDO, Yu Ishima, Elgarhy Helmy Omar, Sarhan A Hatem, Hussein K Amal and Tatsuhiro Ishida : Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products, Journal of Controlled Release, Vol.351, 215-230, Nov. 2022.
Nana Matsuo, Hidenori ANDO, Yusuke Doi, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : The challenge to deliver oxaliplatin (l-OHP) to solid tumors: development of liposomal l-OHP formulations, Chemical & Pharmaceutical Bulletin, Vol.70, No.5, 351-358, May 2022.
Yu Ishima, Toru Maruyama, Masaki Otagiri, G Victor T Chuang and Tatsuhiro Ishida : The new delivery strategy of albumin carrier utilizing the interaction with albumin receptors, Chemical & Pharmaceutical Bulletin, Vol.70, No.5, 330-333, May 2022.
Yu Ishima, Toru Maruyama, Masaki Otagiri and Tatsuhiro Ishida : Drug Delivery System for Refractory Cancer Therapy via an Endogenous Albumin Transport System, Chemical & Pharmaceutical Bulletin, Vol.68, No.7, 583-588, Jul. 2020.
Yu Ishima, U Kragh-Hansen, T Maruyama and M Otagiri : Poly-S-Nitrosated Albumin as a Safe and Effective Multifunctional Antitumor Agent: Characterization, Biochemistry and Possible Future Therapeutic Applications, BioMed Research International, Vol.2013, 353892, Dec. 2013.
(要約)
Nitric oxide (NO) is a ubiquitous molecule involved in multiple cellular functions. Inappropriate production of NO may lead to disease states. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated human serum albumin (Mono-SNO-HSA), and that is why we are testing whether this albumin form can be therapeutically useful. Recently, we developed SNO-HSA analogs such as SNO-HSA with many conjugated SNO groups (Poly-SNO-HSA) which were prepared using chemical modification. Unexpectedly, we found striking inverse effects between Poly-SNO-HSA and Mono-SNO-HSA. Despite the fact that Mono-SNO-HSA inhibits apoptosis, Poly-SNO-HSA possesses very strong proapoptotic effects against tumor cells. Furthermore, Poly-SNO-HSA can reduce or perhaps completely eliminate the multidrug resistance often developed by cancer cells. In this review, we forward the possibility that Poly-SNO-HSA can be used as a safe and effective multifunctional antitumor agent.
Haruka Yamamoto, Hidenori ANDO, Omoto Yasukazu, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Induction of neutralizing antibodies by immunization with inactivated human TNF-alpha in mice, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
2.
Shunto Yamamoto, Hidenori ANDO, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Development of a novel technique for antibody induction against membrane proteins by spleen immunization with membrane protein-loaded PEG-modified liposomes, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
3.
Takaaki Matsuzaki, Taro Shimizu, Hidenori ANDO, Yu Ishima, K Yamanaka, H Hamamoto and Tatsuhiro Ishida : An ionic liquids-based topical antitumor vaccine: a mechanism for induction of antitumor immunity via topical application of cancer-antigen peptides, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
4.
Aiko Hashimoto, Yu Ishima, Hidenori ANDO, Taro Shimizu and Tatsuhiro Ishida : Differential organ-specific distribution of human serum albumin denatured by various modifications, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
5.
Riku Uehara, Hidenori ANDO, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Neutralization of acidic tumor microenvironment by dosing of sodium potassium citrate (K/Na citrate) enhances antitumor effects of Abraxane®, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
6.
Shoichiro Fukuda, Hidenori ANDO, Atsuya Maruyama, Takashi Nakae, Noboru Tatsumi, Hidetoshi Hamamoto, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : The mechanism investigation of intestinal absorption enhancement of drugs using ionic liquids, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
7.
Eslam Mostafa Ramadan Abdelhameed, Hidenori ANDO, Haruka Yamamoto, Mako Fukumoto, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Preparation, optimization, and evaluation of anionic DPPG-based LNPs for delivery of peptide antigens, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
8.
