Masako Ichihara, Naoto Moriyoshi, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Anti-PEG IgM production via a PEGylated nano-carrier system for nucleic acid delivery., Springer, Jun. 2012.
(Summary)
For the systemic application of nucleic acids such as plasmid DNA and small interfering RNA, safe and efficient carriers that overcome the poor pharmacokinetic properties of nucleic acids are required. A cationic liposome that can formulate lipoplexes with nucleic acids has significant promise as an efficient delivery system in gene therapy. To achieve in vivo stability and long circulation, most lipoplexes are modified with PEG (PEGylation). However, we reported that PEGylated liposomes lose their long-circulating properties when they are injected repeatedly at certain intervals in the same animal. This unexpected and undesirable phenomenon is referred to as the accelerated blood clearance (ABC) phenomenon. Anti-PEG IgM produced in response to the first dose of PEGylated liposomes has proven to be a major cause of the ABC phenomenon. Therefore, in a repeated dosing schedule, the detection of anti-PEG IgM in an animal treated with PEGylated lipoplex could be essential to predict the occurrence of the ABC phenomenon. This chapter introduces a method for the evaluation of serum anti-PEG IgM by a simple ELISA procedure, and describes some precautions associated with this method.
(Keyword)
Animals / DNA / Drug Carriers / Enzyme-Linked Immunosorbent Assay / Horseradish Peroxidase / Immunoglobulin M / Liposomes / Male / Mice / Mice, Inbred BALB C / Nanostructures / Polyethylene Glycols / RNA, Small Interfering
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Cationic liposomes and tumour vasculature targeting: a therapeutic approach that has potential for solid tumours., Souto, E.B. (Ed.), i-Smithers - Creative Publishing Solutions, Aug. 2011.
4.
Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Breaking the barriers to tumor-targeting via nanocarrier-based drug delivery to the tumor microenvironment., Nova Science Publishers, 2010.
5.
Jose Mario Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : Complexation of siRNA and pDNA with cationic liposomes: The important aspects in lipoplex preparation., A chapter, Liposomes, Methods Mol. Biol., 2010.
(Summary)
In the last two decades, cationic liposomes have been investigated as vehicles for nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)] delivery in vitro and in vivo. The formation of cationic liposomes-nucleic acids complexes, termed lipoplexes, depends on a number of experimental variables. The quality of the nucleic acid and the cationic liposome as well as the selection of diluents for diluting the concentrated stocks strongly affect the resulting lipoplexes and their efficiency of gene-expression or gene-silencing effect following transfection. In addition, the molar ratio of cationic lipid nitrogen (N) to siRNA or pDNA phosphate (P) (N/P ratio) influences the final characteristics of the lipoplexes, such as size, surface zeta potential, and reproducibility, thereby reflecting their efficiency following transfection. The methods presented in this chapter could be helpful to obtain reliable and reproducible lipoplexes and experimental results.
(Keyword)
Cations / DNA / Gene Silencing / HeLa Cells / Humans / Liposomes / Plasmids / RNA, Small Interfering / Transfection
Tatsuhiro Ishida and Hiroshi Kiwada : Synergistic antitumor activity of metronomic dosing chemotherapy in combination with liposomal anticancer drug., Nova Science Publishers, Apr. 2009.
7.
Tatsuhiro Ishida and Hiroshi Kiwada : Unexpected reactions by in vivo application of PEGylated liposomes., Springer Science + Business Media, New York, USA, 2009.
8.
Tatsuhiro Ishida and Hiroshi Kiwada : 第10章医学薬学でのリポソーム応用 1節リポソームと細胞の相互作用, NTS Inc., Tokyo, Jun. 2005.
9.
Tatsuhiro Ishida and Hiroshi Kiwada : 第10章医学薬学でのリポソーム応用 3節長期血中滞留性リポソーム, NTS Inc., Tokyo, Jun. 2005.
10.
Hiroshi Kiwada : 総合製剤学, NANZANDO Co.,Ltd., Tokyo, Apr. 2000.
Hiroshi Kiwada : Adv. Drug Deliv. Rev. (Theme Editor), --- The pharmacokinetics of liposomes for tumor targeting ---, Sep. 1999.
13.
Hiroshi Kiwada : ライフサイエンスにおけるリポソーム·実験マニュアル, シュプリンガー·フェアラーク東京, Aug. 1992.
14.
Hiroshi Kiwada : ライフサイエンスにおけるリポソーム 実験マニュアル 寺田弘，吉村哲郎編, Springer-Verlag, Tokyo, 1992.
15.
Hiroshi Kiwada : 生物薬剤学ー最近の進歩 伊賀立二，奥村勝彦編, (株) 薬業時報社, Tokyo, 1989.
16.
C Anthony Hunt, Hiroshi Kiwada, M Reader-Shikorr, K Hirano, J Nakamura, R Abra and H Schreier : Liposomes:Are they effective drug carriers?Topics in Pharmaceutical Sciences 1983, 1983.
Academic Paper (Judged Full Paper):
1.
Hiroyuki Nakamura, Lila Selim Ahmed Ali Abu Amr, Miho Nishio, Masao Tanaka, Hidenori ANDO, Hiroshi Kiwada and Tatsuhiro Ishida : Intra-tumor distribution of PEGylated liposome upon repeated injection: No possession by prior dose, Journal of Controlled Release, Vol.220, 406-413, 2015.
(Summary)
Liposomes have proven to be a viable means for the delivery of chemotherapeutic agents to solid tumors. However, significant variability has been detected in their intra-tumor accumulation and distribution, resulting in compromised therapeutic outcomes. We recently examined the intra-tumor accumulation and distribution of weekly sequentially administered oxaliplatin (l-OHP)-containing PEGylated liposomes. In that study, the first and second doses of l-OHP-containing PEGylated liposomes were distributed diversely and broadly within tumor tissues, resulting in a potent anti-tumor efficacy. However, little is known about the mechanism underlying such a diverse and broad liposome distribution. Therefore, in the present study, we investigated the influence of dosage interval on the intra-tumor accumulation and distribution of "empty" PEGylated liposomes. Intra-tumor distribution of sequentially administered "empty" PEGylated liposomes was altered in a dosing interval-dependent manner. In addition, the intra-tumor distribution pattern was closely related to the chronological alteration of tumor blood flow as well as vascular permeability in the growing tumor tissue. These results suggest that the sequential administrations of PEGylated liposomes in well-spaced intervals might allow the distribution to different areas and enhance the total bulk accumulation within tumor tissue, resulting in better therapeutic efficacy of the encapsulated payload. This study may provide useful information for a better design of therapeutic regimens involving multiple administrations of nanocarrier drug delivery systems.
Taro Shimizu, Yu Mima, Yosuke Hashimoto, Masami Ukawa, Hidenori ANDO, Hiroshi Kiwada and Tatsuhiro Ishida : Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells, Immunobiology, Vol.220, No.10, 1151-1160, 2015.
(Summary)
The accelerated blood clearance (ABC) phenomenon makes it crucial to use PEGylated liposomes and micelles to deliver drugs. The ABC phenomenon is an immune response against an initial dose of PEGylated liposome, which causes subsequent doses to be rapidly cleared by macrophages in the liver. We recently found that in the early phase of the ABC phenomenon, subsequent doses of PEGylated liposomes were associated with splenic marginal zone (MZ)-B cells and were transported from the MZ to the follicle (FO). In this study, we investigated the underlying mechanisms behind the association of subsequent doses of PEGylated liposomes with MZ-B cells in the spleen. Serum factors, anti-PEG IgM and complement system, were crucial to the association of PEGylated liposomes with MZ-B cells, while the sensitization of MZ-B cells by the first dose of PEGylated liposomes was not significant. It was the complement receptors (CRs) on the MZ-B cells, rather than either the PEG-specific B-cell receptors or the IgM Fc receptors, that were the main contributors to the association between PEGylated liposomes and MZ-B cells. It appeared that anti-PEG IgM would bind to PEGylated liposomes and causes subsequent complement activation, resulting in the formation of immune complexes of PEGylated liposome-anti-PEG IgM-complement. The MZ-B cells then recognized these immune complexes via their CRs. Such an association via CRs might have triggered the transport of the immune complex by MZ-B cells to the FO in the spleen. The information obtained in this study might be useful in the development of an efficient antigen delivery system to usher PEGylated nanoparticles into FO dendritic cells.
Munehira Kawanishi, Yosuke Hashimoto, Taro Shimizu, Ikuko Sagawa, Tatsuhiro Ishida and Hiroshi Kiwada : Comprehensive analysis of PEGylated liposome-asscociated proteins relating to the accelerated blood clearance phenomenon by combination with shotgun analysis and conventional methods, Biotechnology and Applied Biochemistry, Vol.62, No.4, 547-555, 2015.
(Summary)
PEGylated liposome, sterically stabilized by polyethylene glycol (PEG), results in reduced recognition of the liposome by the mononuclear phagocyte system. Recently, we reported regarding the accelerated blood clearance (ABC) phenomenon that PEGylated liposome is cleared very rapidly from blood circulation upon repeated injection. Anti-PEG IgM production and subsequent complement activation were crucial in causing the ABC phenomenon. However, there still remains the possibility that unknown plasma factors might affect the fate of PEGylated liposome that is subjected to the ABC phenomenon. A label-free approach to shotgun analysis is a great tool for characterizing proteins in a biological system. In this study, therefore, a shotgun analysis was employed to identify plasma protein bound on PEGylated liposome after the ABC phenomenon was induced in the mouse model. The analysis revealed that immunoglobulin and complement components (C1 and C3) are the major proteins. Subsequent analysis with enzyme-linked immunosorbent assay and Western blotting showed that the immunoglobulin was IgM and that the complement system was mainly activated via an anti-PEG IgM-mediated classical pathway. These results support our earlier assumptions-anti-PEG IgM and complement activation were the major causes of the ABC phenomenon. Our proposed analytical strategy would be expected to provide useful information for the development and design of the nanocarrier drug delivery system.
Yu Mima, Yosuke Hashimoto, Taro Shimizu, Hiroshi Kiwada and Tatsuhiro Ishida : Anti-PEG IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein, Molecular Pharmaceutics, Vol.12, No.7, 2429-2435, 2015.
(Summary)
Limited therapeutic efficacy of polyethylene glycol-conjugated (PEGylated) protein drugs has been recently reported in animals and human following repeat injections. Since there are reports that an accelerated blood clearance (ABC) phenomenon is caused by repeated injection of PEGylated liposome, there is an assumption that PEGylated proteins lose their long circulating property when they are injected repeatedly due to the induction of anti-PEG antibody. Although induction of anti-PEG antibody by PEGylated protein has been reported, there is little evidence of accelerated blood clearance of PEGylated protein upon repeated injection. Herein, we investigated the blood concentration of PEGylated ovalbumin (PEG-OVA), a model PEGylated protein, upon its repeated injection. A single intravenous administration of PEG-OVA elicited an anti-PEG IgM response but not anti-PEG IgG response, while the administration did not elicit antibody against OVA. At 24 h postinjection of test PEG-OVA, although control mice showed 41.6% dose of PEG-OVA in blood, the mice pretreated with PEG-OVA showed rapid clearance of test PEG-OVA from blood and undetectable level of PEG-OVA. Interestingly, the anti-PEG IgM induced by PEGylated liposome did not affect the blood concentration of subsequent dose of PEG-OVA. Our result suggests that anti-PEG IgM is a major contributor to the accelerated blood clearance of PEG-conjugated protein, but the presence of anti-PEG IgM in blood circulation does not necessarily affect circulating property of entire PEGylated materials.
Yosuke Hashimoto, Taro Shimizu, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Relationship between the concentration of anti-polyethylene glycol (PEG) in immunoglobulin M (IgM) and the intensity of the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes in mice, Biological & Pharmaceutical Bulletin, Vol.38, No.3, 417-424, 2015.
(Summary)
PEGylation, which is the surface modification of nanocarriers with polyethylene glycol (PEG), has increased the circulation time and reduced the immunogenic responses to nanocarriers. However, many reports have demonstrated that the intravenous injection of sterically stabilized PEGylated liposome (SL) causes an accelerated blood clearance (ABC) of subsequent doses via anti-PEG immunoglobulin M (IgM)-mediated complement activation. In the present study, the relationships between serum anti-PEG IgM concentration, the intensity of complement activation and the hepatic clearance of SL were quantitatively investigated for their role in the ABC phenomenon. Interestingly, with increasing serum anti-PEG IgM concentrations, the intensity of complement activation increased linearly, while the intensity of the hepatic clearance of SL was increased and then saturated. In addition, only 15-17% of anti-PEG IgM in blood circulation induced by SL at different doses was associated with a second dose SL. The present results indicate that it is the hepatic uptake of SL that is the limiting step in the ABC phenomenon, rather than the association of anti-PEG IgM to the SL and a subsequent complement activation.
Takuya Suzuki, Masako Ichihara, Kenji Hyodo, Eiichi Yamamoto, Tatsuhiro Ishida, Hiroshi Kiwada, Hiroshi Kikuchi and Hiroshi Ishihara : Influence of dose and animal species on accelerated blood clearance of PEGylated liposomal doxorubicin, International Journal of Pharmaceutics, Vol.476, 205-212, 2014.
(Summary)
We recently demonstrated that Doxil loses its long-circulating properties when injected repeatedly at doses below 2 mg/m(2) in dogs. In studies using other animal species, PEGylated liposomal doxorubicin has been reported not to induce the accelerated blood clearance (ABC) phenomenon. We investigated the issue of whether Doxil can elicit the ABC phenomenon in several species. In minipigs, the ABC phenomenon was induced at 2 mg/m(2). In other animal species, the ABC phenomenon was not observed at higher doses (>2 mg/m(2)), but was observed at much lower doses (0.2 mg/m(2)). The pharmacokinetic profile of a second dose of Doxil reflected the circulating anti-PEG IgM level induced by the first dose. The ABC phenomenon was not observed at the clinically recommended DXR dose (20 mg/m(2)) in any animal species. These results indicate that Doxil can cause the ABC phenomenon in all animals tested, the extent of induction was dependent on the first dose of Doxil, and a higher Doxil dose lessened the ABC phenomenon. The current study results suggest that a careful study design including selection of animal species is important for preclinical studies using PEGylated liposomal formulations even if they contain anticancer drugs that suppress the host immune response.
Yosuke Hashimoto, Lila Selim Ahmed Ali Abu Amr, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : B cell-intrinsic toll-like receptor 7 is responsible for the enhanced anti-PEG IgM production following injection of siRNA-containing PEGylated lipoplex in mice, Journal of Controlled Release, Vol.184, 1-8, 2014.
(Summary)
Recently, we reported that immunostimulatory siRNA-containing PEGylated lipoplex (PEGylated siRNA-lipoplex) activates the immune system, resulting in the enhanced production of anti-PEG IgM. However, the enhancing mechanism upon anti-PEG IgM production has not been fully elucidated. In this study, we employed toll-like receptor 7 knock out (TLR7 KO) mice, and showed how PEGylated siRNA-lipoplex activates the innate immune system through TLR7 and consequently enhances anti-PEG IgM production. In addition, we showed that SCID mice reconstituted with TLR7-deficient B cells failed to enhance anti-PEG IgM production following the injection of PEGylated siRNA-lipoplex, but that SCID mice reconstituted with wild type B cells did enhance anti-PEG IgM production. These results suggest that immune activation via B cell-intrinsic TLR7, but not other TLR7-expressing cells, contributes predominantly to an enhanced anti-PEG IgM production in response to the intravenous injection of PEGylated siRNA-lipoplexes. A strategy to evade B cell-intrinsic TLR7 activation by siRNA, such as chemical modification, may overcome immunological barriers to PEGylated liposome-based siRNA therapeutics.
(Keyword)
Adoptive Transfer / Animals / B-Lymphocytes / Cytokines / Immunoglobulin M / Liposomes / Male / Membrane Glycoproteins / Mice, Inbred BALB C / Mice, Knockout / Mice, SCID / Polyethylene Glycols / RNA, Small Interfering / Spleen / Toll-Like Receptor 7
Yosuke Hashimoto, Yumi Uehara, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Activation of TLR9 by incorporated pDNA within PEG-coated lipoplex enhances anti-PEG IgM production, Gene Therapy, Vol.21, No.6, 593-598, 2014.
(Summary)
Cationic liposome represents a promising alternative to viral vectors for the delivery of therapeutic genes. For in vivo use, surface modification of the liposome with polyethylene glycol (PEG) is frequently applied to achieve gene-expression in the targeted tissue. However, we have reported that PEG-coated liposomes have induced anti-PEG IgM, which has caused subsequent doses of PEG-coated liposome to be rapidly cleared from blood circulation, and the complexation of pDNA electrostatically associated with liposome surface has enhanced this antibody response. In this study, we investigated how a Toll-like receptor (TLR) might enhance anti-PEG IgM production. PEG-coated pDNA-lipoplex (PDCL) was injected into either wild type, MyD88 (all TLR adaptor protein, independent of TLR3) knock out (KO) or TLR9 KO mice, and the anti-PEG IgM production levels were detected. Attenuated anti-PEG IgM production following the injection of PDCL was observed in both MyD88 and TLR9 KO mice compared to wild type mice, probably due to the abolished induction of cytokines in both MyD88 and TLR9 KO mice. Our results suggest that TLR, exclusively TLR9, signaling plays a potential role in the enhanced anti-PEG IgM production following the injection of PDCL. This result may have important implications for the design and development of an efficient PEG-coated non-viral gene vector.
Yosuke Hashimoto, Taro Shimizu, Yu Mima, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs, Toxicology and Applied Pharmacology, Vol.277, No.1, 30-38, 2014.
(Summary)
PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs--HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG2000 conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH2CH2) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics.
Hiroyuki Nakamura, Yusuke Doi, Lila Selim Ahmed Ali Abu Amr, Ai Nagao, Tatsuhiro Ishida and Hiroshi Kiwada : Sequential treatment of oxaliplatin-containing PEGylated liposome together with S-1 improves intratumor distribution of subsequent doses of oxaliplatin-containing PEGylated liposome, European Journal of Pharmaceutics and Biopharmaceutics, Vol.87, No.1, 142-151, 2014.
(Summary)
We recently reported that combination therapy with metronomic S-1 dosing and oxaliplatin (l-OHP)-containing PEGylated liposomes improved antitumor activity in a murine colorectal tumor model. However, little is known about the mechanism underlying such improved therapeutic efficacy. Here we investigated the impact of combined treatment on biodistribution, tumor accumulation and intratumor distribution of test PEGylated liposomes and on the structure of tumor vasculature in a solid tumor. The combined treatment clearly enhanced tumor accumulation and intratumor distribution of a subsequent test dose of PEGylated liposome as a result of on the one hand prolonging blood circulation of test liposome and on the other hand the alteration in tumor microenvironment. The l-OHP-containing PEGylated liposomes contributed predominantly to the enhanced tumor accumulation and altered tumor distribution of test liposome. On the other hand, metronomic S-1 dosing contributed to the altered tumor distribution but not the tumor accumulation of test liposome. The antitumor effect of the combined treatment, reflected by the proportion of apoptotic cells in the tumor, was approximately equally accounted for by each of the two treatments, leading to a roughly additive effect. In conclusion, 1-OHP-containing PEGylated liposome together with S-1 enhanced intratumor influx, leading to improved antitumor activity of subsequently injected 1-OHP-containing PEGylated liposomes and/or S-1. This strategy we propose, which is clinically applicable, may overcome the problems related to the use of EPR effect-based nanocarrier systems.
(Keyword)
Animals / Antineoplastic Agents / Apoptosis / Cell Line, Tumor / Colorectal Neoplasms / Drug Administration Schedule / Drug Carriers / Drug Combinations / Drug Therapy, Combination / Liposomes / Liver / Male / Mice, Inbred BALB C / Organoplatinum Compounds / Oxonic Acid / Polyethylene Glycols / Tegafur / Tissue Distribution
Lila Selim Ahmed Ali Abu Amr, Yumi Uehara, Tatsuhiro Ishida and Hiroshi Kiwada : Application of polyglycerol-coating to pDNA lipoplex for the evasion of the accelerated blood clearance (ABC) phenomenon in nucleic acid delivery., Journal of Pharmaceutical Sciences, Vol.103, No.2, 557-566, 2014.
(Summary)
Cationic liposomes (CLs) have shown promise as nonviral delivery systems. To achieve in vivo stability and long circulation, most liposomes are modified with hydrophilic polymer polyethylene glycol (PEG). However, we have reported that repeated administration of PEG-coated CLs containing plasmid DNA (pDNA; PEGylated lipoplexes) induces what is referred to as "the accelerated blood clearance (ABC) phenomenon" and, consequently, subsequently administered lipoplexes lose their prolonged circulation characteristics. Anti-PEG IgM produced in response to the first dose of PEG-coated pDNA-lipoplexes (PEG-DCL) has proven to be a major cause of the ABC phenomenon. In this study, to evade and/or attenuate this unexpected immune response, we modified the surface of a lipoplex with polyglycerol (PG)-derived lipid. The PG-coated pDNA-lipoplex (PG-DCL) attenuated the production of anti-polymer IgM, whereas PEG-coated pDNA-lipoplex (PEG-DCL) did not. In addition, a second dose of PG-DCL maintained the accumulation level in the tumor tissue of a tumor-bearing mouse model, comparable to that of the first dose, whereas the tumor accumulation level of a second dose of PEG-DCL was significantly compromised, compared with the first dose of PEG-DCL. Our results indicate that surface modification of lipoplex with PG represents a viable means for the attenuation, and/or evasion, of the ABC phenomenon that is encountered upon repeated administrations of nucleic acids containing PEG-coated nanocarriers.
(Keyword)
Animals / Antimetabolites / B-Lymphocytes / Bromodeoxyuridine / Cell Proliferation / Cytokines / DNA / Drug Carriers / Drug Delivery Systems / Excipients / Fluorescent Dyes / Genetic Therapy / Glycerol / Immunoglobulin M / Liposomes / Male / Mice / Mice, Inbred BALB C / Neoplasm Transplantation / Plasmids / Polyethylene Glycols / Polymers / Rats / Rats, Wistar
Lila Selim Ahmed Ali Abu Amr, Masako Ichihara, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Ex-Vivo/In-Vitro Anti-Polyethylene Glycol (PEG) IgM Production From Murine Splenic B Cells Stimulated by PEGylated Liposome, Biological & Pharmaceutical Bulletin, Vol.36, No.11, 1842-1848, 2013.
(Summary)
We have reported that PEGylated liposomes lose their long-circulating properties when injected twice into the same animal within a certain interval (the accelerated blood clearance (ABC) phenomenon). We assumed that this phenomenon was triggered via the abundant secretion of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) in response to the first dose of PEGylated liposomes and that the spleen played an important role in the production of anti-PEG IgM. However, no direct evidence has yet confirmed this suspicion. In the current study, we verified, both in vitro and ex vivo, that spleen cells are indeed responsible for the production of anti-PEG IgM in response to PEGylated liposomes. In this study, spleen cells obtained from either naïve mice or mice pre-treated with PEGylated liposomes induced the production of anti-PEG IgM in a dose- and time-dependent manner, upon incubation with PEGylated liposomes. In addition, we confirmed that among the different fractions of splenic B cells, IgM-positive B cells, rather than CD45R-positive or CD19-positive splenic B cells, which are presumed to be the marginal zone B (MZB) cells, are the major cells producing anti-PEG IgM in the response to stimulation by PEGylated liposomes. These results may provide new insights into the mechanisms underlying the anti-PEG IgM production in response to the stimulation by PEGylated liposomes.
Takako Nakashima, N Uematsu, M Shibamori, K Sakurai, Tatsuhiro Ishida and Hiroshi Kiwada : Establishment of an X-ray irradiation-induced glossitis model in rats: biphasic elevation of proinflammatory cytokines and chemokines, The Journal of Pharmacology and Experimental Therapeutics, Vol.347, No.3, 660-668, 2013.
(Summary)
Oral mucositis is a frequent and serious side effect in patients who receive radiotherapy for head and neck cancer. The purpose of this study was to develop a noninvasive and quantitative model of oral mucositis in rats, investigate the pathophysiology, and evaluate the efficacy of pharmacological interventions. Rats received a single dose of 15 Gy of X-rays to the snout after shielding of the remainder of the rat body with lead plates to protect the body from irradiation (day 0). After irradiation, the macroscopic area of tongue injury gradually increased. The total area of injury and the ulcer-like area reached a maximum on day 7 and then gradually decreased until disappearance on day 28. Expression of proinflammatory cytokines and chemokines occurred transiently within 1-4 hours after irradiation and returned to a normal level at 24 hours. This expression was again observed from days 3 to 5 and increased significantly on day 7, which approximately coincided with the histologic severity of tissue damage. Subcutaneous administration of palifermin at 3 mg/kg per day for 3 consecutive days before irradiation completely prevented ulcer formation in this model. In conclusion, we established a novel model of glossitis in rats, induced by X-ray irradiation, in which biphasic elevations of expression of proinflammatory cytokines and chemokines could be monitored. This model is considered useful to investigate the pathophysiology of oral mucositis and evaluate the preventive effect of pharmacological interventions on oral mucositis induced by X-ray irradiation.
Lila Selim Ahmed Ali Abu Amr, Kousuke Nawata, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration, International Journal of Pharmaceutics, Vol.456, No.1, 235-242, 2013.
(Summary)
The accelerated blood clearance (ABC) phenomenon accounts for the rapid systemic clearance of PEGylated nanocarriers upon repeated administrations. IgM production against the polyethylene glycol (PEG) coating in PEGylated liposomes is now known to be responsible for such unexpected pharmacokinetical alterations. The ABC phenomenon poses a remarkable clinical challenge by reducing the therapeutic efficacy of encapsulated drugs and causing harmful effects due to the altered tissue distribution pattern of the drugs. In this study, we investigated the in vivo performance of liposomes modified with polyglycerol (PG) upon repeated injection, and the in vivo therapeutic efficacy of such liposomes when they encapsulated a cytotoxic agent, doxorubicin (DXR). Repeated injection of PEG-coated liposomes in rats induced the ABC phenomenon, while repeated injection of PG-coated liposomes did not. In addition, DXR-containing PG-coated liposomes showed antitumor activity that was superior to that of free DXR and similar to that of DXR-containing PEG-coated liposomes upon repeated administration. These results indicate that polyglycerol (PG) might represent a promising alternative to PEG via enhancing the in vivo performance of liposomes by not eliciting the ABC phenomenon upon repeated administration.
E Alaaeldin, Lila Selim Ahmed Ali Abu Amr, Naoto Moriyoshi, H Sarahan, A Khaled, Tatsuhiro Ishida and Hiroshi Kiwada : The co-delivery of oxaliplatin abrogates the immunogenic response to PEGylated siRNA-lipoplex., Pharmaceutical Research, Vol.30, No.9, 2344-2354, 2013.
(Summary)
In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.
(Keyword)
Animals / Antineoplastic Agents / Cytokines / Immunoglobulin M / Liposomes / Male / Mice / Mice, Inbred BALB C / NF-kappa B / Organoplatinum Compounds / Polyethylene Glycols / RNA, Small Interfering
Tatsuhiro Ishida and Hiroshi Kiwada : Development of siRNA Delivery Strategy by Active Control of Tumor Microenvironment, Journal of the Pharmaceutical Society of Japan, Vol.133, No.3, 379-386, 2013.
(Summary)
Efficient systemic siRNA delivery to cells in the target tissue is a current critical challenge in the drug delivery field. Several studies have demonstrated that nanoparticles such as polyethylene glycol (PEG)-coated siRNA-lipoplexes may enhance the systemic delivery of siRNA to tumor. However, the disordered tumor microenvironment still poses a potential impediment with respect to the efficient delivery of PEG-coated siRNA-lipoplexes. We recently showed that metronomic S-1 dosing (daily oral administration) enhanced the accumulation of PEG-coated liposome containing anticancer drug in solid tumor tissue and thereby increased therapeutic efficacy in tumor-bearing mouse model. To extend this work, we tried to investigate the effect of metronomic S-1 dosing on the intratumoral accumulation of PEG-coated siRNA-lipoplex and, thereby, their therapeutic efficacy in solid tumor-bearing mouse model. Results showed that metronomic S-1 dosing improved systemic delivery of intravenously injected PEG-coated siRNA-lipoplexes into solid tumor tissue. In addition, the combined therapy of S-1 and PEG-coated siRNA-lipoplexes showed potent tumor growth suppressive effect. Our proposed strategy may pose a promising therapeutic one to conquer cancer progression with siRNA.<br>
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon., Immunobiology, Vol.218, No.5, 725-732, 2013.
(Summary)
The accelerated blood clearance (ABC) phenomenon has been reported to enhance the clearance of PEGylated liposomes from the blood circulation when the liposomes are injected into the same animal repeatedly. We have shown that anti-PEG IgM production from splenic B cells is crucial in the ABC phenomenon. In this study, we describe the crucial role of marginal zone (MZ) B cells in the anti-PEG IgM production and recognition of PEGylated liposomes in the induction phase of ABC phenomenon. Suppression of the anti-PEG IgM production was correlated with the disappearance of IgM(high) cells in the MZ, particularly MZ-B cells, following cyclophosphamide (CPA)-treatment, confirming that splenic MZ-B cells are responsible for anti-PEG IgM production. The MZ-B cells stimulated by a first dose of PEGylated liposomes internalized the second dose of PEGylated liposomes in a PEG modification-dependent manner and transported the liposomes into the follicle (FO) region. To the best of our knowledge, this is the first report showing that PEGylated liposome is recognized by MZ-B cells and transported to the FO region like blood-borne antigens or immune complexes. It is likely that PEGylated liposomes are recognized as a TI-2 antigen by the first line of defense against life-threatening infections by blood-borne organisms. Our study may have implications for immunogenicity of synthesized polymer-grafted therapeutics including nanocarriers, nucleic acids and proteins.
K Ichikawa, T Asai, K Shimizu, S Yonezawa, T Urakami, H Miyauchi, H Kawashima, Tatsuhiro Ishida, Hiroshi Kiwada and N Oku : Suppression of immune response by antigen-decorated liposomes encapsulating model agents: A novel strategy for the treatment of allergy., Journal of Controlled Release, Vol.167, No.3, 284-289, 2013.
(Summary)
A specific antigen-sensitized animal has antigen-specific immune cells that recognize the antigen. Therefore, an antigen-modified drug carrier would be recognized by the immune cells. When such a carrier encapsulates certain drugs, these drugs should be specifically delivered to the immune cells. To examine this strategy, ovalbumin (OVA) was used as model antigen, and mice were presensitized with 100 μg of OVA with Alum. For preparing OVA-modified liposomes (OVA-lipo), OVA was incubated with DSPE-PEG-NHS and resulting DSPE-PEG-OVA was inserted into liposomes. OVA-specific IgG was produced 6-fold higher by intravenous injection of OVA-lipo thrice (10 μg as OVA in each injection) in OVA-sensitized mice, than that by the injection of control liposomes, suggesting that OVA-lipo was recognized by the antigen-specific immune cells. Moreover, intra-splenic accumulation of OVA-lipo was observed in OVA-sensitized mice, but not in naive mice. To achieve the delivery of a drug to specific immune cells, OVA-lipo encapsulated low dose of doxorubicin (DOX) as a model drug (20 μg DOX/mouse, Ca. 1 mg/kg) was injected in the sensitized mice. The injection of OVA-lipo encapsulating DOX suppressed the production of IgE against OVA, suggesting that the specific delivery of the drug to immune cells responsible for OVA recognition was achieved and that these immune cells were removed by the drug treatment. This strategy would be useful for the fundamental treatment of allergy by the use of immunosuppressing agents.
Ai Nagao, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Abrogation of the accelerated blood clearance phenomenon by SOXL regimen: Promise for clinical application., International Journal of Pharmaceutics, Vol.441, No.1-2, 395-401, 2013.
(Summary)
We recently proposed an S-1 combined with oxaliplatin (SOXL) regimen, a combination treatment consisting of oral metronomic S-1 dosing and intravenous administration of oxaliplatin (l-OHP) containing PEGylated liposomes, which showed potent antitumor activity in vivo. PEGylated liposomes induce what is referred to as the "accelerated blood clearance (ABC) phenomenon" upon repeated administration and consequently lose their long-circulating characteristics. This phenomenon seems to pose an impediment for the clinical application and use of PEGylated liposomal formulations. In the present study, l-OHP-containing PEGylated liposomes in the SOXL regimen significantly attenuated the ABC phenomenon in a dose-dependent manner through suppression of the anti-PEG IgM response, which allowed an enhanced hepatic uptake of subsequently injected test PEGylated liposomes. In tumor-bearing mice, the abrogation of the ABC phenomenon restored intratumor accumulation of subsequently injected PEGylated liposomes. Consequently, the therapeutic efficacy of the SOXL regimen over the combination of the free form of the drugs was credited not only with the selective delivery of drugs to the tumor tissue but also with ensuring an adequate accumulation of subsequent doses within the tumor tissue. The SOXL regimen we proposed may hold promise as a safe and effective treatment regimen for advanced colorectal cancer.
(Keyword)
Administration, Metronomic / Animals / Antineoplastic Combined Chemotherapy Protocols / Colonic Neoplasms / Colorectal Neoplasms / Dose-Response Relationship, Drug / Drug Combinations / Drug Delivery Systems / Liposomes / Liver / Male / Mice / Mice, Inbred BALB C / Organoplatinum Compounds / Oxonic Acid / Polyethylene Glycols / Tegafur / Tissue Distribution
Takuya Suzuki, Masako Ichihara, Kenji Hyodo, Eiichi Yamamoto, Tatsuhiro Ishida, Hiroshi Kiwada, Hiroshi Ishihara and Hiroshi Kikuchi : Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs., International Journal of Pharmaceutics, Vol.436, No.1-2, 636-643, 2012.
(Summary)
The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.
Lila Selim Ahmed Ali Abu Amr, N Eldin Essam, Masako Ichihara, Tatsuhiro Ishida and Hiroshi Kiwada : Multiple administration of PEG-coated liposomal oxaliplatin enhances its therapeutic efficacy: a possible mechanism and the potential for clinical application., International Journal of Pharmaceutics, Vol.438, No.1-2, 176-183, 2012.
(Summary)
We previously developed a PEG-coated cationic liposome that enabled dual targeting delivery of oxaliplatin (l-OHP) to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation consequently elicited potent antitumor efficacy in a murine solid tumor model after 3 sequential injections. However, the probable mechanism(s) for this enhanced antitumor activity has not been fully elucidated. In the present study, therefore, the changes in tumor microenvironment induced by sequential administration of liposomal l-OHP were investigated, with emphasis on its impact to the intratumoral localization of the subsequently injected dose. In addition, the potential for anti-PEG IgM production upon repeated administration of liposomal l-OHP-containing PEGylated lipid was clearly revealed. Two sequential injections of liposomal l-OHP induced superior apoptotic activity in tumor tissue and thus resulted in broader intratumor distribution of the subsequent test dose of PEG-coated cationic liposomes, compared with a single injection of liposomal l-OHP. In addition, it was confirmed that repeated administration of liposomal l-OHP did not induce a significant anti-PEG IgM response, indicating that l-OHP encapsulated in PEG-coated liposomes was efficient in abrogating the ABC phenomenon. These results suggest that sequential treatment strategies with liposomal cytotoxic agents might be superior to mono-treatment strategies in achieving alterations in the tumor microenvironment and maintaining/restoring the pharmacokinetics of the formulation, and, therefore, would result in substantial therapeutic efficacy.
Taro Shimizu, Masako Ichihara, Yasuo Yoshioka, Tatsuhiro Ishida, Shinsaku Nakagawa and Hiroshi Kiwada : Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response., Biological & Pharmaceutical Bulletin, Vol.35, No.8, 1336-1342, 2012.
(Summary)
A single intravenous administration of polyethylene glycol-coated (PEGylated) bovine serum albumin (BSA) and ovalbumin (OVA) elicited an anti-PEG immunoglobulin M (IgM) response, similar to that from PEGylated liposomes, although the administration did not elicit specific neutralizing antibodies to BSA and OVA. A cross-reactivity was observed between anti-PEG IgMs elicited by PEG-BSA and PEGylated liposomes. The anti-PEG IgM level induced by PEGylated proteins (BSA and OVA) reached the maximum at day 5 following intravenous injection. This production pattern was consistent with that induced by PEGylated liposomes. Splenectomy suppressed the anti-PEG IgM response against PEG-BSA and PEGylated liposomes. These observations relating PEG-BSA and PEGylated liposomes indicate that PEGylated proteins might promote the immune responses against PEG with a mechanism similar to that of PEGylated liposomes. In addition, a single intravenous administration of PEGylated adenovirus (PEG-Ad) also elicited an anti-PEG IgM response in a PEG-modification ratio dependent manner. To the best of our knowledge, this is the first report showing that an intravenous administration can elicit an anti-PEG IgM response against PEGylated substances. It appears that anti-PEG IgMs can be produced by the systemic administration of a PEGylated substance and may limit the efficacy of PEGylated substances such as proteins, Ad vector and nanoparticles, due to a cross-reactivity seen in some patients. The immunogenicity of PEGylated substances is usually tested against those very substances, rather than against covalently attached PEG. Our study suggests that the PEG immunogenicity of PEGylated therapeutic agents and particles merits further investigation.
(Keyword)
Adenoviridae / Animals / Cattle / Immunoglobulin M / Injections, Intravenous / liposomes / Male / Ovalbumin / Polyethylene Glycols / Rats / Rats, Wistar / Serum Albumin / Splenectomy
Lila Selim Ahmed Ali Abu Amr, Haruna Matsumoto, Yusuke Doi, Hiroyuki Nakamura, Tatsuhiro Ishida, Hiroshi Kiwada and Hiroshi Kiwada : Tumor type dependent vascular permeability constitutes a potential impediment to the therapeutic efficacy of liposomal oxaliplatin., European Journal of Pharmaceutics and Biopharmaceutics, Vol.81, No.3, 524-531, 2012.
(Summary)
The delivery of anticancer agents to solid tumors is problematic. Nanomolecular drug carriers represent an attractive alternative strategy for efficient anticancer drug delivery to tumor tissue, because they appear to target tumors and have limited toxicity in normal tissue. However, inadequate and heterogeneous distribution of nanocarriers in tumor tissue is a major impediment for their efficient use in clinical cancer therapy. In the present study, we examined the effect of tumor type on the intratumor accumulation and distribution of polyethylene glycol (PEG)-coated liposomes using in vivo mouse models of three cancer cell lines: colon adenocarcinoma (C26), Lewis lung carcinoma (LLC), and B16BL6 melanoma (B16BL6). The tumor growth inhibition and the apoptotic response of oxaliplatin (l-OHP) encapsulated in the PEG-coated liposomes were tumor type dependent and correlated with a tendency toward tumor accumulation and intratumor distribution of PEG-coated liposome, in contrast to in vitro cytotoxicity of l-OHP. A potent antitumor effect observed in both C26 and LLC tumor-bearing mice was attributed to the enhanced extravasation with subsequent preferential accumulation of PEG-coated liposomes through tumor vasculature with high permeability. Our results suggest that the permeability of tumor vasculature constitutes a potential impediment to tumor localization and thereby to the antitumor efficacy of PEG-coated liposomal anticancer drugs.