Nana Matsuo, Hidenori ANDO, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Oral sodium bicarbonate enhances accumulation and antitumor effects of PEGylated liposomal doxorubicin (Doxil®), 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
9.
Yoshino Kawaguchi, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Transfusion of mouse B cells, embedded antigens with hydroxyl PEG-modified liposomes in vitro, induces cellular immune responses in mice, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
10.
A S Gaballa, Taro Shimizu, Hidenori ANDO, Yu Ishima, Sherif Abdallah Emam Emam, M Ibrahim, M F Mady, W Y Naguib, A K Khaled and Tatsuhiro Ishida : Accelerated blood clearance of PEGylated liposomal antitumor agents after topical application of PEG derivatives containing cosmetics in a mouse model, 15th International Symposium on Nanomedicine, Tokushima, Dec. 2022.
11.
Takeru Hirai, Nanami Tasaka, Hidenori ANDO, Taro Shimizu, Tatsuhiro Ishida and Yu Ishima : Biological roles of supersulfide in human hair, Redox Week in Sendai 2022, Sendai, Oct. 2022.
12.
Yamade Rina, Taro Shimizu, Hirakawa Naoki, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Human serum albumin (HSA)-based nanocarriers efficiently deliver antigens to the spleen and induce antigen-specific humoral immunity, 14th International Symposium on Nanomedicine, Nov. 2021.
13.
Haruka Takata, Taro Shimizu, Ueda Hiro, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : The induction level of anti-PEG IgM by PEGylated liposomes depends on the route of administration and the presence of nucleic acids, 14th International Symposium on Nanomedicine, Nov. 2021.
14.
Hitomi Okuyama, Mayumi Ikeda, Yu Ishima, Miyu Nishikawa, Shinichi Ikushiro and Rie Mukai : Binding profile of quercetin and its derivatives to human serum albumin measured by multiplex drug-site mapping, ICoFF2019/ISNFF2019/ICPH2019, Kobe, Nov. 2019.
15.
Mayumi Ikeda, Yu Ishima, T Maruyama, T Akaike, M Otagiri and Tatsuhiro Ishida : A novel S-sulfhydrated human serum albumin suppresses reactive oxygen species induced by kidney injuries, 17th International Symposium on Blood Substitutes & Oxygen Therapeutics, Nara, Nov. 2019.
16.
Taro Shimizu, Kohga Miyahara, G Kozma, Hidenori ANDO, Yu Ishima, J Szebeni and Tatsuhiro Ishida : Pre-treatment with Doxebo suppresses anti-PEG IgM immune responses through PEG-specific immune tolerance, 17th International Symposium on Blood Substitutes & Oxygen Therapeutics, Nara, Nov. 2019.
17.
M Otagiri, Yu Ishima, K Taguchi and T Maruyama : Human albumin based drug delivery: SNO-albumin dimer for cancer therapeutic application, 17th International Symposium on Blood Substitutes & Oxygen Therapeutics, Nara, Nov. 2019.
18.
Taro Shimizu, Shunsuke Takese, Yu Ishima and Tatsuhiro Ishida : Combination of cancer vaccines with liposomal oxaliplatin increased anti-cancer therapeutic efficacy by stimulating antitumor immunity, Liposome Research Days 2019, Sapporo, Sep. 2019.
19.
Yuna Shimazaki, Hidenori ANDO, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Immunization method to obtain antigen-specific antibodies by antigen delivery to splenic marginal zone B cells using PEGylated liposomes, Liposome Research Days 2019, Sapporo, Sep. 2019.
20.
Yu Ishima : Quantitative determination of polysulfide in plasma proteins and biological human fluids using a novel Sulfide elimination from polysulfide (SEP) method, The 1st International Conference on Persulfide and Sulfur Metabolism in Biology and Medicine, Sendai, Sep. 2019.
21.
Mayumi Ikeda, Yu Ishima, H Watanabe, T Maruyama, M Otagiri and Tatsuhiro Ishida : Functional Elucidation of Polysulfides In Serum Albumin and Development of Biomimetic Donor of Polysulfides, 2019 CRS Annual Meeting & Exposition, Valencia, Jul. 2019.