Jose Mario Barichello, Shinji Kizuki, Tatsuaki Tagami, Soares Alberto Lira Luiz, Hiroshi Kikuchi, Tatsuhiro Ishida and Hiroshi Kiwada : Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes., International Journal of Pharmaceutics, Vol.430, No.1-2, 359-365, 2012.
(Summary)
We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.
(Keyword)
Cations / Circular Dichroism / Ethanolamines / Factor Analysis, Statistical / Fluorescence Resonance Energy Transfer / Gene Knockdown Techniques / HeLa Cells / Humans / Liposomes / Luciferases / Motion / Myristates / Nucleic Acid Conformation / Phosphatidylethanolamines / RNA Interference / RNA, Small Interfering / Time Factors / Transfection
Tatsuaki Tagami, Lila Selim Ahmed Ali Abu Amr, Mariko Matsunaga, Naoto Moriyoshi, Hiroyuki Nakamura, Kazuya Nakamura, Takuya Suzuki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Improved intratumoral delivery of PEG-coated siRNA-lipoplexes by combination with metronomic S-1 dosing in a murine solid tumor model, Drug Delivery and Translational Research, Vol.2, No.2, 77-86, 2012.
Lila Selim Ahmed Ali Abu Amr, Tomoko Okada, Yusuke Doi, Masako Ichihara, Tatsuhiro Ishida and Hiroshi Kiwada : Combination therapy with metronomic S-1 dosing and oxaliplatin-containing PEG-coated cationic liposomes in a murine colorectal tumor model: Synergy or antagonism?, International Journal of Pharmaceutics, Vol.426, 263-270, 2012.
(Summary)
Combination therapy with 2 or more drugs with different mechanisms of action has been considered a promising strategy for the effective treatment of advanced and metastatic cancers. However, the rational design of combination therapy represents a potential prerequisite for its effectiveness. Recently, we showed that the combination of oral metronomic S-1 dosing with oxaliplatin (l-OHP)-containing PEG-coated "neutral" liposomes exerted excellent antitumor activity. In addition, we recently designed a PEG-coated "cationic" liposome for dual-targeting delivery of l-OHP to tumor endothelial cells and tumor cells in a solid tumor. This targeted liposomal l-OHP formulation showed efficient antitumor activity in a murine tumor model, compared with l-OHP-containing PEG-coated "neutral" liposomes. In the present study, we investigated the issue of whether metronomic S-1 dosing with l-OHP-containing PEG-coated "cationic" liposomes creates synergy. Unfortunately, metronomic S-1 dosing resulted in impaired delivery of PEG-coated "cationic" liposomes into tumor tissue, presumably by decreasing the binding sites on tumor blood vessels available for the liposomes. The anticipated cytotoxic synergistic effect of the combination treatment was not achieved. Instead, the combination treatment showed lower antitumor efficacy than l-OHP-containing PEG-coated "cationic" liposomes alone. These results suggest that the combined treatment of S-1 and l-OHP-containing PEG-coated "cationic" liposomes seems to be antagonistic rather than synergistic.
Tatsuaki Tagami, Takuya Suzuki, Mariko Matsunaga, Kazuya Nakamura, Naoto Moriyoshi, Tatsuhiro Ishida and Hiroshi Kiwada : Anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery., International Journal of Pharmaceutics, Vol.422, No.1-2, 280-289, 2012.
(Summary)
siRNA has been touted as a therapeutic molecule against genetic diseases, which include cancers. But several challenging issues remain in order to achieve efficient systemic siRNA delivery and a sufficient therapeutic effect for siRNA in vivo. Cationic liposome shows promise as a carrier for nucleic acids, as it can selectively bind to angiogenic tumor blood vessels. In this way, anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery could be achieved in cancer therapy. In the present study, we proved our assumption by preparing various kinds of polyethylene glycol (PEG)-coated siRNA/cationic liposome complexes (siRNA-lipoplexes) and screening the avidity of these siRNA-lipoplexes upon angiogenic tumor blood vessels by means of a murine dorsal air sac (DAS) model. The lipoplex, having a lipid composition of DC-6-14/POPC/CHOL/DOPE/mPEG(2000)-DSPE=20/30/30/20/5 (molar ratio) and a charge ratio of cationic liposome and siRNA=3.81 (+/-), showed a higher binding index to newly formed blood vessels. Systemic injection with the lipoplex containing siRNA for the Argonaute2 gene (apoptosis-inducible siRNA) resulted in significant anti-tumor effect without severe side effects in mice with Lewis lung carcinoma. Our results indicate that the PEGylated cationic liposome-mediated systemic delivery of cytotoxic siRNA achieves anti-angiogenesis, resulting in the suppression of tumor growth.
Kazuya Nakamura, Lila Selim Ahmed Ali Abu Amr, Mariko Matsunaga, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : A double-modulation strategy in cancer treatment with a chemotherapeutic agent and siRNA., Molecular Therapy, Vol.19, No.11, 2040-2047, 2011.
(Summary)
5-Fluorouracil (5-FU) is broadly considered the drug of choice for treating human colorectal cancer (CRC). However, 5-FU resistance, mainly caused by the overexpression of antiapoptotic proteins such as Bcl-2, often leads ultimately to treatment failure. We here investigated the effect of Bcl-2 gene silencing, using small interfering RNA (siRNA) (siBcl-2), on the efficacy of 5-FU in CRC. Transfection of siBcl-2 by a Lipofectamine2000/siRNA lipoplex effectively downregulated Bcl-2 expression in the DLD-1 cell line (a CRC), resulting in significant cell growth inhibition in vitro upon treatment with 5-FU. For in vivo treatments, S-1, an oral formulation of Tegafur (TF), a prodrug of 5-FU, was used to mimic 5-FU infusion. The combined treatment of polyethylene glycol (PEG)-coated siBcl-2-lipoplex and S-1 showed superior tumor growth suppression in a DLD-1 xenograft model, compared to each single treatment. Surprisingly, daily S-1 treatment enhanced the accumulation of PEG-coated siBcl-2-lipoplex in tumor tissue. We propose a novel double modulation strategy in cancer treatment, in which chemotherapy enhances intratumoral siRNA delivery and the delivered siRNA enhances the chemosensitivity of tumors. Combination of siRNA-containing nanocarriers with chemotherapy may compensate for the limited delivery of siRNA to tumor tissue. In addition, such modulation strategy may be considered a promising therapeutic approach to successfully managing 5-FU-resistant tumors.
Tatsuaki Tagami, Takuya Suzuki, Kiyomi Hirose, Jose Mario Barichello, Naoshi Yamazaki, Tomohiro Asai, Naoto Oku, Tatsuhiro Ishida and Hiroshi Kiwada : Argonaute2 is a potential target for siRNA-based cancer therapy for HT1080 human fibrosarcoma., Drug Delivery and Translational Research, Vol.1, No.4, 277-288, 2011.
Atsushi Saito, Hiroaki Shimizu, Yusuke Doi, Tatsuhiro Ishida, Miki Fujimura, Takashi Inoue, Hiroshi Kiwada and Teiji Tominaga : Immunoliposomal drug delivery system targeting lectin-like oxidized low density lipoprotein receptor 1 for carotid plaque lesion in rats., Journal of Neurosurgery, Vol.115, No.4, 720-727, 2011.
(Summary)
Targeted drug delivery with immunoliposomes has been applied to various in vivo animal models and is newly focused as a novel therapeutic target. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) is a potent regulator of systemic atherosclerosis, and the authors focused on its effect on carotid plaques. The authors developed a LOX1-targeted liposomal rho-kinase inhibitor and examined the therapeutic effect on carotid intimal hypertrophy in rats. LOX1-targeted rho-kinase inhibitor fasudil-containing liposomes, composed of hydrogenated soy phosphatidylcholine/cholesterol/PEG(2000)-DSPE, were prepared by conjugating anti-LOX1 antibodies on the surface and by remote loading of fasudil. Carotid intimal hypertrophy was induced by balloon injury, and the drugs were intravenously administered on Day 3 postinjury. The rats were divided into 4 groups: nontreatment, treatment with intravenous fasudil (2 mg), treatment with liposomal fasudil (2 mg), and treatment with LOX1-targeted liposomal fasudil (2 mg). The authors compared intimal hypertrophy, atherosclerotic factor, and matrix metalloproteinase-9 expression among groups. DiI-labeled LOX1-targeted liposomes were prominently observed in the lesions on Day 7 after the surgery. The intimal thickness was significantly reduced in the LOX1-targeted liposomal fasudil-treated group (mean 81.6 ± 13.9 m) compared with the other groups (no treatment 105.4 ± 16.8 m; fasudil treatment 102.4 ± 20.0 m; and liposomal fasudil treatment 102.8 ± 22.2 m; p = 0.046). Matrix metalloproteinase-9 expression was also significantly reduced in the LOX1-targeted liposomal fasudil group. Liposomes conjugated with anti-LOX1 antibody effectively reached carotid artery lesions, and liposomal rho-kinase significantly inhibited intimal hypertrophy. The new liposomal drug delivery system targeting LOX1 may become a therapeutic strategy for atherosclerotic diseases.
(Keyword)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Animals / Carotid Artery Diseases / Carotid Artery, Common / Drug Delivery Systems / Liposomes / Male / Plaque, Atherosclerotic / Rats / Rats, Sprague-Dawley / Scavenger Receptors, Class E
Jose Mario Barichello, Shinji Kizuki, Tatsuaki Tagami, Tomohiro Asai, Tatsuhiro Ishida, Hiroshi Kikuchi, Naoto Oku and Hiroshi Kiwada : Agitation during lipoplex formation improves the gene knockdown effect of siRNA., International Journal of Pharmaceutics, Vol.410, No.1-2, 153-160, 2011.
(Summary)
The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.
Tatsuaki Tagami, Yumi Uehara, Naoto Moriyoshi, Tatsuhiro Ishida and Hiroshi Kiwada : Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA., Journal of Controlled Release, Vol.151, No.2, 149-154, 2011.
(Summary)
We recently reported that the prolonged circulation property of PEGylated cationic liposomes containing nucleic acids disappears, if the second dose is injected within a few days later, due to the production of anti-PEG IgM. This accelerated blood clearance is a concern for treating diseases which require repeated treatment with a PEGylated formulation containing nucleic acids. In this study, we investigated the effect of encapsulation of siRNA in a recently introduced PEGylated lipid nanocarrier for which the term "wrapsome" (PEGylated wrapsome, PEG-WS) was proposed as well as the sequence of the encapsulated siRNA on anti-PEG IgM production. siRNA encapsulated in PEG-WS produced little anti-PEG IgM relative to siRNA in conventional PEGylated lipoplexes. The sequence of siRNA in the PEG-WL dramatically affected the anti-PEG IgM production; a potent immune stimulatory siRNA induced a higher anti-PEG IgM production. Such enhanced effect was abrogated by incorporation of 2'-O-methyl (2'-OMe) uridine into the sequence of siRNA, probably via inhibiting cytokine induction such as IL-6 and TNF-. Our results strongly indicate that the use of an encapsulation-type lipid nanocarrier with a low immuno-stimulatory siRNA may allow repeated dosing of siRNA containing PEGylated formulations without the induction of a strong immune reaction against PEG and thus may advance synthetic siRNA into a broad range of therapeutic applications.
(Keyword)
Animals / Base Sequence / Drug Carriers / Immunoglobulin M / Liposomes / Male / Mice / Nanocapsules / Nanoparticles / Polyethylene Glycols / RNA, Small Interfering
Yusuke Doi, Tomoko Okada, Haruna Matsumoto, Masako Ichihara, Tatsuhiro Ishida and Hiroshi Kiwada : Combination therapy of metronomic S-1 dosing with oxaliplatin-containing PEG-coated liposome improves antitumor activity in a murine colorectal tumor model., Cancer Science, Vol.101, No.11, 2470-2475, 2010.
(Summary)
Metronomic chemotherapy has been advocated recently as a novel chemotherapeutic regimen. Polyethylene glycol (PEG)-coated liposomes are well known to accumulate in solid tumors by virtue of the highly permeable angiogenic blood vessels characteristic for growing tumor tissue, the so-called "enhanced permeability and retention (EPR) effect". To expand the range of applications and investigate the clinical value of the combination strategy, the therapeutic benefit of metronomic S-1 dosing in combination with oxaliplatin (l-OHP)-containing PEG-coated liposomes was evaluated in a murine colon carcinoma-bearing mice model. S-1 is an oral fluoropyrimidine formulation and metronomic S-1 dosing is a promising alternative to infused 5-FU in colorectal cancer therapy. Therefore, the combination of S-1 with l-OHP may be an alternative to FOLFOX (infusional 5-FU/leucovorin (LV) in combination with l-OHP), which is a first-line therapeutic regimen of a colorectal carcinoma. The combination of oral metronomic S-1 dosing with intravenous administration of liposomal l-OHP formulation exerted excellent antitumor activity without severe overlapping side-effects, compared with either metronomic S-1 dosing, free l-OHP or liposomal l-OHP formulation alone or metronomic S-1 dosing plus free l-OHP. We confirmed that the synergistic antitumor effect is due to prolonged retention of l-OHP in the tumor on account of the PEG-coated liposomes, presumably via alteration of the tumor microenvironment caused by the metronomic S-1 treatment. The combination regimen proposed here may be a breakthrough in treatment of intractable solid tumors and an alternative to FOLFOX in advanced colorectal cancer therapy with acceptable tolerance and preservation of quality of life (QOL).
T Ishihara, T Maeda, H Sakamoto, N Takasaki, M Shigyo, Tatsuhiro Ishida, Hiroshi Kiwada, Y Mizushima and T Mizushima : Evasion of the accelerated blood clearance phenomenon by coating of nanoparticles with various hydrophilic polymers., Biomacromolecules, Vol.11, No.10, 2700-2706, 2010.
(Summary)
The accelerated blood clearance (ABC) phenomenon is induced upon repeated injections of poly(ethylene glycol) (PEG)-coated colloidal carriers. It is essential to suppress this phenomenon in a clinical setting because the pharmacokinetics must be reproducible. In this study, we evaluated the induction of the ABC phenomenon using nanoparticles coated with various hydrophilic polymers instead of PEG. Nanoparticles encapsulating prostaglandin E1 were prepared by the solvent diffusion method from a blend of poly(lactic acid) (PLA) and block copolymers consisting of various hydrophilic polymers and PLA. Coating of nanoparticles with poly(N-vinyl-2-pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) led to extended residence of the nanoparticles in blood circulation in rats, although they had a shorter half-life than the PEG-coated nanoparticles. The ABC phenomenon was not induced upon repeated injection of PVP-coated nanoparticles at various time intervals, dosages, or frequencies, whereas it was elicited by PEG-coated nanoparticles. In addition, anti-PVP IgM antibody, which is estimated to be one of the crucial factors for induction of the ABC phenomenon, was not produced after injection of PVP-coated nanoparticles. These results suggest that the use of PVP, instead of PEG, as a coating material for colloidal carriers can evade the ABC phenomenon.
(Keyword)
Animals / Blood Circulation Time / Drug Carriers / Enzyme-Linked Immunosorbent Assay / Hydrophobic and Hydrophilic Interactions / Immunoglobulin M / Injections, Intravenous / Male / Metabolic Clearance Rate / Nanoparticles / Polymers / Polyvinyls / Pyrrolidines / Rats / Rats, Wistar
Y Eto, Y Yoshioka, Tatsuhiro Ishida, X Yao, T Morishige, S Narimatsu, H Mizuguchi, Y Mukai, N Okada, Hiroshi Kiwada and S Nakagawa : Optimized PEGylated adenovirus vector reduces the anti-vector humoral immune response against adenovirus and induces a therapeutic effect against metastatic lung cancer., Biological & Pharmaceutical Bulletin, Vol.33, No.9, 1540-1544, 2010.
(Summary)
Application of adenovirus vectors (Adv) in metastatic cancer treatment is limited. We previously demonstrated that covalent conjugation of polyethleneglycol (PEG) to Adv enhances therapeutic effects and decreases toxic side-effects after systemic administration, but the level of immune response to PEGylated Adv (PEG-Ad) was not examined. Here, we examined the effect of PEGylation of Adv on the production of anti-Adv antibodies and antitumor response. We constructed a set of PEG-Ad using 5-kDa PEG, with modification rates of 30%, 45% and 90%. After systemic administration of Advs to rats, we examined the level of anti-Adv immunoglobulin (Ig)G and IgM in serum. The levels of anti-Adv IgG and anti-Adv IgM in rats treated with unmodified Adv were higher than those in control group. Rats treated with PEG-Ad that had a 90% modification rate showed lower level of anti-Adv IgG and anti-Adv IgM than those treated with unmodified Adv, whereas rats treated with PEG-Ad that had a 30% or 45% modification rate showed a similar level of anti-Adv IgG and IgM to those treated with unmodified Adv. Systemic administration of PEG-Ad that had a 90% modification rate, and expressed tumor necrosis factor-alpha, significantly reduced the number of metastatic colonies in the lung compared to unmodified Adv, with negligible side effects. These results suggest that systemic administration of PEG-Ad with an appropriate PEG modification rate has the potential to reduce the production of antibodies against Adv and increase the therapeutic response against metastatic cancer.
Hiroyuki Yamazaki, Masateru Miyake, Naoki Kamada, Toru Nishibayashi, Tadashi Mukai, Masaaki Odomi, Tatsuhiro Ishida and Hiroshi Kiwada : Co-administration of tacrolimus suppresses pharmacokinetic modulation of multiple subcutaneously administrated human interferon-alpha in beagle dogs., Drug Metabolism and Pharmacokinetics, Vol.25, No.2, 149-154, 2010.
(Summary)
Specific antibody production is an important issue in crossover pharmacokinetic (PK) studies of protein-based formulations. We recently reported that intravenous co-administration of tacrolimus with multiple human interferon-alpha (h-IFN) administrations successfully suppressed the production of anti-h-IFN antibodies in rats. Since crossover PK studies are preferentially carried out using larger animals such as dogs or monkeys that are capable of accepting the same dosage formulations as those for clinical use, we extended our study of co-administration of tacrolimus with multiple h-IFN administrations to beagle dogs in the present study. Beagle dogs were subcutaneously administered 0.5 million IU/kg of h-IFN once a week for 4 weeks. In some experiments, tacrolimus at 0.01 or 0.1 mg/kg was intravenously co-administered at the same time as the h-IFN administration. Co-administration of the lower dose of tacrolimus (0.01 mg/kg) failed to suppress the anti-h-IFN IgG responses, while co-administration of the higher dose (0.1 mg/kg) successfully suppressed these responses. Moreover, co-administration of tacrolimus had little effect on the serum creatinine concentrations, suggesting that multiple administrations of tacrolimus at the concentrations examined did not cause severe renal disorders. Taken together, the present data confirm that co-administration of tacrolimus is a promising way to assess crossover PK studies of human or humanized proteinic formulations in beagle dogs.
H Koide, T Asai, K Hatanaka, S Akai, T Ishii, E Kenjo, Tatsuhiro Ishida, Hiroshi Kiwada, H Tsukada and N Oku : T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes., International Journal of Pharmaceutics, Vol.392, No.1-2, 218-223, 2010.
(Summary)
Repeated injection of polyethyleneglycol-modified (PEGylated) liposomes causes a rapid clearance of them from the bloodstream, this phenomenon is called accelerated blood clearance (ABC). In the present study, we focused on the immune system responsible for the ABC phenomenon. PEGylated liposomes were preadministered to BALB/c mice and [(3)H]-labeled ones were then administered to them 3 days after the preadministration. Consistent with our previous results, the preadministration with PEGylated liposomes triggered the rapid clearance of [(3)H]-labeled PEGylated liposomes from the bloodstream, but that with PEGylated liposomes encapsulating doxorubicin (Dox) did not. In addition, we found that the ABC phenomenon was observed when a mixture of free Dox and PEGylated liposomes was preadministered. These data indicate that immune cells responsible for the ABC phenomenon might be selectively damaged by the Dox encapsulated in PEGylated liposomes. The ABC phenomenon was also observed in BALB/c nu/nu mice, but not in BALB/c SCID mice. The amount of anti-PEG IgM antibody induced by the stimulation with the PEGylated liposomes was significantly increased in the BALB/c nu/nu mice, but not in the BALB/c SCID ones. These data indicate that a T cell-independent B cell response would play a significant role in the ABC phenomenon. Furthermore, the present study suggests that PEGylated liposomes might be recognized by B cells as a thymus-independent type 2 (TI-2) antigen. The present study provides important information for the future development of liposomal medicines.
(Keyword)
Animals / Antigens, T-Independent / B-Lymphocytes / Dose-Response Relationship, Drug / Doxorubicin / Immunoglobulin M / Injections, Intravenous / Liposomes / Male / Metabolic Clearance Rate / Mice / Mice, Inbred BALB C / Mice, SCID / Polyethylene Glycols / T-Lymphocytes / Tissue Distribution
Tatsuaki Tagami, Kazuya Nakamura, Taro Shimizu, Naoshi Yamazaki, Tatsuhiro Ishida and Hiroshi Kiwada : CpG motifs in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNA-lipoplexes., Journal of Controlled Release, Vol.142, No.2, 160-166, 2010.
(Summary)
Gene therapy is largely dependent on the development of efficient delivery vehicles. To prolong their circulating time, PEGylation of the surface of a delivery vehicle is frequently applied. However, we have reported previously that anti-PEG IgM produced by intravenous injection of PEG-coated liposome is responsible for enhanced clearance of second dose PEG-coated liposomes, which is known as the "accelerated blood clearance (ABC) phenomenon." A similar phenomenon has been observed with PEG-coated pDNA-lipoplexes (PDCLs) upon their repeated injection. But the effect of the sequence of pDNA in PDCLs on inducing the ABC phenomenon has not been thoroughly investigated. Here, we focus on CpG motifs in pDNA, which are known to have a potent immune-stimulatory activity. PDCLs with non-CpG pDNA (PNDCL) diminished the anti-PEG IgM response, resulting in significant accumulation of a second dose in tumor tissue, comparable to that of a single injection, but not in enhanced accumulation in liver. In addition, PDCL induced proliferation of IgM(+) splenic cells including B cells. These results suggest that the CpG motif is a major cause of the induction of the ABC phenomenon when PDCLs are repeatedly injected. Immunogenicity is a relevant point of concern for non-viral delivery systems. Our results indicate that the use of non-CpG pDNA may allow meaningful repeated dosing of pDNA formulations without the induction of a strong immune reaction and thus may have important implications for therapeutic use of liposomal formulations of nucleic acids.
(Keyword)
Animals / B-Lymphocytes / Cell Proliferation / CpG Islands / DNA / Immunoglobulin M / Liposomes / Male / Mice / Mice, Inbred BALB C / Mice, Nude / Polyethylene Glycols
Lila Selim Ahmed Ali Abu Amr, Yusuke Doi, Kazuya Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor., Journal of Controlled Release, Vol.142, No.2, 167-173, 2010.
(Summary)
Recently, we designed a PEG-coated cationic liposome to achieve dual targeting delivery of l-OHP to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation showed an efficient antitumor activity in a murine tumor model after three sequential liposomal l-OHP injections. This led us to assume that prior dosing with liposomes might enhance the intra-tumoral accumulation of a subsequent dose, and hence improve the therapeutic efficacy of entrapped l-OHP. The present study shows that while a single liposomal l-OHP injection does not enhance tumor accumulation of subsequent test-PEG-coated cationic liposomes, two sequential injections of liposomal l-OHP do. Cumulative cytotoxic effects of l-OHP delivered by PEG-coated cationic liposomes led to deep diffusion of a subsequent dose of liposomal l-OHP in solid tumor presumably as a result of the enlarged intra-tumoral interstitial space. Our study suggests that sequential injections of a targeted liposomal anticancer drug is of significant clinical and practical importance in enhancing the delivery of adequate quantities of anticancer agents into intractable solid tumors, and thereby may achieve a significant anticancer efficacy.
We recently developed prostaglandin E(1) (PGE(1))-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw = 17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats. The plasma levels of PGE(1) and anti-PEG IgM antibody were determined by EIA and ELISA, respectively. Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw = 28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE(1) than NP-L20. This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE(1).
Hiroyuki Yamazaki, Masateru Miyake, Toru Nishibayashi, Tadashi Mukai, Masaaki Odomi, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of co-administration of tacrolimus on the pharmacokinetics of multiple subcutaneous administered interferon-alpha in rats., Pharmaceutical Research, Vol.26, No.8, 1832-1837, 2009.
(Summary)
Repeated administration of exogenous proteinic compounds triggers the production of specific antibodies. This reaction is limits not only pharmacokinetic studies in animal but also development of human or humanized proteins as drugs. We investigated the effect of co-administration of tacrolimus on pharmacokinetic of human interferon-alpha (h-IFN) following multiple subcutaneous administration in rats. h-IFN was administered at a dose of 5 million IU/kg. For some experiments, tacrolimus was also either subcutaneously or intravenously injected in rats at a dose of 0.001 or 0.5 mg/kg as well as with administration of h-IFN. Multiple administration of h-IFN without co-administration of tacrolimus induced IgG response at 2 or 3 weeks following first administration in the short dosing interval (daily, once per 3 days, or once per a week), irrespective of the dosing interval. Both intravenous and subcutaneous administration of tacrolimus (0.5 mg/kg) with multiple h-IFN injections successfully suppressed IgG response against h-IFN. Interestingly, in lower doses (0.001 mg/kg), intravenous co-administration of tacrolimus showed much stronger suppressive effect than subcutaneous co-administration. Intravenous co-administration of tacrolimus (0.001 mg/kg) may be a promising way to assess crossover pharmacokinetic study of human or humanized proteinic formulations with multiple dosing schedules in an experimental animal.
Tatsuaki Tagami, Kazuya Nakamura, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of siRNA in PEG-coated siRNA-lipoplex on the anti-PEG IgM production., Journal of Controlled Release, Vol.137, No.3, 234-240, 2009.
(Summary)
For efficient delivery of small interfering RNA (siRNA) in vivo, it is important to control the blood circulation of the delivery vehicle. Surface modification of the siRNA/cationic liposome complex (siRNA-lipoplex) with polyethylene glycol (PEG) is expected to enhance circulation time in blood. However, we have recently reported that anti-PEG IgM production after the first injection of PEG-coated liposome is responsible for a reduction in the blood circulation of the second dose of the liposome, which is known as the accelerated blood clearance (ABC) phenomenon. It is unknown whether a PEG-coated siRNA-lipoplex (PSCL) can cause the ABC phenomenon and anti-PEG IgM production. In this study, an anti-PEG IgM response to PSCL was detected and was inversely related to the PSCL dose. Interestingly, the anti-PEG IgM response was significantly lower for PSCL than it was for PEG-coated naked cationic liposomes (PCL). The studies with splenectomized mice and nude mice indicated that anti-PEG IgM production was closely related to an interaction of PSCL and PCL with the spleen, which is associated with a T cell-independent mechanism. In addition, PSCL induced apoptosis on IgM-expressing splenic cells more strongly than PCL did, which suggests that accumulation in the spleen and the apoptotic effect of PEG-coated substances on splenic B cells could affect the potency of anti-PEG IgM production.
(Keyword)
Animals / Apoptosis / B-Lymphocytes / Dose-Response Relationship, Immunologic / Immunoglobulin G / Immunoglobulin M / Liposomes / Male / Mice / Mice, Inbred BALB C / Mice, Nude / Polyethylene Glycols / RNA, Small Interfering / Spleen / Splenectomy
Tatsuhiro Ishida, Emi Shiraga and Hiroshi Kiwada : Synergistic antitumor activity of metronomic dosing of cyclophosphamide in combination with doxorubicin-containing PEGylated liposomes in a murine solid tumor model., Journal of Controlled Release, Vol.134, No.3, 194-200, 2009.
(Summary)
Cyclophosphamide (CPA) and doxorubicin (DXR)-containing sterically stabilized liposomes (DXR-SL) have a proven clinical activity. We propose that a metronomic CPA dosing schedule enhances accumulation of DXR-SL in solid tumors, because it causes apoptosis in the endothelial cells of the growing tumor vasculature and thereby may increase the permeability of the tumor microvessels. To establish the validity of this hypothesis we investigated the therapeutic benefits of metronomic CPA dosing (p.o.) combined with DXR-SL (i.v.) in a Lewis lung carcinoma, subcutaneously growing in C57BL/6 mouse. The metronomic CPA dosing clearly promoted accumulation and subsequent deep diffusion of SL in the solid tumor as a result of rather a transient increase in the density of CD31(+)-microvessels, which shows high permeability to SL. It appears that the enhancing effect of metronomic CPA dosing is strongly dependent on the dose of CPA as well as on the time at which the treatment was initiated. Our study indicates that the use of metronomic chemotherapy combined with nanocarriers may be of significant clinical and practical importance in treating intractable solid tumors.
(Keyword)
Administration, Oral / Animals / Antineoplastic Combined Chemotherapy Protocols / Carcinoma, Lewis Lung / Cell Line, Tumor / Cyclophosphamide / Dose-Response Relationship, Drug / Doxorubicin / Drug Administration Schedule / Drug Synergism / Injections, Intravenous / Male / Mice / Mice, Inbred C57BL / Neovascularization, Pathologic / Polyethylene Glycols / Treatment Outcome / Xenograft Model Antitumor Assays
Lila Selim Ahmed Ali Abu Amr, Shinji Kizuki, Yusuke Doi, Takuya Suzuki, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin encapsulated in PEG-coated cationic liposomes induces significant tumor growth suppression via a dual-targeting approach in a murine solid tumor model., Journal of Controlled Release, Vol.137, No.1, 8-14, 2009.
(Summary)
We recently designed a PEG-coated cationic liposome targeted to angiogenic vessels and showed, in a murine dorsal air sac model, potent anti-angiogenic activity of an oxaliplatin (l-OHP) formulation of this liposome. In the present study, we extended the l-OHP formulation to a murine tumor-xenograft model. Following three injections, l-OHP containing PEG-coated cationic liposomes showed substantial tumor growth suppression and increased survival time of tumor-bearing mice without apparent side effects, compared with other l-OHP containing PEG-coated neutral liposomes and free l-OHP. In vivo imaging showed a preferential tumor accumulation and a broader distribution of PEG-coated cationic liposomes, compared with PEG-coated neutral liposomes. In addition, PEG-coated cationic liposomes delivered larger amounts of l-OHP into the tumor tissue than other l-OHP formulations, correlating with its antitumor efficiency. In vitro studies indicated that PEG-coated cationic liposomes were internalized not only by tumor cells but also by endothelial cells, and consequently its l-OHP formulation displayed higher cytotoxicity towards both cell types as compared with l-OHP containing PEG-coated neutral liposomes. In summary, l-OHP containing PEG-coated cationic liposomes induced significant tumor growth suppression, presumably by delivering encapsulated l-OHP into both tumor endothelial cells and tumor cells. Such dual targeting approach, i.e. vascular-targeting and tumor-targeting with a single liposomal l-OHP formulation, may have great potential for overcoming some major limitations in conventional chemotherapy.
(Keyword)
Angiogenesis Inhibitors / Animals / Carcinoma, Lewis Lung / Cations / Coated Materials, Biocompatible / Dosage Forms / Drug Carriers / Drug Delivery Systems / Excipients / Liposomes / Male / Mice / Mice, Inbred C57BL / Neovascularization, Pathologic / Organoplatinum Compounds / Polyethylene Glycols / Xenograft Model Antitumor Assays
Yoshimasa Isakari, Shinji Sogo, Tatsuhiro Ishida, Takuma Kawakami, Toshihide Ono, Takao Taki and Hiroshi Kiwada : Gene expression analysis during platelet-like particle production in phorbol myristate acetate-treated MEG-01 cells., Biological & Pharmaceutical Bulletin, Vol.32, No.3, 354-358, 2009.
(Summary)
A comprehensive gene-expression analysis during platelet (PLT) production from megakaryocytes may give important information on genes involved in the PLT production process. However, the low abundance of primary megakaryocytes makes the gene expression analysis difficult. Therefore, we employed MEG-01 cells, a human megakaryocytic cell line, and confirmed that the cell line produces PLT-like particles by treatment with phorbol myristate acetate (PMA). After treatment of MEG-01 cells with PMA for 8 or 24 h, comprehensive gene expression analysis was carried out using a microarray and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). From the microarray analysis, 141 genes were up-regulated (>2-fold) and 164 genes were down-regulated (<1/2-fold). However, known PLT-related genes were not included in the up- or down-regulated genes. On the other hand, RT-PCR analysis detected increased expression of beta1-tubulin, CD62P, gpIbalpha and gpIII, which are related to PLT function and megakaryocyte differentiation, following PMA treatment for 24 h. These results indicate that the MEG-01 cell may be an alternative model system to study the process of human PLT production from megakaryocytes. The gene-expression analysis might be a powerful tool for identifying genes related to PLT production, if the experimental conditions are optimized.
Lila Selim Ahmed Ali Abu Amr, Takuya Suzuki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin targeting to angiogenic vessels by PEGylated cationic liposomes suppresses the angiogenesis in a dorsal air sac mouse model., Journal of Controlled Release, Vol.134, No.1, 18-25, 2009.
(Summary)
Oxaliplatin (trans-l-diaminocyclohexane oxalatoplatinum, l-OHP) is a third-generation platinum analogue with proven anti-tumor activity against many tumor cell lines, however it does not show sufficient anti-tumor activity in vivo when used alone. In order to overcome this problem and to achieve an anti-angiogenic therapy with l-OHP, the drug was encapsulated into PEG-coated cationic liposomes, which were designed to target the newly formed vessels, and its anti-angiogenic activity was evaluated in an in vivo mouse dorsal air sac (DAS) assay. For the DAS assay, chambers filled with tumor cells were implanted underneath the dorsal skin. l-OHP encapsulated in PEG-coated cationic liposomes (5 mg/kg mice) was intravenously injected once on day 1, 2, 3 or 4 after chamber implantation. On the fifth day after chamber implantation, animals were sacrificed and tumor-angiogenesis was evaluated. Liposome-encapsulated l-OHP completely suppressed angiogenesis in the skin when it was administered day 3 after chamber implantation. Under similar experimental conditions, neither l-OHP encapsulated in PEG-coated neutral liposomes, nor free l-OHP, nor "empty" (no drug containing) PEG-coated cationic liposomes showed such strong suppressive effect. The present study suggests that the liposomal formulation of l-OHP, which targeted to angiogenic vessels, has a remarkable in vivo anti-angiogenic activity and the formulation may become a promising novel approach to achieve anti-angiogenic therapy.
Tatsuaki Tagami, Kiyomi Hirose, Jose Mario Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : Global gene expression profiling in cultured cells is strongly influenced by treatment with siRNA-cationic liposome complexes., Pharmaceutical Research, Vol.25, No.11, 2497-2504, 2008.
(Summary)
The purpose of this study is to determine if the treatment with siRNA-lipoplexes significantly influences on global gene expression in the treated cells. We investigated global gene expression in a HT1080 cell line by a cDNA microarray. We also evaluated the effect of lipofection on global gene expression by determining the change of the expression of an exogenous gene, green fluorescence protein (GFP), and also determined treatment-related cytotoxicity. Treatment of the cells with either siRNA-lipoplexes or cationic liposomes altered the expression of approximately 2,500 genes. When lipoplexes containing non-specific siRNAs were used, GFP expression was enhanced. In this case the effect was independent on the dose and type of siRNA in the formulation. By contrast, when lipoplexes containing a specific siRNA against GFP was used, GFP expression was markedly diminished. These results clearly indicate that an efficient reduction of a targeted gene expression by a specific siRNA is accompanied by a significant alteration of the expression of numerous non-targeted genes. In addition, treatment-related cytotoxicity increased with siRNA- and cationic lipid-doses, but was not dependent on siRNA type. Non-specific effects of siRNA-lipoplexes may either enhance, attenuate or even fully mask the desired outcomes of siRNA-based biochemical studies and therapies.
Hiroyuki Koide, Tomohiro Asai, Kentaro Hatanaka, Takeo Urakami, Takayuki Ishii, Eriya Kenjo, Masamichi Nishihara, Masayuki Yokoyama, Tatsuhiro Ishida, Hiroshi Kiwada and Naoto Oku : Particle size-dependent triggering of accelerated blood clearance phenomenon., International Journal of Pharmaceutics, Vol.362, No.1-2, 197-200, 2008.
(Summary)
A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2 nm in diameter) or PEGylated liposomes (119, 261 or 795 nm) were preadministered into BALB/c mice. Three days after the preadministration [(3)H]-labeled PEGylated liposomes (127 nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2-795 nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7-31.5 nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.
(Keyword)
Animals / Dose-Response Relationship, Drug / Liposomes / Male / Metabolic Clearance Rate / Mice / Mice, Inbred BALB C / Particle Size / Phospholipids / Polyethylene Glycols / Tissue Distribution
Emi Shiraga, Jose Mario Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : A metronomic schedule of cyclophosphamide combined with PEGylated liposomal doxorubicin has a highly antitumor effect in an experimental pulmonary metastatic mouse model., International Journal of Pharmaceutics, Vol.353, No.1-2, 65-73, 2008.
(Summary)
Metronomic chemotherapy is a novel approach to the control of advanced cancer, as it appears to preferentially inhibit endothelial cell activity in the growing vasculature of tumors. Doxorubicin-containing sterically stabilized liposomes (DXR-SL) accumulate in large amounts in tumor tissue, resulting in enhanced antitumor effects of the encapsulated DXR. In the present study, it was hypothesized that metronomic chemotherapy may further augment the accumulation of DXR-SL, improving its therapeutic efficacy. This study tests the antitumor efficacy for the combination of a metronomic cyclophosphamide (CPA)-dosing schedule with sequential intravenous injections of DXR-SL in the treatment of lung metastatic B16BL6 melanoma-bearing mice. Three dosing schedules for the combination of metronomic CPA injections (s.c. 170 mg/kg every 6 days) plus either a low or a high dose of DXR-SL (i.v. 1 or 5 mg/kg every 6 days) were set: Schedule I, DXR-SL was given 3 days before the first CPA treatment; Schedule II, DXR-SL and CPA were given simultaneously; and, Schedule III, DXR-SL was given 3 days after the first CPA treatment. Lung weight and median survival time (MST) were evaluated. As expected, both the dosing schedule as well as the dose of DXR-SL improved therapeutic efficacy. Schedule I with the low DXR dose and Schedule II with the low or high DXR dose significantly increased MST, compared with regular metronomic CPA therapy. Under the dosing schedules (Schedule I with the low DXR dose and Schedule II with the high DXR), there was a strong relationship between increased MST and decreased lung weight. However, Schedule I with high DXR dose resulted in significantly lower lung weights, but did not increase MST, suggesting that chemotherapy may result in increased toxicity in some conditions. Although treatment regimens require optimization, the results of the present study may prove useful in further explorations of combining metronomic chemotherapy with liposomal anticancer drugs in the treatment of solid tumors.