22.
Mayumi Ikeda, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : A Novel S-Sulfhydrated Serum Albumin Preparation Suppresses Melanin Synthesis, AAPS PharmSci 360, Washington, D.C., Nov. 2018.
23.
Emam Emam Abdallah Sherif, Hidenori ANDO, Lila Selim Ahmed Ali Abu Amr, Yu Ishima, A M Mahdy, S F Ghazy and Tatsuhiro Ishida : The effect of liposome co-incubation with cancer cells on the secretion, uptake propensity and expression of certain surface proteins of cancer cell-derived exosomes (extracellular vesicles), 2018 CRS Annual Meeting & Exposition, New York, Jul. 2018.
24.
Nanami Tasaka, Mayumi Ikeda, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : Reactive Sulfur Species in Human Hair Keratin Respond To Oxidative Stress, 5th World Congress on Hydrogen Sulfide in Biology&Medicine, Toronto, Jun. 2018.
25.
Mayumi Ikeda, Nanami Tasaka, Taro Shimizu, Yu Ishima and Tatsuhiro Ishida : A Novel S-Sulfhydrated Human Serum Albumin Preparation Suppresses Melanin Synhtesis, 5th World Congress on Hydrogen Sulfide in Biology&Medicine, Toronto, Jun. 2018.
26.
Kinoshita Ryo, Yu Ishima, Ikeda Mayumi, Nakamura Hideaki, Fang Jun, Maeda Hiroshi, Watanabe Hiroshi, Otagiri Masaki and Maruyama Toru : Development of the next-generation nab-paclitaxel therapy by using a novel nano EPR effect accelerator, S-nitrosated human serum albumin dimer, Controlled Release Society 2017, Boston, Jul. 2017.
27.
Ryo Kinoshita, Yu Ishima, Mayumi Ikeda, Jun Fang, Hiroshi Maeda, Masaki Otagiri and Toru Maruyama : S-Nitrosated human serum albumin dimer as a novel nano EPR enhancer applied to nanotechnology based anticancer drugs, 2016Controlled Release Society Annual Meeting & Exposition, Washington, D.C., Jul. 2016.
28.
Yu Ishima, Shinagawa Takuya, Yoneshige Shinji, Otagiri Masaki and Maruyama Toru : UW SOLUTION IMPROVED WITH HIGH ANTI-APOPTOTIC ACTIVITY BY S-NITROSATED HUMAN SERUM ALBUMIN, The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, May 2016.
29.
Kinoshita Ryo, Yu Ishima, Ikeda Mayumi, Fang Jun, Maeda Hiroshi, Otagiri Masaki and Maruyama Toru : S-Nitrosated human serum albumin dimer as a novel nano EPR enhancer applied to nanotechnology-based anticancer drug, The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, May 2016.
30.
Ikeda Mayumi, Yu Ishima, Kinoshita Ryo, Watanabe Hiroshi, Ikeda Tsuyoshi, Otagiri Masaki and Maruyama Toru : Anti-tumor effect of Poly-S-nitrosated Human Serum Albumin is enhanced by phosphodiesterase 5 inhibitors, The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, May 2016.
31.
Ikeda Mayumi, Yu Ishima, Shibata Motonori, Watanabe Hiroshi, Xian Ming, Ouchi Yuya, Akaike Takaaki and Maruyama Toru : Sulfide Anti-Oxidant Buffer Enables Polysulfur to Be Detected by Methylene Blue Assay, The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, May 2016.
32.
Matsusaka Kotaro, Yu Ishima, Watanabe Kaori, Watanabe Hiroshi, Otagiri Masaki and Maruyama Toru : Therapeutic potential of S-nitrosated 1-acid glycoprotein as a novel antibacterial agent, The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide, Sendai, May 2016.
33.