Kuniko Matsumura-Takeda, Tatsuhiro Ishida, Shinji Sogo, Yoshimasa Isakari, Takao Taki, Toshiki Sudo and Hiroshi Kiwada : Lactoferrin inhibits platelet production from human megakaryocytes in vitro., Biological & Pharmaceutical Bulletin, Vol.31, No.4, 569-573, 2008.
(Summary)
The mechanism of megakaryopoiesis, proplatelet formation (PPF) and platelet (PLT) production is not fully elucidated. Lactoferrin (LF) has been reported to have many biological functions including cell proliferation and differentiation, and the LF receptor is present on megakaryocytic cells. In the present study, we examined the effect of human LF (hLF) on PLT production from primary megakaryocytes (MKs). At first, we developed a PLT production system derived from human CD34+ cells by thrombopoietin (TPO) stimulation. Because the number of proplatelets, PLTs and CD41+ MKs was remarkably increased after day 5, we employed the TPO-induced CD34+ cells on day 5. Then, the effect of hLF on PLT production from human primary MKs was examined. In the range of 3-30 micrg/ml, hLF significantly inhibited PLT production up to about 60%. However, it did not significantly change the intensity of CD41 expression in MKs and the ploidy of MKs. In addition, it did not inhibit MK progenitors. These results suggest that LF directly inhibits PLT production from matured MKs, but does not inhibit megakaryopoiesis, including proliferation/maturation processes.
Tatsuhiro Ishida, Shuntarou Kashima and Hiroshi Kiwada : The contribution of phagocytic activity of liver macrophages to the accelerated blood clearance (ABC) phenomenon of PEGylated liposomes in rats., Journal of Controlled Release, Vol.126, No.2, 162-165, 2008.
(Summary)
We earlier reported that PEGylated liposomes lose their long-circulating property when they are administered twice in the same animal within certain intervals. We recently proposed that anti-PEG IgM elicited by the first dose PEGylated liposomes selectively binds to the surface of a second dose, subsequently leading to substantial complement activation and complement-receptor mediated uptake of the second dose by hepatic Kupffer cells. In this study we found, by using a single-pass liver perfusion technique, that the first dose does not increase the intrinsic phagocytic activity of the Kupffer cells. It was also found that only serum obtained from rats that had received a first dose is able to enhance the hepatic uptake of test dose. The conditioned-serum-dependent hepatic uptake was completely abolished by pre-treatment of the serum at 56 degrees C for 30 min, which inhibits the complement activity. Conclusively, our results strongly support our earlier proposal that complement activation caused by anti-PEG IgM elicited by the first dose is a major cause of the initiation of the accelerated blood clearance of a subsequent dose PEGylated liposome in the ABC phenomenon.
Mario José Barichello, Noriko Yamakawa, Masatoshi Kisyuku, Hiroshi Handa, Taiki Shibata, Tatsuhiro Ishida and Hiroshi Kiwada : Combined effect of liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate in rat skin., International Journal of Pharmaceutics, Vol.357, No.1-2, 199-205, 2008.
(Summary)
In this report, we investigated the combined effect of drug liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate (ISN) in rat abdominal skin in vitro. Occlusive application of both liposomal and aqueous ISN solution, with and without addition of 5% glycerol, showed that drug liposomalization and addition of glycerol has far-reaching implications for ISN permeation and accumulation in 4 and 8 weeks old rat abdominal skin. Using 8 weeks old rat abdominal skin, the optimal concentration of glycerol to be added to liposomal ISN was found to be 5%. The ISN mean values permeated through and accumulated in stripped 8 weeks old rat abdominal skin from those formulations described above were not significant different, which might indicate the combined effect of glycerol and liposomal ISN resides solely in the stratum corneum (SC). Based on previous reports, the enhancement effect of glycerol might be due to an increase in the SC hydration, and perhaps due to subtle changes in the lipid organization caused by penetration of liposomal lipids within the SC intercellular spaces. These data might provide evidence that glycerol action on SC is useful to facilitate skin permeation and accumulation of drugs formulated in liposome.
T Asai, Y Suzuki, S Matsushita, S Yonezawa, J Yokota, Y Katanasaka, Tatsuhiro Ishida, T Dewa, Hiroshi Kiwada, N Nango and N Oku : Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown., Biochemical and Biophysical Research Communications, Vol.368, No.2, 243-248, 2008.
(Summary)
Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.
Tatsuhiro Ishida, Wang Xinyu, Taro Shimizu, Kousuke Nawata and Hiroshi Kiwada : PEGylated liposomes elicit an anti-PEG IgM response in a T-cells independent manner., Journal of Controlled Release, Vol.122, No.3, 349-355, 2007.
(Summary)
We recently reported that intravenous injections of "empty" PEGylated liposomes without encapsulated or surface coupled proteins elicit a PEG-specific IgM response in rats. In the present study, simultaneous weak anti-PEG IgG and strong anti-PEG IgM responses were detected following intravenous injections of "empty" PEGylated liposomes. The pattern of immune response appears to differ from the classic primary response against T cell-dependent (TD) antigens. The anti-PEG IgM response was detected in T-cell deficient nude BALB/c mice following intravenous injection of "empty" PEGylated liposomes, suggesting that "empty" PEGylated liposomes initiate the immune response against PEG in a T cell-independent manner. In vitro splenic lymphocytes-proliferation assay indicated that TNP-LPS, a typical type 1 T cell-independent (TI) antigen (TI-1 antigen), significantly primed the proliferation, while TNP-Ficoll, a typical type 2 TI antigen (TI-2 antigen), and "empty" PEGylated liposomes did not prime any proliferation under these experimental conditions. In addition, in splenic marginal zone (MZ) B-cell-depleted rats, the anti-PEG IgM response was diminished, while the immune reactions against TNP-BSA (a TD antigen) and TNP-LPS (TI-1 antigen) were not diminished. These results demonstrate that "empty" PEGylated liposomes may promote the immune response against PEG as a result of priming the activation of MZ B cells, as TI-2 antigen promotes a specific IgM response. In conclusion, although the mechanistic details behind the immune reaction against "empty" PEGylated liposomes are not yet clear, the liposomes elicit an anti-PEG IgM response in a T cell-independent manner and appear to be a TI-2 antigen, and splenic MZ B cells may be essential for the immune response against "empty" PEGylated liposomes.
(Keyword)
Animals / Antigens, T-Independent / Cell Proliferation / Cyclophosphamide / Immunoglobulin G / Immunoglobulin M / Injections, Intravenous / liposomes / Male / Mice / Mice, Inbred BALB C / Mice, Nude / Polyethylene Glycols / Rats / Rats, Wistar / Spleen / T lymphocytes
Hiroto Hatakeyama, Hidetaka Akita, Emi Ishida, Koichi Hashimoto, Hideo Kobayashi, Takanori Aoki, Junko Yasuda, Kenichi Obata, Hiroshi Kikuchi, Tatsuhiro Ishida, Hiroshi Kiwada and Hideyoshi Harashima : Tumor targeting of doxorubicin by anti MT1-MMP antibody-modified PEG liposomes., International Journal of Pharmaceutics, Vol.342, No.1-2, 194-200, 2007.
(Summary)
Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.
(Keyword)
Animals / Antibiotics, Antineoplastic / Antibodies, Monoclonal / Doxorubicin / Drug Compounding / Drug Delivery Systems / Excipients / Immunochemistry / Immunoglobulin Fab Fragments / Liposomes / Male / Matrix Metalloproteinase 14 / Mice / Mice, Inbred BALB C / Neoplasms, Experimental / Polyethylene Glycols
Kazutaka Atobe, Tatsuhiro Ishida, Emi Ishida, Kouichi Hashimoto, Hideo Kobayashi, Jyunko Yasuda, Takanori Aoki, Ken-ichi Obata, Hiroshi Kikuchi, Hidetaka Akita, Tomohiro Asai, Hideyoshi Harashima, Naoto Oku and Hiroshi Kiwada : In vitro efficacy of a sterically stabilized immunoliposomes targeted to membrane type 1 matrix metalloproteinase (MT1-MMP)., Biological & Pharmaceutical Bulletin, Vol.30, No.5, 972-978, 2007.
(Summary)
The poor selective cytotoxicity of anticancer drugs lead to dose-limiting adverse effects which compromise the clinical outcome. Solid tumors recruit new blood vessels to support their growth, and epitopes that are uniquely expressed on tumor cells and tumor endothelial cells (ECs) can function as targets for immunoliposomal anticancer drugs. Membrane type 1 matrix metalloproteinase (MT1-MMP), an important protein related to tumor growth and angiogenesis, is expressed on malignant tumor cells and is activated ECs. Selective delivery could be achieved by targeting MT1-MMP, as well as other angiogenic ECs. In this regard, an anti-MT1-MMP Fab' antibody was used to prepare a MT1-MMP targeted sterically stabilized immunoliposomes (SIL[anti-MT1-MMP(Fab')]). The binding and intracellular distribution of SIL[anti-MT1-MMP(Fab')] and a non-targeted sterically stabilized liposomes (SL) were examined using human fibrosarcoma HT-1080 cells. SIL[anti-MT1-MMP(Fab')] was taken up by the cells in a lipid concentration, temperature, and time dependent manner, ultimately accumulating in the lysosomes. The cytotoxicity of doxorubicin (DXR)-containing SIL[anti-MT1-MMP(Fab')] (DXR-SIL[anti-MT1-MMP(Fab')]) was significantly higher than that of DXR-containing SL. The cellular internalization of SIL[anti-MT1-MMP(Fab')] was inhibited by endocytosis inhibitors, suggesting that their internalization was mediated via clathrin- or caveolae-dependent endocytosis. Furthermore, the efficient binding of SIL[anti-MT1-MMP(Fab')] was observed on human umbilical vein endothelial cells (HUVEC). Based on these results, it would be expected that DXR-SIL[anti-MT1-MMP(Fab')] may achieve direct tumor cell kill and indirect tumor cell kill via the destruction of the tumor endothelium in vivo. This strategy may have the potential for overcoming some major limitations in conventional chemotherapy in vivo.
Lap Thi Nguen, Kazutaka Atobe, Mari Jose Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes., Biological & Pharmaceutical Bulletin, Vol.30, No.4, 751-757, 2007.
(Summary)
Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.
Tatsuaki Tagami, Mari Jose Barichello, Hiroshi Kikuchi, Tatsuhiro Ishida and Hiroshi Kiwada : The gene-silencing effect of siRNA in cationic lipoplexes is enhanced by incorporating pDNA in the complex., International Journal of Pharmaceutics, Vol.333, No.1-2, 62-69, 2007.
(Summary)
Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.
Xinyu Wang, Tatsuhiro Ishida and Hiroshi Kiwada : Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes., Journal of Controlled Release, Vol.119, No.2, 236-244, 2007.
(Summary)
Earlier we reported that PEGylated liposomes lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). We proposed that anti-PEG IgM, induced by the PEGylated liposomes, is responsible for the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of PEGylated liposomes, subsequently leading to substantial complement activation. Interestingly, we found that under certain circumstances administration of conventional liposomes without PEG-coating also caused a strong ABC response upon injection of a second dose of PEGylated liposomes, but not of conventional liposomes. This suggests that also conventional liposomes not modified with PEG can promote an IgM response against PEG. We report here that, irrespective of the presence or absence PEG-coating, a single first dose of liposomes is capable of inducing a strong anti-PEG IgM response and, under certain circumstances, also weak responses against other lipid components. A good correspondence was observed between the amount of IgM associating with both PEGylated and conventional liposomes, concomitant complement activation triggered by those liposomes and the magnitude of the ABC phenomenon against those liposomes. Taken together, our results demonstrate that the ABC phenomenon is fully attributable to production of anti-PEG IgM by the first dose of liposomes and the subsequent complement activation upon a second dose of PEGylated but not conventional liposomes. Although the responsible immunogenic epitopes of the liposomes remain to be determined, the immunogenicity of 'empty' liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. Furthermore, our findings as described here raise important concerns with regard to the safety and efficiency of liposomes currently under development for clinical use.
Yoshimasa Isakari, Yasuo Harada, Dai Ishikawa, Kuniko Matsumura-Takeda, Shinji Sogo, Tatsuhiro Ishida, Takao Taki and Hiroshi Kiwada : Efficient gene expression in Megakaryocytic cell line using Nucleofection., International Journal of Pharmaceutics, Vol.338, No.1-2, 157-164, 2007.
(Summary)
To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.
Tomotaka Kobayashi, Tatsuhiro Ishida, Yurie Okada, Saori Ise, Hideyoshi Harashima and Hiroshi Kiwada : Effect of Transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells., International Journal of Pharmaceutics, Vol.329, No.1-2, 94-102, 2007.
(Summary)
The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.
(Keyword)
Antibiotics, Antineoplastic / Cell Line, Tumor / Doxorubicin / Drug Delivery Systems / Drug Resistance, Neoplasm / Humans / Liposomes / Neoplasms / P-Glycoprotein / Receptors, Transferrin
Tatsuhiro Ishida, Kazutaka Atobe, Xinyu Wang and Hiroshi Kiwada : Accelerated blood clearance of PEGylated liposomes upon repeated injections: Effect of doxorubicin-encapsulation and high-dose first injection., Journal of Controlled Release, Vol.115, No.3, 251-258, 2006.
(Summary)
The "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection, has been reported to occur in rodents and rhesus monkeys. This rapid clearance was reported to be caused by the binding of PEG-specific IgM, which was generated by the first dose of injected liposomes, to the second dose of liposomes and the subsequent activation of complement, serving in turn as an opsonin. Although there are several PEGylated liposomal formulations, such as Doxil/Caelyx loaded with doxorubicin (DXR), in clinical use, the rapid clearance phenomenon has never been reported for such formulations. In the present article, we report that a first injection of PEGylated liposomes containing encapsulated DXR failed to induce the ABC phenomenon. Likewise, no rapid clearance of the test dose was observed when the first dose of "empty" PEGylated liposomes (without DXR) exceeded 5 micromol phospholipids/kg. By contrast, "empty" PEGylated liposomes at a low dose (1 micromol phospholipids/kg) induced the phenomenon as before. Western blot analysis revealed abundant binding of IgM to PEGylated liposomes when these were incubated in serum from rats that had received "empty" PEGylated liposomes. Substantially less binding of IgM was found when the liposomes were incubated in serum from rats treated with DXR-loaded PEGylated liposomes. For both the empty and the DXR-containing liposomes the amounts of IgM binding to the liposomes decreased with an increasing dose of injected liposomes. Serum obtained from rats following injection of empty PEGylated liposomes caused complement activation by addition of PEGylated liposomes in an inversely dose-dependent manner: the lower the dose, the higher the complement activation. By contrast, no complement activation was detected with serum from rats that had been treated with DXR-loaded PEGylated liposomes. These findings suggest that encapsulation of DXR as well as a relatively high lipid dose abrogate the immune response against PEGylated liposomes which is observed with the same liposomes but without DXR and at low doses. Our observations may thus have important implications for the development, evaluation and therapeutic use of liposomal cytotoxic drug formulations requiring multiple injection schemes.
Tatsuhiro Ishida, Masako Ichihara, Xinyu Wang and Hiroshi Kiwada : Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes., Journal of Controlled Release, Vol.115, No.3, 243-250, 2006.
(Summary)
It is well known that steric stabilization of the surface of liposomes by a polyethyleneglycol (PEG) conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended circulation times of the liposomes (t1/2 approximately 20 h in rat). Recently, we reported on the "accelerated blood clearance (ABC) phenomenon", causing PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. We also reported that abundant binding of IgM, secreted into the blood stream after the first dose and, to PEGylated liposomes, plays an essential role in the induction of the ABC phenomenon. Spleen is well known to play a central role in the immune reaction and to produce IgM following a bacterial infection. The aim of the present study was to determine whether spleen contributes to the induction of the ABC phenomenon and to unravel its role in the phenomenon. In rats that were splenectomized (surgical removal of spleen) prior to the first injection of liposomes (0.001 micromol phospholipids/kg), the ABC phenomenon was totally abolished. In these rats serum IgM concentrations as well as the amounts of IgM bound to PEGylated liposomes were substantially reduced. Splenectomy attenuated the ABC phenomenon when performed until 3 days post-first injection. Removal of the spleen 4 days post-first injection left the ABC phenomenon unchanged. This finding indicates that the immune reaction in the spleen against the PEGylated liposomes occurs during at least 2-3 days following the first administration and then IgM reactive to PEGylated liposomes is produced. The present study proves that the spleen plays a critical role in the induction phase of the ABC phenomenon. For effective clinical application, many liposomal drug formulations will require multiple injections. The ABC phenomenon described in this and several preceding papers therefore has important implications for the development and evaluation of therapeutically useful liposomal formulations requiring multiple-dose administration.
Mario Jose Barichello, M. Handa, M. Kisyuku, Taiki Shibata, Tatsuhiro Ishida and Hiroshi Kiwada : Inducing effect of liposomalization on the transdermal delivery of hydrocortisone: creation of a drug supersaturated state., Journal of Controlled Release, Vol.115, No.1, 94-102, 2006.
(Summary)
In order to investigate the effect of liposomal drugs on skin delivery, it was postulated that the process of liposomalization might lead the drug to an overpredicted solubility state which has far-reaching implications for drug skin permeation and accumulation. In this regard, conventional (CL) and flexible liposomes (FL) were prepared by the lipid film hydration method and the particles were downsized by sonication using hydrocortisone (HC) as a poorly water soluble model drug. The solutions derived from the whole CL and FL suspensions eluted on a Sephadex G-50 column (SG-50) demonstrated that most part of HC not only resides solely in the water phase but also it might exist in an improved solubility state. The results of the in vitro study using rat abdominal skin and occlusive application indicated that HC penetrated and accumulated much better solely than when associated with CL or FL. In regard to the penetration of the non-entrapped HC associated to liposomes bilayer fragments, a very small amount of phospholipids in the non-liposomal part eluted on SG-50 was found that could not justify by itself the penetration of HC associated to liposome bilayer fragments. It was proposed that all the steps of the liposomes preparation process might contribute for the increased HC solubility state, but definitively the presence of phospholipids played a crucial role on improving the HC solubility in the absence of sodium cholate. In comparison with commercially available ointments, the non-entrapped HC solution derived from the whole CL suspension eluted on SG-50 showed a higher concentration of HC accumulated and more uniformly distributed as well in the epidermis and dermis compartments. In addition, the thermodynamic activity of the non-entrapped HC solutions maintaining a driving force of the drug across the skin barrier pointed out that the level of HC solubility achieved during liposome preparation has far-reaching implication for drug skin permeation and accumulation in the experimental conditions used. The findings also indicated that the non-entrapped drug solutions obtained on the process of liposomalization could be useful on transdermal drug delivery systems, particularly for improving the permeation and accumulation capacity of poorly soluble drugs.
Foi Dang, Wenhao Li, LiGo Zhang, M Japasini, Tatsuhiro Ishida, Hiroshi Kiwada, N. Kaji, M. Tokeshi and Yoshinobu Baba : Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes., Journal of Chromatography. A, Vol.1118, No.2, 218-225, 2006.
(Summary)
In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.
Tatsuhiro Ishida, Masako Ichihara, Xinyu Wang, Kenji Yamamoto, Jyunji Kimura, Eiji Majima and Hiroshi Kiwada : Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes., Journal of Controlled Release, Vol.112, No.1, 15-25, 2006.
(Summary)
Steric stabilization of the surface of liposomes by a PEG conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended their circulation times (t(1/2) approximately 20h in rat). Recently, we reported on the "accelerated blood clearance phenomenon", causing "invisible" PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. In addition, we reported that certain serum factor(s) secreted into the blood after the first dose of PEGylated liposomes play an essential role in the phenomenon. The aim of the present study was to identify the major serum protein(s) responsible for the phenomenon and to unravel their mode of action. The amount of protein binding to PEGylated liposomes during incubation with serum hardly correlated with the extent of the phenomenon. PEGylated liposomes incubated with serum obtained from rats pre-injected 5 days before with the same liposomes showed a much more complex pattern of bound proteins than when incubated with naïve serum, as revealed by 2D-PAGE and SDS-PAGE. Subsequent analysis with LC-MS/MS and Western blot showed that the major pre-treated serum protein binding to PEGylated liposomes was IgM. Semi-quantitative analysis showed that larger amount of IgM bound to PEGylated liposomes compared to conventional liposomes. It was further demonstrated that PEGylated liposomes could activate the complement system due to IgM binding when incubated in serum from rats pre-injected with PEGylated liposomes, while conventional liposomes were not. These findings suggest that the selective binding of IgM to the second injected PEGylated liposomes and the subsequent complement activation by IgM resulted in the accelerated clearance and enhanced hepatic uptake of the second injected PEGylated liposomes. Based on the results described here, we are drawing attention to the potential occurrence of unexpected immune reactions upon intravenous administration of PEGylated liposomes or other particles and, by extension, PEGylated proteins and DNAs.
Tatsuhiro Ishida, Wenhao Liu, Zhung Li and Hiroshi Kiwada : Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection., The Journal of Gene Medicine, Vol.8, No.3, 324-334, 2006.
(Summary)
Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.
Tatsuhiro Ishida, Yurie Okada, Tomotaka Kobayashi and Hiroshi Kiwada : Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood., International Journal of Pharmaceutics, Vol.309, No.1-2, 94-100, 2006.
(Summary)
We have reported that targeted, pH-sensitive sterically stabilized liposomes are able to increase the cytotoxicity of DXR in vitro against B lymphoma cells, but the rate of release of DXR in plasma was too rapid to permit the results to be extended to in vivo applications. The purpose of the study reported here is two-fold. First, to understand the mechanism of the rapid release of DXR from pH-sensitive sterically stabilized liposomes (PSL) in human plasma. Second, to reformulate the above liposomes to improve their drug retention, while retaining their pH sensitivity. The stability of the PSL formulations in human plasma was evaluated by comparing the rate of release of encapsulated DXR with that of HPTS, a water-soluble fluorescent marker. Since DXR, but not HPTS, a water soluble-less membrane permeable fluorescence marker, was rapidly released from liposomes in the presence of plasma, the rapid release of DXR is likely caused by the diffusion of DXR molecules through the lipid bilayer, not by the disruption of the membrane. In order to develop more stable PSL formulations, various molar ratios of the membrane rigidifying lipid, hydrogenated soy HSPC and/or CHOL, were added to the lipid composition and the rate of release of encapsulated solutes and pH-sensitivity were evaluated. The compositions that showed the best drug retention and pH-sensitivity were a mixture of DOPE/HSPC/CHEMS/CHOL/mPEG(2000)-DSPE at a molar ratio of 4:2:2:2:0.3 and DOPE/HSPC/CHEMS/CHOL at a molar ratio of 4:2:2:2. Our formulations, if targeted to internalizing antigens on cancer cells, may increase intracellular drug release rates within acidic compartment, resulting in a further increase in the therapeutic efficacy of targeted anticancer drug-containing liposomes.
Lap Thi Nguyen, Tatsuhiro Ishida and Hiroshi Kiwada : Gene Expression in Primary Cultured Mouse Hepatocytes with a Cationic Liposomal Vector, TFL-3: Comparison with Rat Hepatocytes, Biological & Pharmaceutical Bulletin, Vol.28, No.8, 1472-1475, 2005.
(Summary)
We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.
Tatsuhiro Ishida, Masae Harada, Wang Xinyu, Masako Ichihara, Kenji Irimura and Hiroshi Kiwada : Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: Effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes, Journal of Controlled Release, Vol.105, No.3, 305-317, 2005.
(Summary)
We recently reported that a second dose of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG2000-liposomes) is rapidly cleared from the blood and accumulates in the liver when injected twice in the same rat or mouse at several-day intervals (referred to as the "accelerated blood clearance (ABC) phenomenon"). In the present study we observed that a high dose (5 micromol/kg) of conventional liposomes (CL: without a PEG-coating) can induce the same phenomenon, while a low lipid dose (0.001 micromol/kg) did not. The induction of the phenomenon by mPEG2000-liposomes decreased with increasing first dose (0.001-5 micromol/kg). We observed a strong inverse relationship between the dose of initially injected PEG2000-liposomes and the extent to which the ABC phenomenon was induced: the higher the dose the smaller the phenomenon. Increasing the PEG density at the liposome surface beyond 5 mol% attenuated rather than induced the induction of the phenomenon, but elongation of the PEG chain length up to M.W. 5000, had no effect. In a series of hematological, serum-biochemical and histopathological safety evaluations we observed neither acute toxicity nor any signs of hepatic damage during the induction of the ABC phenomenon. Morphological examination of the liver by transmission electron microscopy (TEM) showed extensive accumulation of the second dose of mPEG2000-liposomes in the Kupffer cells, even already after 15 min, suggesting that the PEG liposomes had somehow lost the protective effect of the surface-grafted PEG against rapid clearance. The observations reported in this paper may have a considerable impact on the design and engineering of PEGylated liposomal formulations for use in multiple drug therapy.
Wang Xinyu, Tatsuhiro Ishida, Masako Ichihara and Hiroshi Kiwada : Influence of the physicochemical properties of liposomes on the accelerated blood clearance phenomenon in rat, Journal of Controlled Release, Vol.104, No.1, 91-102, 2005.
(Summary)
We have recently reported that PEGylated liposomes (PL) are cleared rapidly from the blood circulation when they are administered twice in the same rat at certain intervals, even if the liposomes are sterically stabilized by a surface modification with PEG (referred to as the accelerated blood clearance (ABC) phenomenon, J. Control. Release, 88, 35-42 (2003)). Now we report on the influence of physicochemical properties (PEG-modification, size and surface charge) of either the first or the second dose of liposomes on the ABC phenomenon. When, for the first dose, conventional liposomes (CL; without a PEG coating) of 110-nm diameter were injected, only a very slight ABC phenomenon was observed, irrespective of the liposomal surface charge: both clearance rate and hepatic accumulation of the second injected PL were only slightly enhanced compared to those of a single dose of PL. Interestingly, when for the first injection small-size liposomes (60 nm) were used, either charged or PEG-modified, but not neutral, the ABC phenomenon was clearly manifest. Apparently, the induction of the ABC phenomenon is not only determined by the PEG coating but also by the size and surface charge of the first dose of liposomes. Also when for the second dose small-size PEGylated liposomes were used, the ABC phenomenon was observed after induction by a first injection of PL, whereas plasma kinetics and organ uptake of a second dose of negatively charged CL (NCL, 110 nm) or small-sized NCL (SNCL, 60 nm) were not altered. Apparently, the PEG coating on the second dose is essential for the liposomes to be susceptible to the ABC phenomenon. The results reported here suggest that the physicochemical properties of both the first and second dose of liposomes are important either for the induction of the phenomenon or for its expression. Our observations may have a considerable impact on the clinical application and engineering of liposomal formulations for use in multiple drug therapy.
Wenhao Li, Tatsuhiro Ishida, Yurie Okada, Naoto Oku and Hiroshi Kiwada : Increased Gene Expression by Cationic Liposomes (TFL-3) in Lung Metastases Following Intravenous Injection, Biological & Pharmaceutical Bulletin, Vol.28, No.4, 701-706, 2005.
(Summary)
We recently showed that size, not surface charge, is a major determinant of the in vitro lipofection efficiency of pDNA/TFL-3 complex (lipoplex), even in the presence of serum. In this study, the effect of lipoplex size as a result of interaction with serum proteins on in vitro lipofection and the relationship of this with in vivo lipofection was examined in a murine lung metastasis model. As previously described, the pDNA to lipid ratio (P/L ratio) affected both the size and zeta potential of the lipoplex. In vitro studies also indicated that transgene expression in B16BL6 cells was largely dependent on the size of the lipoplex, both in the absence or presence (50% (v/v)) of serum. An in vivo lipofection experiment showed that predominant gene expression in lungs occurred only in tumor-bearing mice, not in normal mice. Based on the in vitro study, this tumor-related gene expression was not related to lipoplex size in the presence of serum (50% (v/v)), suggesting that the size alteration, as the result of interactions with serum proteins in the blood stream may not play an important role in the case of systemic injections. In addition, the efficient gene expression in tumor-bearing lung was not related to the progression of lung metastases. The area-specific gene expression in tumor-bearing lungs, which was largely dependent on the P/L ratio of the lipoplexes, was observed by fluorescent microscopy. Although the underlying mechanism for the area-specific transgene expression is not clear, it may be related to the interaction of lipoplexes with tumor cells, vascular endothelial cells under angiogenesis and normal cells in the lungs. The possibility that TFL-3 is a useful utility to the targeted delivery of pDNA to lungs and tumor-related lipofection is demonstrated. This result suggests that area-specific gene expression in lung metastases may be achieved by controlling the physicochemical properties of the lipoplex, i.e. the P/L ratio.
Wenhao Li, Tatsuhiro Ishida, Rieko Tachibana, Mohamad Radwan Almofti, Xinyu Wang and Hiroshi Kiwada : Cell type-specific gene expression, mediated by TFL-3, a cationic liposomal vector, is controlled by a post-transcription process of delivered plasmid DNA, International Journal of Pharmaceutics, Vol.276, No.1-2, 67-74, 2004.
(Summary)
The issue of whether the TFL-3, a recently developed cationic liposome, achieves efficient gene expression in different mammalian cell lines (NIH/3T3, LLC, A431 and HeLa cells) was examined. The issue of whether gene expression is related to the amount of plasmid DNA (pDNA) delivered in cells or nuclei following transfection was also examined. The cells were transfected for 1h with pDNA/TFL-3 lipoplexes, and the transfection efficiency was determined by means of a luciferase activity assay. The amount of intracellular and intranuclear pDNA following the transfection was also quantitatively determined. Successful transgene expressions in all cell lines we tested were observed under our experimental conditions, suggesting that the TFL-3 represents a suitable nonviral vector system for the successful gene expression in mammalian cells in vitro. The degree and rate of gene expression were dependent on the type of cells used as well as the incubation time after transfection, but these parameters were independent of the amount of gene delivered to cells and nuclei. These results suggest that TFL-3 mediated gene expression is largely controlled by the process of post-transcription of the delivered pDNA, and not by the process of cellular entry of pDNA and cytoplasmic trafficking of pDNA into nuclei, which is dependent on the cell type. Therefore, the results obtained here clearly suggest that the cell type-specific improvement in transcription efficiency of pDNA and translation of the derived mRNA, together with an improved delivery system to enhance the nuclear delivery of pDNA, is necessary to achieve efficient transgene expression in mammalian cells.
Tatsuhiro Ishida, Takako Ichikawa, Masako Ichihara, Yasuyuki Sadzuka and Hiroshi Kiwada : Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice., Journal of Controlled Release, Vol.95, No.3, 403-412, 2004.
(Summary)
Using mice as a model, we recently reported that the long-circulating properties of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG(2000)-liposomes) disappeared when they were intravenously injected at certain intervals [referred to as the "accelerated blood clearance (ABC) phenomenon"]. Herein, we report on a study of issue of whether physicochemical properties of a prior dose of liposomes such as degree of PEGylation, PEG chain length, lipid dose, surface charge, size, play a role in inducing this phenomenon. The injection of conventional liposomes (without a PEG-coating) significantly induced the phenomenon. The PEGylation of conventional liposomes attenuated the induction of the phenomenon somewhat with increasing molar content of PEG derivative and PEG chain length. These findings clearly suggest that the PEGylation of liposomes are not the major cause of the ABC phenomenon but, rather, played a role in preventing it. In addition, increasing the lipid dose in a prior dose of mPEG(2000)-liposomes (0-25 micromol/kg) increased the induction of the phenomenon in a sigmoid manner. The surface charge and size of the liposomes were not critical for the induction of the phenomenon, although generally these serve as determinants in the biodistribution of liposomes. The results reported here clearly indicate that the physicochemical properties of a prior dose of liposomes strongly affect the pharmacokinetic behavior of a subsequent injection of mPEG(2000)-liposomes: The extent of PEGylation and the lipid dose had an effect, but the surface charge and size did not. The results reported herein have a considerable impact on the design and engineering of liposomal formulations for use in multiple drug therapy as well as in therapy that involves the use of liposomal drugs.
(Keyword)
Animals / physical chemistry / Drug Administration Schedule / Injections, Intravenous / Japan / liposomes / Liver / Male / Metabolic Clearance Rate / Mice / Mice, Inbred Strains / Polyethylene Glycols / Radiopharmaceuticals / Time Factors
Rieko Tachibana, Naoko Ide, Yasuo Shinohara, Hideyoshi Harashima, C. Anthony Hunt and Hiroshi Kiwada : An assessment of relative transcriptional availability from nonviral vectors, International Journal of Pharmaceutics, Vol.270, No.1-2, 315-321, 2004.
(Summary)
To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.
(Keyword)
Animals / Chloramphenicol O-Acetyltransferase / Drug Carriers / Drug Delivery Systems / Gene Expression / Gene Transfer Techniques / Genes, Reporter / Genetic Vectors / Liposomes / Mice / NIH 3T3 Cells / Plasmids / Polyethyleneimine / Time Factors / Transcription, Genetic
Lap Thi Nguyen, Tatsuhiro Ishida, Sachiko Ukitsu, Wen Hao Li, Rieko Tachibana and Hiroshi Kiwada : Culture Time-Dependent Gene Expression in Isolated Primary Cultured Rat Hepatocytes by Transfection with the Cationic Liposomal Vector TFL-3, Biological & Pharmaceutical Bulletin, Vol.26, No.6, 880-885, 2003.
(Summary)
The development of a carrier system that enables the transfer of a functional exogenous gene to non- or less frequently dividing mammalian cells is essential for increasing the available options for the treatment of various diseases. The issue of whether TFL-3, a recently developed cationic liposome, can be successfully used to achieve gene expression in primary cultured rat hepatocytes was examined. The hepatocytes were transfected for 4 h with plasmid DNA (pDNA) in TFL-3 at various time points after 4-h preculture. The transfection efficiency was determined at various times posttransfection with pDNA coding for chloramphenicol acetyltransferase (CAT), luciferase, or beta-galactosidase. The amount of intranuclear pDNA present, as a consequence of the lipofection, was also quantitatively determined. Successful lipofections were observed for all pDNA tested, and the efficiencies were superior to that of commercially available LIPOFECTAMINE under our experimental conditions. The degree and rate of gene expression were dependent on incubation time prior to lipofection as well as on the density of the cells per dish, but this relationship did not hold for the amount of gene delivered to the nuclei. These results indicate that TFL-3 could be a useful vector for achieving sufficient gene expression in rat hepatocytes and suggest that the culture time prior to and following lipofection, which is related to the biological condition of the cells, may be one major factor affecting efficient gene expression in nondividing cells.
Tatsuhiro Ishida, Kaori Masuda, Takako Ichikawa, Masako Ichihara, Kenji Irimura and Hiroshi Kiwada : Accelerated clearance of a second injection of PEGylated liposomes in mice., International Journal of Pharmaceutics, Vol.255, No.1-2, 167-174, 2003.
Mohamad Radwan Almofti, Hideyoshi Harashima, Yasuo Shinohara, Almofti Ammar, Yoshinobu Baba and Hiroshi Kiwada : Cationic liposome-mediated gene delivery:Biophysical study and mechanism of internalization Arch, Archives of Biochemistry and Biophysics, Vol.410, No.2, 246-253, 2003.
(Summary)
To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.
Tatsuhiro Ishida, Ryuhei Maeda, Masako Ichihara, Kenji Irimura and Hiroshi Kiwada : Accelerated clearance of PEGylated liposomes in rats after repeated injections., Journal of Controlled Release, Vol.88, No.1, 35-42, 2003.
(Summary)
Polyethylene glycol-modified liposomes (PEGylated liposomes) represent promising carrier systems for therapeutic agents. Herein, we report on a study of the effect of repeated injection of PEGylated liposomes on their pharmacokinetics in rats. The first dose resulted in a reduction in the circulation time and an increase in hepatic accumulation of the second dose in a time-interval of injection-dependent manner. No significant increases in the number of Kupffer cells were detectable, although the liver most likely played an important role in the accelerated clearance. Interestingly, the acceleration in clearance became less pronounced, when the third dose was injected at 4, 7 or 14 days after the second injection (the second dose was given 5 weeks after the first injection). An accelerated clearance was evoked in normal rats by the transfusion of serum from rats that had received PEGylated liposomes 5 days earlier, indicating that humoral serum factor(s) are also involved in causing the accelerated clearance. A complement consumption assay indicated that the complement system is not the factor. In summary, multiple injections of PEGylated liposomes clearly altered their pharmacokinetic behavior in rats. It is likely that cellular immunity (Kupffer cells) and humoral immunity are required to cause the phenomenon. The results reported here have a considerable impact in and important implications on the clinical application, design and engineering of PEGylated liposomes for use in repeated intravenous administration.
Mohamad Radwan Almofti, Hideyoshi Harashima, Yasuo Shinohara, Ammar Almofti, Wenhao Li and Hiroshi Kiwada : Lipoplex size determines lipofection efficiency with or without serum., Molecular Membrane Biology, Vol.20, No.1, 35-43, 2003.
(Summary)
In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.
(Keyword)
Cationic Liposomes / Dna / Lipofection / Fusion / Uptake
Rieko Tachibana, Hideyoshi Harashima, Naoko Ide, Sachiko Ukitsu, Yasuko Ohta, Norio Suzuki, Hiroshi Kikuchi, Yasuo Shinohara and Hiroshi Kiwada : Quantitative Analysis of Correlation between Nunber of Nuclear Plasmids and Gene Expression Activity after Transfection with Cationic Liposomes, Pharmaceutical Research, Vol.19, No.4, 377-381, 2002.
(Summary)
A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.
(Keyword)
Animals / Cations / Cell Nucleus / Dose-Response Relationship, Drug / Drug Delivery Systems / Drug Evaluation, Preclinical / Gene Expression Regulation / Liposomes / Plasmids / Polymerase Chain Reaction / Rats / Transfection / Tumor Cells, Cultured
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12033367
Rieko Tachibana, Hideyoshi Harashima, Tatsuhiro Ishida, Yasuo Shinohara, Mari Hino, Hiroshi Terada, Yoshinobu Baba and Hiroshi Kiwada : Effect of Cationic Liposomes in an in Vitro Transcription and Translation System, Biological & Pharmaceutical Bulletin, Vol.25, No.4, 529-531, 2002.
(Summary)
The effects of cationic liposomes complexed with plasmid DNA on the process of transcription was examined using a recently developed rapid cell free translation system. The findings indicate that the liposome itself inhibited the process when the ratio of DNA/liposome typically used in transfection studies was used.
Tatsuhiro Ishida, Yoshihiro Takanashi, Hisako Doi, Isao Yamamoto and Hiroshi Kiwada : Encapsulation of an antivasospastic drug, fasudil, into liposomes, and in vitro stabiliy of the fasudil-loaded liposomes gradient., International Journal of Pharmaceutics, Vol.232, No.1-2, 59-67, 2002.