Ikeda Mayumi, Yu Ishima, Kinoshita Ryo, Watanabe Hiroshi, Ikeda Tsuyoshi, Otagiri Masaki and Maruyama Toru : Anti-tumor effect of Poly-S-nitrosated Human Serum Albumin is enhanced by phosphodiesterase 5 inhibitors, Asian Federation for Pharmaceutical Sciences 2015, Bangkok, Nov. 2015.
34.
Kinoshita Ryo, Yu Ishima, Ikeda Mayumi, Fang Jun, Maeda Hiroshi, Otagiri Masaki and Maruyama Toru : S-Nitrosated human serum albumin dimer as novel nano EPR enhancer applied to nano-anticancer drugs, Asian Federation for Pharmaceutical Sciences 2015, Bangkok, Nov. 2015.
35.
Maeda Hitoshi, Ichimizu Shota, Watanabe Hiroshi, Yu Ishima, Suenaga Ayaka, Otagiri Masaki and Maruyama Toru : Polythiolated- And Recombinant Mannosylated-Albumin As A Novel Cd68+/Cd206+ Kupffer Cell-Targeted Nano-Antioxidant For The Treatment Of Acute And Chronic Hepatitis Models, 19th North American ISSX Meeting and 29th JSSX Meeting, San Francisco, Oct. 2014.
36.
Watanabe Hiroshi, Sakaguchi Yoshiaki, Sugimoto Ryusei, Yu Ishima, Kaneko Ken-ichi, Iwata Hiroshi, Otagiri Masaki and Maruyama Toru : Human organic anion transporters function as a high-capacity transporter for p-cresyl sulfate, 8th International Society on Uremia Research and Toxicity, Okinawa, Mar. 2014.
37.
Watanabe Kaori, Yu Ishima, Kuroda Teruo, Ogawa Wakano, Watanabe Hiroshi, Suenaga Ayaka, Kai Toshiya, Otagiri Masaki and Maruyama Toru : S-nitrosated a-1-acid glycoprotein kills drug-resistant bacteria and aids survival in sepsis, Asian Federation for Pharmaceutical Sciences 2013, Cheju, Nov. 2013.
38.
Tanaka Ryota, Maeda Hitoshi, Kodama Azusa, Watanabe Hiroshi, Yu Ishima, Maruyama Toru and Otagiri Masaki : Therapeutic impact of human serum albumin-thioredoxin fusion protein, long-acting anti-oxidative and anti-inflammatory modulator, against acetaminophen-induced acute liver failure, Asian Federation for Pharmaceutical Sciences 2013, Cheju, Nov. 2013.
39.
Watanabe Hiroshi, Kodama Azusa, Tanaka Ryota, Tanaka Hisae, Chuang Tuan Giam Victor, Yu Ishima, Fukagawa Masafumi, Otagiri Masaki and Maruyama Toru : A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy, Asian Federation for Pharmaceutical Sciences 2013, Cheju, Nov. 2013.
40.
Yu Ishima, Hara Marie, Watanabe Hiroshi, Otagiri Masaki and Maruyama Toru : Poly-S-nitrosated Human Serum Albumin inhibits the Expression of P-glycoprotein Transporter in Human Multidrug-resistant Tumor, The 5th Asian Arden conference, 愛知, Aug. 2013.
41.
Maeda Hitoshi, Watanabe Hiroshi, Yu Ishima, Otagiri Masaki and Maruyama Toru : Kupffer cell selective delivery of thiols by genetically engineered mannosylated-albumin as a new therapeutic strategy for hepatitis, The 5th Asian Arden conference, 愛知, Aug. 2013.
Rina Yamade, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Investigation of the usefulness of human serum albumin nanoparticles as antigen delivery carriers to the spleen, 日本薬剤学会第2回英語セミナー, Feb. 2023.
Yoshino Kawaguchi, Taro Shimizu, Hidenori ANDO, Yu Ishima and Tatsuhiro Ishida : Enhancement of the anti-tumor effect of B cell-based vaccines via increasing the loading amount of antigens by utilizing novel antigen carrier system, 2021 Tokushima Bioscience Retreat, Sep. 2021.