(Summary)
The objectives of this work were to develop a liposomal fasudil, an antivasospastic drug, as a possible means to deliver the encapsulated drug to the brain, and to characterize the stability of the liposomal formulation in vitro. Transmembrane electrochemical gradients of H+ or ammonium sulfate were created, and their effect on the uptake of fasudil into preformed hydrogenated soy phosphatidylcholine/cholesterol (HSPC/CHOL) liposomes were examined. Fasudil was successfully loaded into preformed liposomes in response to sulfate ion (SO4(2-)) and, in part, by H+. Encapsulation levels approaching 100% could be achieved up to a drug to lipid ratio of 0.364 (mol/mol). A stability study of the fasudil-loaded liposomes was performed by storage at 4 degrees C in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffer (pH 7.4) and by incubation in human cerebrospinal fluid (CSF) at 37 degrees C. The formulations were stable with respect to drug retention as well as size alteration, for the period studied. A leakage study clearly showed the sustained release properties of the fasudil-loaded liposomes in human CSF. We recently reported that the intrathecal administration of liposomal fasudil significantly decreased ischemia, with no obvious adverse effect in a rat model [Neurol. Med. Chir. 41 (2001) 109]. Taken together, efficient encapsulation of fasudil into preformed liposomes, their long-term stability at 4 degrees C and the sustained release characteristics in CSF indicate that fasudil-loaded liposomes could be potential candidates for further clinical evaluation.
Ryotaro Azuma, Masahito Komuro, Yasuro Kawaguchi, Kazuho Okudaira, Masahiro Hayashi and Hiroshi Kiwada : Comparative Analysis of In Vitro and In Vivo Pharmacokinetic Parameters Related to Individual Variability of GTS-21 in Canine, Drug Metabolism and Pharmacokinetics, Vol.17, No.1, 75-82, 2002.
(Summary)
To clarify the cause of the canine individual variability in plasma concentration after oral administration of GTS-21, we evaluated in vitro the metabolism to 4-OH-GTS-21 in liver microsomes of the same individuals from in vivo pharmacokinetic study. First, we applied to the Michaelis-Menten kinetic parameters to a dispersion model, and compared hepatic availability (F(H)) and hepatic clearance (CL(H)) values from in vitro with bioavailability (F), hepatic plasma flow (Q(PH)), and plasma clearance (CL(P)) values from in vivo. The ratios of CL(H) to Q(PH) were ranged 0.74 to 0.94, suggesting that GTS-21 is a hepatic plasma flow-limiting drug. A significant correlation of F(H) and F in the four dogs (r=0.995, p=0.005) indicates that the variability is predominantly caused by GTS-21 O4-demethylase activity. Second, we specified the cytochrome P450 (CYP) enzymes that are involved with the metabolism by chemical inhibition. alpha-Naphthoflavone, furafylline, quinidine, quinine, and troleandomycin significantly inhibited GTS-21 O4-demethylase activity. Thus CYP1A, CYP2D15, and CYP3A12 were involved with O4-demethylation. The variability in control activity decreased on addition of alpha-naphthoflavone and furafylline. Third, we quantified the contents of CYP1A and CYP3A12 by enzyme-linked immunosorbent assay. The content of CYP1A was consistent with GTS-21 O4-demethylase activity. We concluded that canine liver CYP1A causes the individual variability in GTS-21 plasma concentration after oral administration.
Ryotaro Azuma, Tomio Hirota, Hiroshi Manabe, Masahito Komuro and Hiroshi Kiwada : First-pass of GTS-21 on canine gut wall and liver determined by portal-systemic concentration difference, European Journal of Pharmaceutical Sciences, Vol.14, No.2, 159-165, 2001.
(Summary)
To clarify the cause of the canine individual variability of plasma concentration after oral administration of GTS-21 [(E)-3-(2,4-dimethoxybenzylidene)-3,4,5,6-tetra-hydro-2,3'-bipyridine dihydrochloride], we evaluated the absorption ratio (F(A)), intestinal availability (F(G)), and hepatic availability (F(H)). The bioavailability (F) was evaluated from the ratio of the area under the plasma concentration versus time curves after oral and intravenous administration. Three isoflurane anaesthetised dogs were fitted with an electromagnetic flow probe attached to the portal vein and cannulated through the portal and the femoral veins. After intraduodenal administration of GTS-21, both plasma concentrations were determined simultaneously. F(A) x F(G) was calculated from the portal-systemic concentration difference taking into consideration the blood-plasma partition ratio. F(A) was calculated from the residual drug contents of the small intestine. F(H) was calculated by dividing F by F(A) x F(G). The F values were 0.072, 0.021, and 0.037, indicating an individual variability of ca. threefold. The F(A) values were close to 1, and the F(G) values ranged from 0.449 to 0.461. Accordingly, the F(H) values were estimated at 0.170, 0.047, and 0.083. GTS-21 was completely absorbed but lost by first-pass effects of passage through the gut wall and liver. The first-pass effect of liver is larger than that of the gut wall, and dominates the individual variability in plasma concentration.
Tatsuhiro Ishida, Kazumi Yasukawa, Hiroko Kojima, Hideyoshi Harashima and Hiroshi Kiwada : Effect of cholesterol content in activation of the classical versus the alternative pathway of rat complement system induced by hydrogenated egg phosphatidylcholine-based liposomes, International Journal of Pharmaceutics, Vol.224, No.1-2, 69-79, 2001.
(Summary)
Liposomes composed of hydrogenated egg phosphatidylcholine (HEPC) and cholesterol (CHOL) were found to activate the rat complement (C) system in a CHOL content-dependent manner. Liposomes containing 22 or 33 mol% CHOL activated the C system in a Ca(2+)-dependent manner, suggesting that C activation occurred via the classical pathway. Liposomes containing 44 mol% CHOL activated the C system in a Ca(2+) independent manner, suggesting that C activation occurred via the alternative pathway. The CHOL content appeared to dictate the pathway by which the C system was activated. This C activation was inhibited by removal of serum component(s), which adsorb to the liposomes. Activation of the alternative pathway, induced by the liposomes, was reduced by the depletion of IgG and IgM, whereas the classical pathway activation was reduced by the depletion of IgG, but not IgM. In addition, the removal of adsorbed serum component(s) by treatment with 44 mol% CHOL-containing liposomes decreased serum IgG and IgM levels that adsorb to the same liposomes, whereas the removal of adsorbed serum component(s) by treatment with 22 mol% CHOL-containing liposomes only slightly decreased serum IgG levels, which adsorbs to the same liposomes. Collectively, both IgG and IgM, which are specifically adsorbed to the liposomes in a CHOL-content dependent manner, were responsible for C activation via the alternative pathway induced by the 44 mol% CHOL containing liposomes. IgG alone would be partially responsible for C activation via the classical pathway induced by 22 or 33 mol% CHOL-containing liposomes. The discovery of this unique C-activating property of liposomes will be of value in attempts to decipher the underlying mechanism of C activation by providing a useful model membrane system.
(Keyword)
Liposomes / Drug delivery system / Complement activation / Antibodies
Tran Minh Huong, Tatsuhiro Ishida, Hideyoshi Harashima and Hiroshi Kiwada : Species Difference in Correlation between in Vivo/ in Vitro LiposomeComplement Interactions, Biological & Pharmaceutical Bulletin, Vol.24, No.4, 439-441, 2001.
(Summary)
The objective of this study was to investigate the correlation between in vitro and in vivo liposome-complement interactions. Third component of the complement (C3) fragments associated with hydrogenated egg phosphatidylcholine (HEPC)-based liposomes in vivo and complement-dependent destabilization in vitro were determined as an indication of liposome-complement interaction in vivo and in vitro, respectively. C3 fragments on the liposomes were detected in both rats and guinea pigs. Pretreatment with K76COOH (K76), a complement inactivating agent, reduced the binding of C3 fragments. These findings indicated that the liposomes remarkably activated the complement system in both animals in vivo. Interestingly, significant complement-dependent liposome destabilization was observed in rat serum, but not in guinea pig serum, indicating that the liposomes activated the complement system in rats, but not in guinea pigs in vitro. Taken together, it is apparent that in vitro complement activation by the liposomes is not in agreement with in vivo complement activation in ginea pigs. This discrepancy in the liposome-complement interaction would suggest the need for further investigation to utilize the information obtained from the liposome-complement interaction to predict in vivo behavior of the liposomes.
(Keyword)
liposome / complement system / liposome destabilization / rat / guinea pig
Yoshihiro Takanashi, Tatsuhiro Ishida, Toshinari Meguro, Hiroshi Kiwada, John H Zhang and Isao Yamamoto : Efficacy of Intrathecal Liposomal Fasudil for Experimental Cerebral Vasospasm after Subarachnoid Hemorrhage, Neurosurgery, Vol.48, No.4, 894-901, 2001.
(Summary)
To investigate the safety and efficacy of liposomal fasudil in a sustained-release form for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). Eighteen rats were divided into three groups, each of which received 2.5 mg/kg of liposomal fasudil, 5 mg/kg of liposomal fasudil, or drug-free liposomes after SAH. Next, experimental SAH was induced in 15 dogs by injection of autologous arterial blood into the cisterna magna twice after baseline vertebral angiography. In six dogs, 0.94 mg/kg of liposomal fasudil was injected into the cisterna magna (treatment group). In four dogs, drug-free liposomes were similarly injected (placebo group), and the remaining five dogs were not treated with liposomal injection after SAH (control group). Angiography was repeated on Day 7, and cerebrospinal fluid was collected before the dogs were killed. A high dose of liposomal fasudil caused no significant changes in mean arterial blood pressure and did not induce seizures during the observation period. Gross and microscopic examination of the brains revealed no abnormalities, but severe vasospasm was noted in the rat basilar artery, mainly in the group treated with drug-free liposomes. Likewise, in the canine placebo and control groups, significant vasospasm occurred in the basilar artery on Day 7. In the treatment group, vasospasm in the basilar artery was significantly ameliorated (P < 0.01). In vivo, 90% of fasudil was released from liposomes in the cerebrospinal fluid. A single injection of intrathecal liposomal fasudil is safe and effective for the prevention of vasospasm in experimental SAH.
Tran Minh Huong, Tatsuhiro Ishida, Hideyoshi Harashima and Hiroshi Kiwada : The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig, International Journal of Pharmaceutics, Vol.215, No.1-2, 197-205, 2001.
(Summary)
In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.
(Keyword)
Liposomes / Biodistribution / Complement system / Rat / Guinea pig
Tatsuhiro Ishida, Hiroko Kojima, Hideyoshi Harashima and Hiroshi Kiwada : Biodistribution of liposomes and C3 fragments associated with liposomes:evaluation of their relationship, International Journal of Pharmaceutics, Vol.205, No.1-2, 183-193, 2000.
(Summary)
The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.
(Keyword)
Liposomes / Complement / C3 / Cholesterol / Drug delivery system (DDS)
Hideyoshi Harashima, Shinya Iida, Yumiko Urakami, Mari Tsuchihashi and Hiroshi Kiwada : Optimization of antitumor effect of liposomally encapsulated doxorubicin based on simulations by pharmacokinetic/pharmacodynamic modeling, Journal of Controlled Release, Vol.61, No.1-2, 93-106, 1999.
(Summary)
It has been reported that long circulating liposomes enhanced the antitumor effect of doxorubicin (DOX) by increasing delivery of DOX to tumor tissues. However, there is no quantitative information on the relationship between the antitumor effect and liposomal characteristics governing the release rate of entrapped drugs, although the importance of drug release-rate control from liposomes has been pointed out. Here, we developed a physiological model for free and liposomal DOX to calculate the time course of free DOX in the extracellular space and linked this with a cell kill kinetic model to quantify the antitumor effect of DOX. Simulations were performed to clarify the relationship between antitumor effect and pharmacokinetic or physicochemical parameters of liposomes, as well as pharmacological or physiological parameters of tumor tissues. The importance of long circulation time of liposomes was confirmed. The optimum rate of drug release from long circulating liposomes was found at the release rate constant of around 0.06 h(-1). This optimum value was not dependent on the tumor proliferation time, sensitivity of tumor cells to DOX, or the tumor blood flow-rate. This simulation indicated that the optimization of the delivery to tumor tissue by long circulating liposomes could be possible by changing the release rate of DOX for the maximum antitumor effect.
(Keyword)
drug delivery system / simulation / liposomes / Doxorubicin / optimization
Mari Tsuchihashi, Hideyoshi Harashima and Hiroshi Kiwada : Development of a pharmacokinetic/pharmacodynamic(PK/PD)-simulation system for doxorubicin in long circulating liposomes in mice using peritoneal P388, Journal of Controlled Release, Vol.61, No.1-2, 9-19, 1999.
(Summary)
The objective of this study was to develop a simulation system that optimizes the pharmacokinetic parameters of drug carriers for anticancer agents in order to maximize their anticancer effects. The pharmacokinetic/pharmacodynamic (PK/PD) model of doxorubicin (DOX) encapsulated into liposomes has been developed for mice and each parameter required for simulations was obtained in the peritoneally inoculated P388 leukemia model in mice. PK parameters, which describe the dispositions of free and liposomally encapsulated DOX, were obtained by kinetic analysis of experimental data in this study, as well as from literature. PD parameters, which describe the growth and death rate of cancer cells in vivo, were also determined. The PK/PD model developed in this study is capable of simulating the time course of the number of cancer cells quantitatively and evaluating the significance of each parameter on the carrier system for DOX. Simulations based on the PK/PD model predict the optimum rate of drug release from long circulating liposomes as 0.06 h(-1) for maximum anticancer effect. Thus, this simulation system provides useful information relative to the optimization of drug carriers for DOX.
(Keyword)
pharmacokinetics / Pharmacodynamics / simulation / drug delivery system
Tran Minh Huong, Hideyoshi Harashima and Hiroshi Kiwada : In Vivo Studies on the Role of Complement in the Clearance of Liposomes in Rats and Guinea Pigs, Biological & Pharmaceutical Bulletin, Vol.22, No.5, 515-520, 1999.
(Summary)
The ability of complement (C) system to remove liposomes from blood circulation was examined in vivo using rat and guinea pig as models. Although the liposomes were not degraded in guinea pig serum in vitro, they were degraded remarkably in guinea pig circulation, as assessed by the urinary excretion of [3H]inulin released from liposomes. The suppression of rat C system to 64% normal C hemolytic activity by treating animals with K76COOH agent resulted in a significant decrease in both the uptake of liposomes by liver and the release of [3H]inulin, providing in vivo evidence for C-mediated clearance of liposomes in rats via uptake by macrophages and degradation in blood circulation, respectively. On the other hand, the K76COOH-induced suppression of C (70% normal hemolytic activity) in guinea pigs slightly increased both the hepatic uptake and the release of [3H]inulin. In addition, the hepatic uptake and in vivo degradation in guinea pigs varied in an opposite manner when the animals were preloaded by empty liposomes or when the liposome size and cholesterol content varied. These results suggest there is a difference between the factors involved in liposome degradation and the factors involved in hepatic uptake and also support the likelihood that there is no C-mediated degradation in guinea pigs.
Hisao Nemoto, Junya Kikuishi, Shiho Yanagida, Tomoaki Kawano, Mizuki Yamada, Hideyoshi Harasima, Hiroshi Kiwada and Masayuki Shibuya : Design and synthesis of cholestane derivatives bearing a cascade-type polyol and the effect of their property on a complement system in rat serum, Bioorganic & Medicinal Chemistry Letters, Vol.9, No.2, 205-208, 1999.
97.
土橋 麻理, 原島 秀吉 and Hiroshi Kiwada : 血中滞留性リポソーム封入ドキソルビシンの腹腔癌における抗腫瘍効果防御システムの構築, Drug Delivery System, Vol.14, 109-113, 1999.
98.
Rieko Tachibana, Hideyoshi Harashima, Masayuki Shono, Makiko Azumano, Mineo Niwa, Siroh Futaki and Hiroshi Kiwada : Intracellular Regulation of Macromolecules using pH-senstive Liposomes and Nuclear Localization Signal : Qualitative and Quantitative Evaluation of intracellular Trafficking., Biochemical and Biophysical Research Communications, Vol.251, No.2, 538-544, 1998.
(Summary)
The objective of this study is to present a rational strategy to target macromolecules to the nucleus via the endocytic pathway. The two major barriers in this route to the nucleus are known as endosomal escape and nuclear transport. pH-sensitive liposomes were used in order to achieve endosomal escape under the conditions of low pH in endosomes. Bovine serum albumin (alb) served as a model compound to be delivered to nucleus and was encapsulated into the pH-sensitive liposomes. The liposomes are composed of dioleoyl phosphatidyl ethanolamine: cholesterylhemisuccinate. They were taken up by rat peritoneal macrophages via endocytosis and subsequently underwent degradation, principally by lysosomal enzymes. By using pH-sensitive liposomes, intracellular degradation was reduced by a significant extent, as expected, via endosomal escape. Cytosolic delivery of FITC-labelled alb was also detected by confocal microscopy. Selective targeting to the nucleus was performed by adding the nuclear localization signal (NLS) of the SV-40 large T antigen to the FITC-alb, which were then encapsulated into the pH-sensitive liposomes. Confocal microscopy revealed that FITC-alb, in the presence of NLS was successfully delivered into nucleus, while no transport was observed in the absence of NLS. These results provide a useful strategy for the nuclear targeting of macromolecules using pH-sensitive liposomes in conjunction with NLS.
Tran Minh Huong, Hideyoshi Harashima and Hiroshi Kiwada : Complement Dependent and Independent Liposome Uptake by Peritoneal Macrophages:Cholesterol Content Dependency, Biological & Pharmaceutical Bulletin, Vol.21, No.9, 969-973, 1998.
(Summary)
The uptake mechanisms of liposomes by rat peritoneal macrophages (PMs) were investigated. Incubation of liposomes with fresh rat serum enhanced the uptake of liposomes depending on the liposome size and cholesterol (CH) content. The binding of liposomes was also enhanced by serum, and this increase depended on the size and CH content as in the case of liposome uptake, which suggested that the binding of opsonized liposomes with PMs govern the extent in liposome uptake. The rate constant for the internalization (k(int)) was calculated by measuring both uptake and binding. The k(int) cannot explain the variation of liposome uptake for different sizes and CH contents. The kint values for liposomes with high (44%) and medium (33%) CH contents were constant (2.5 h(-1)) , while those for liposomes with low (22%) CH content were significantly elevated (5-9 h(-1)). These results indicate the presence of at least two kinds of uptake mechanisms of liposomes. Treatment of serum with anti-C3 antibody completely inhibited the enhanced uptake of CH-high, large liposomes, which suggested that complement receptor-mediated phagocytosis may be an uptake mechanism for CH-high and -medium liposomes. In addition, complement-independent enhanced uptake was suggested for CH-low liposomes, since no inhibition was observed for CH-low liposomes by anti-C3 antibody and these liposomes were disintegrated in serum via complement-independent pathway. These results provided evidence that PMs take up liposomes via complement-dependent and independent mechanisms depending on the CH content of the liposomes.
Mizuki Yamada, Hideyoshi Harashima and Hiroshi Kiwada : Kinetic Analysis of the Interaction between Liposomes and the Complement System in Rat Serum:Re-evaluation of Size-Dependency, Biological & Pharmaceutical Bulletin, Vol.21, No.9, 964-968, 1998.
(Summary)
The size of liposomes is considered to be an important factor in determining the liposome-complement interaction. In this study, the release of carboxyfluorescein (CF) from liposomes was measured continuously for three different diameters (800, 400 and 200 nm) by changing the liposome concentration from 1 to 1000 nmol/ml. At a low liposome concentration range (1-10 nmol/ml), small liposomes (200 nm) released CF to a similar extent (approximately 35%) as in the medium (400 nm) and large (800 nm) liposomes. The affinity (Km) and capacity (Lmax) of a complement system to release liposomally encapsulated CF were estimated by kinetic analysis of the liposome-complement interaction. Surprisingly, there was no remarkable size dependency in the Km and Lmax in terms of liposome number, although these parameters depended on the size of liposomes in terms of lipid concentration. These results indicated the possibility that the complement system does not discriminate according to liposome size.
Naomi Mizuno, Yukio Kato, Kazuhiko Shirota, Yuki Izumi, Tatsuro Irimura, Hideyoshi Harashima, Hiroshi Kiwada, Naomi Motoji, Akiyo Shigematsu and Yuichi Sugiyama : Mechanism of initial distribution of blood-borne colon carcinoma cells in the liver, Journal of Hepatology, Vol.28, No.5, 878-885, 1998.
(Summary)
The distribution characteristics of a human colon carcinoma cell line, KM12-HX cells, were examined. After intraportal vein (i.p.v.) or intravenous (i.v.) injection into rats, almost all the injected tumor cells are distributed to liver or lung, respectively, both after 30 s and 30 min. Our previous kinetic analysis of the fate of tumor cells revealed that the cumulative amount of tumor cells distributed in the liver is a factor determining the degree of metastasis. Thus, we examined the mechanism of initial efficient trapping of tumor cells by the liver in more detail. Thirty minutes after tumor cells were injected into the left ventricle of the heart, the distribution of tumor cells was more restricted in several tissues (kidney, small intestine, large intestine and spleen), as compared with the distribution of microspheres undergoing 100% extraction, indicating that the first-pass extraction of KM12-HX cells is incomplete in these organs. The hepatic first-pass distribution of these tumor cells was unaffected by pretreatment of liposomes, such that the preinjected amount was sufficient to saturate the phagocytotic function of macrophages. Thus, the mechanism of initial distribution of the tumor cells to the liver is different from the mechanism of liposome uptake by macrophages. Considering that the diameter of microvessels in sinusoid and KM12-HX cells is approximately 7 and 12 microm, respectively, it is possible that these tumor cells are trapped physically in hepatic microvessels. In fact, after i.p.v. injection of microspheres 5 microm in diameter, only 20% of the dose was distributed to liver and the rest to other tissues. In contrast, almost 100% of microspheres 10 microm in diameter were distributed to the liver. These results support the hypothesis that the initial organ distribution of blood-borne tumor cells is determined by mechanical and physical properties of the cells.
Tatsuhiro Ishida, Shinya Iida, Kouichi Funato and Hiroshi Kiwada : A novel plasma factor initiating complement activation on cetylmannoside-modified liposomes in human plasma, International Journal of Pharmaceutics, Vol.164, No.1-2, 91-102, 1998.
小島 弘子, Tatsuhiro Ishida, 原島 秀吉 and Hiroshi Kiwada : リポソーム膜中のコレステロール含量が及ぼす補体第3成分(C3)の結合と体内動態への影響, Drug Delivery System, Vol.13, 55-61, 1998.
105.
Tatsuhiro Ishida, Kouichi Funato, Shigeo Kojima, Ritsuko Yoda and Hiroshi Kiwada : Enhancing effect of cholesterol on the elimination of liposomes from circulation is mediated by complement activation, International Journal of Pharmaceutics, Vol.156, No.1, 27-37, 1997.
(Keyword)
Drug delivery system / Liposome / Cholesterol / Complement activating factor / Complement
S Liu, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of Serum Components from Different Species on Destabilizing Hydrogenated Phosphatidylcholine-Based Liposomes, Biological & Pharmaceutical Bulletin, Vol.20, No.8, 874-880, 1997.
島田 貴浩, 原島 秀吉 and Hiroshi Kiwada : In vivo実験系による異種リポソーム間の肝取込における競合機構の解明, 薬剤学, Vol.57, No.4, 197-203, 1997.
110.
K Yachi, H Harashima, H Kikuchi, R Sudo, H Yamauchi, K Ebihara, H Matsuo, K Funato and Hiroshi Kiwada : Biopharmaceutical evaluation of the liposomes prepared by rehydration of freeze-dried empty liposomes(FDELs)with an aqueous solution of a drug, Biopharmaceutics & Drug Disposition, Vol.17, No.7, 589-605, 1996.
(Summary)
We have evaluated a method for preparation of a dispersion of liposomes encapsulating a drug, namely rehydration of freeze-dried empty (not containing drug) liposomes with an aqueous drug solution (FDEL method). In the present study, we characterized and compared this method with the conventional method using a lipid composition of DPPC-DPPG-cholesterol in a molar ratio of 27:3:20. Two hydrophilic compounds, [3H]-inulin and [3H]-mannitol, were used as model drugs. Liposomal preparations by the FDEL method had an encapsulation efficiency of 2.9 and 6.7% for [3H]-inulin and [3H]-mannitol, respectively, when rehydrated and incubated at 70 degrees C. Since non-specific adsorption of these markers to liposomal membrane is negligible, this method produces liposomes which encapsulate a drug in the intravesicular space. One-tenth of the marker encapsulated in the liposomes prepared by the FDEL method (F-liposomes) was released very rapidly on incubation with rat plasma, followed by the slow release of the remaining fraction thereafter. No such rapid-release phase was observed for the liposomes prepared by the conventional method (C-liposomes). This suggests the existence of two types of encapsulation, loose encapsulation and tight encapsulation, in F-liposomes at least. Pharmacokinetic parameters of marker encapsulated tightly in F-liposomes were comparable to those in C-liposomes. It is likely that amphipathic drugs such as doxorubicin are incorporated into liposomes more easily than inulin and mannitol when formulated by the FDEL method. These results therefore suggest that the FDEL method is useful in the preparation of a liposomal formulation of a drug.
(Keyword)
1,2-Dipalmitoylphosphatidylcholine / Animals / Area Under Curve / Biopharmaceutics / Diuretics, Osmotic / Drug Carriers / Drug Stability / Evaluation Studies as Topic / Fluid Therapy / Freeze Drying / Injections, Intravenous / Inulin / liposomes / Male / Mannitol / Particle Size / Rats / Rats, Wistar / Solutions / Tissue Distribution
T Yamamoto, M Naito, H Moriyama, H Umezu, H Matsuo, Hiroshi Kiwada and M Arakawa : Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate, The American Journal of Pathology, Vol.149, No.4, 1271-1286, 1996.
(Summary)
Kupffer cells were selectively eliminated in mice by the intravenous administration of liposome-entrapped dichloromethylene diphosphonate. At 5 days, small peroxidase-negative and acid-phosphatase-weakly-positive macrophages appeared, increased in number, and differentiated into peroxidase- and acid-phosphatase-positive Kupffer cells. Repopulating small macrophages actively proliferated, and the number of Kupffer cells returned to the normal level by day 14. The numbers of macrophage precursors in the liver as detected by the monoclonal antibodies ER-MP20 and ER-MP58 increased after liposome-entrapped dichloromethylene diphosphonate injection. ER-MP58-positive cells proliferated and differentiated into ER-MP20-positive cells and eventually into BM8-positive Kupffer cells in the liver. Bone-marrow-derived ER-MP58-positive cells were also detectable in the liver and differentiated into ER-MP20-positive cells, but they did not become BM8-positive macrophages. Macrophage colony-stimulating factor mRNA expression was enhanced in the liver 1 day after injection. The administration of macrophage colony-stimulating factor did not shorten the period of Kupffer cell depletion but increased the number and the proliferative capacity of repopulating Kupffer cells. These findings implied that repopulating Kupffer cells are derived from a macrophage precursor pool in the liver rather than from bone-marrow-derived monocytes. Local production of macrophage colony-stimulating factor in the liver plays a crucial role in the differentiation, maturation, and proliferation of Kupffer cells.
H Harashima, T M Huong, Tatsuhiro Ishida, Y Manabe, H Matsuo and Hiroshi Kiwada : Synergistic Effect Between Size and Cholesterol Content in the Enhanced Hepatic Uptake Clearance of Liposomes Through Complement Activation in Rats, Pharmaceutical Research, Vol.13, No.11, 1704-1709, 1996.
AJ Ferdous, Tatsuhiro Ishida, M Shinohara, H Harashima and Hiroshi Kiwada : Size-dependent release of carboxyfluorescein from cetylmannoside-modifide liposomes in human plasma, Biopharmaceutics & Drug Disposition, Vol.17, No.2, 145-154, 1996.
H Harashima, S Komatsu, H Kojima, C Yanagi, Y Morioka, M Naito and Hiroshi Kiwada : Species difference in the disposition of liposomes among mice, rats, and rabbits: Allometric relationship and species dependent hepatic uptake mechanism, Pharmaceutical Research, Vol.13, No.7, 1049-1054, 1996.
(Summary)
The species difference in the pharmacokinetics of liposomes was investigated in mice, rats and rabbits. Liposomes were intravenously injected at doses of 1, 10 and 100 (nmol/g body weight), and the time courses of liposomes in blood, liver and spleen were measured. Pharmacokinetic parameters were regressed as a function of body weight (BW) and dose of liposomes (D). The uptake mechanism of liposomes was also examined with the isolated perfused liver between rats and mice. Mean residence time increased with the increase of BW and D of liposomes. This increase of mean residence time resulted from the decreased total body clearance, which was principally explained by the species difference in the hepatic uptake clearance (CLh) of liposomes. The parameter CLh was regressed well by a multiple regression as a function of BW and D. In this analysis, an exponent for BW was around 0.5, which clearly indicates that smaller animals have higher uptake clearance per unit BW. Immunohistochemical analysis revealed that there was no significant difference in the density of Kupffer cells among these species. This suggest that the species difference in CLh resulted not from the density of Kupffer cells but from the uptake ability of Kupffer cells among species. In the isolated perfused liver, the hepatic uptake of liposomes was mainly explained by opsonin dependent uptake in rats, while opsonin independent uptake in mice. These quantitative and qualitative information on the species difference of liposome disposition will provide an useful information for constructing a drug delivery system using liposomes.
(Keyword)
Animals / Body Weight / Dose-Response Relationship, Drug / immunohistochemistry / In Vitro Techniques / Kupffer Cells / liposomes / Liver / Mice / Opsonin Proteins / Rabbits / Rats / Rats, Wistar / Species Specificity / Spleen
Atsushi Nagayasu, Kazuko Uchiyama, Tomoyo Nishida, Yumiko Yamagiwa, Yumiko Kawai and Hiroshi Kiwada : Is control of distribution of liposomes between tumors and bone marrow possible?, Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1278, No.1, 29-34, 1996.
(Summary)
The objective of this study is to clarify to what extent the accumulation of liposomes from the blood into the tumor and bone marrow can be controlled by liposome size and membrane fluidity. Liposomes with different diameters (50-400 nm) and different membrane fluidity were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol (Ch) and dicetylphosphate in various molar ratios. These liposomes were injected intravenously into rats bearing Yoshida sarcoma, and the ratios of the accumulation of liposomes in the tumor to those in the bone marrow, liver and spleen were compared. The tumor-to-bone marrow accumulation ratio increased with the decrease in liposome size from 400 to 50 nm. This ratio was greater than those for the liver and spleen at all sizes. Although tumor-to-liver accumulation ratios of 50- and 100-nm HEPC-containing liposomes were higher than those of EPC-containing liposomes, no obvious difference in tumor-to-bone marrow or tumor-to-spleen accumulation ratios was found between these liposomes. Tumor-to-bone marrow accumulation ratio of HEPC-containing liposomes increased remarkably with the decrease in Ch content from 40 to 30 or 20 mol% compared with ratios for the liver and spleen. Interestingly, the tumor uptake clearance of liposomes of the same size was constant regardless of their membrane fluidity. These findings show that the increases in these accumulation ratios are due to their decreased uptake clearance by the bone marrow. Furthermore, the uptake of 50-nm HEPC-containing liposomes by the bone marrow was specifically inhibited by preinjection of other liposomes, but not when they were exposed in advance to in vivo components. These observations suggest the involvement of in vivo component(s) in the uptake of these liposomes by the bone marrow. We conclude that small HEPC-liposomes with low Ch content show their significantly decreased uptake by the bone marrow due to their decreased recognition by this tissue.
(Keyword)
liposomes / Distribution / Size, Membrane fluidity / Tumor / Bone marrow
Kazuko Uchiyama, Atsushi Nagayasu, Yumiko Yamagiwa, Tomoyo Nishida, Hideyoshi Harashima and Hiroshi Kiwada : Effects of the size and fluidity of liposomes on their accumulation in tumors:A presumption of their interaction with tumors, International Journal of Pharmaceutics, Vol.121, No.2, 195-203, 1995.
(Keyword)
liposomes / AUC / Tumor uptake clearance / Angiotensin II / Yoshida sarcoma
Atsushi Nagayasu, Takashi Shimooka and Hiroshi Kiwada : Effect of vesicle size on in vivo release of daunorubicin from hydrogenated egg phosphatidylcholine-based liposomes into blood circulation, Biological & Pharmaceutical Bulletin, Vol.18, No.7, 1020-1023, 1995.
(Summary)
The effect of the vesicle size on in vivo release of daunorubicin, an anthracycline anticancer drug, from liposomes into the circulation was studied in rats. The liposomes were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol and dicetylphosphate at a molar ratio of 5:4:1, and their mean vesicle sizes were adjusted to about 50 and 100 nm. The drug retained in the liposomes and the drug that leaked were separated from plasma by gel filtration. On the assumption that the lipid content does not change, the drug release from each liposome preparation was estimated from the latency (%) calculated from the drug/lipid concentration ratio of the liposome preparation. From HEPC-liposomes with a diameter of 50 nm, the drug was released gradually after intravenous administration, and the cumulative percent release of the drug reached 40% after 240 min. However, from EPC-liposomes with the same size, 50% of the drug was released within 5 min, and more than 90% of the drug was released within 60 min. From HEPC-liposomes with a diameter of 100 nm, no drug release was observed for 240 min after administration. These findings indicate that the vesicle size and the lipid composition of liposome preparation affect the in vivo drug release.
(Keyword)
Animals / Daunorubicin / Drug Carriers / In Vitro Techniques / liposomes / Male / Ovum / Particle Size / Phosphatidylcholines / Rats
永易 篤, 内山 和子, 越智 美代子, 山際 有美子, 武市 陽一郎 and Hiroshi Kiwada : Effects of Membrane Fluidity on Accumulation of Small Unilamellar Vesicles in Liver and Tumor, 薬剤学, Vol.55, No.2, 81-87, 1995.
Hirotami Matsuo, Chikamasa Yamashita, Kazue Akiyama and Hiroshi Kiwada : Effect of cetylmannoside modification on the alternative complement pathway activation by liposomes in rat serum, Biological & Pharmaceutical Bulletin, Vol.18, No.4, 581-585, 1995.
(Summary)
Complement activation is important for removing foreign substances by the mononuclear phagocyte system in vivo. The interaction between liposomes and complement components is considered to affect the clearance of liposomes from the circulation. It has been previously demonstrated in our laboratory that multilamellar vesicles (MLV) with surfaces modified by cetylmannoside (Man) were eliminated from the circulation rapidly and showed an approximately 2-fold higher hepatic accumulation compared with control MLV (PC-MLV) (Yamashita et al., Int. J. Pharmaceut., 70, 225, 1991). In this study, we investigated the effect of Man-modification on complement system activation. As far as elimination from the blood is concerned, the initial values of blood liposome concentration were decreased and liposomes were removed from the circulation rapidly in accordance with the extent of the Man content into their membranes. The Man-modification also affected the organ distribution of injected liposomes and their stability in rat serum. Except for MLV containing 50 mol% Man, it was observed that the hepatic uptake of liposomes was enhanced according to the increasing Man content, whereas splenic uptake was decreased and the splenic clearance was comparable. The stability of liposomes in rat serum decreased with increasing Man content. Liposomal instability in rat serum was significantly reduced by preheating the serum at 56 degrees C for 30 min, the treatment with anti-C3 antiserum and with EDTA but not abolished in serum treated with EGTA/MgCl2. Thus, it is considered that the activation of the complement system through the alternative pathway is facilitated as a result of increasing the Man content in the liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)
(Keyword)
Animals / Complement Pathway, Alternative / In Vitro Techniques / liposomes / Male / Mannosides / Perfusion / Rats / Rats, Wistar / surface properties / Tissue Distribution
Hideyoshi Harashima, Noriko Hirai and Hiroshi Kiwada : Kinetic modelling of liposome degradation in peritoneal macrophages, Biopharmaceutics & Drug Disposition, Vol.16, No.2, 113-123, 1995.
(Summary)
The objective of this study was to quantify and model the degradation process of liposomes in peritoneal macrophages (PMs). Iodinated albumin (125I-alb) was chosen to be the marker of liposome degradation. The time course of the degradation of free 125I-alb after pinocytosis by PMs followed first-order kinetics with a half-life of 23 min. The degradation of liposomally encapsulated 125I-alb was also quantified. Kinetic modelling of liposome degradation indicated the existence of two kinetically different processes, one with a half-life of 13 min and the other with a half-life of 7.5 h. Comparing the degradation of liposomal and free 125I-alb suggested that 125I-alb was delivered to lysosomes much faster through phagocytosis than pinocytosis. These results indicate that the intracellular degradation kinetics of pinosomes and phagosomes is different. This method can quantify the rate and extent of liposomal degradation in macrophages and provide kinetic information on the intracellular destiny of liposomally encapsulated compounds.
H Harashima, T Hiraiwa, Y Ochi and Hiroshi Kiwada : Size dependent liposome degradation in blood:In vivo/in vitro correlation by kinetic modeIing, Journal of Drug Targeting, Vol.3, No.4, 253-261, 1995.
(Summary)
The degradation of liposomes in blood circulation is important in regulating the releasing rate of encapsulated compounds. In this study, the effect of liposome size--one of the principal determining factors in liposome disposition--on their degradation in serum/blood was evaluated quantitatively both in vitro and in vivo. In the in vitro study, the time courses of the degradation of liposomes in fresh rat serum were measured continuously using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker and were described by the kinetic model with the lag time (tau), first order degradation rate constant (k), and the maximum degradation (alpha). Both k and alpha increased with the increase of liposome size, which indicated a higher affinity of larger liposomes for complement activation. In the in vivo study, the degradation of liposomes was evaluated sensitively by a first order degradation rate constant (kd) in blood circulation. The kd was obtained by kinetically modeling the liposome degradation in vivo using 3H-inulin as an aqueous phase marker. The size dependent kd correlated well with the hepatic uptake clearance, which suggests an underlying complement activation mechanism common to both degradation and hepatic uptake of liposomes. There was a good correlation in the degradation rate constant between in vitro and in vivo trials. These kinetic analyses validate the quantitative evaluation of liposome degradation in blood circulation and provide a useful way to predict the degradation of liposomes in vivo from in vitro experiments.
A Nagayasu, T Shimooka and Hiroshi Kiwada : Solubilization by Triton X-100 makes possible complete recoverery of lipids from liposomes in enzymatic assay, Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1194, No.1, 12-16, 1994.
(Summary)
We have developed an accurate and sensitive method for enzymatically determining phosphatidylcholine (PC) and cholesterol (CHOL) in liposomes. Solubilizing liposomes with a high concentration (80%) of Triton X-100 at 65 degrees C for 5 min led to the complete recovery of the lipids by current assay using commercial kits. The method had good linearity in a range of 0.004-0.4 mumol PC. Using this method, PC and CHOL were completely recovered from various liposomes. We conclude that PC and CHOL in liposomes can be determined accurately and sensitively by this method.
Yoko Matsuki, Rie Bandou, Hiroshi Kiwada, Harumi Maeda and Tsuyoshi Goromaru : Effects of ascorbic acid on iproniazid-induced hepatitis in phenobarbital-treated rats, Biological & Pharmaceutical Bulletin, Vol.17, No.8, 1078-1082, 1994.
(Summary)
The effects of ascorbic acid (AA) on hepatic injury induced by iproniazid (IPN) in phenobarbital-treated rats were investigated by the evaluation of hepatic function using the clearance of aminopyrine (AM). Either IPN or isopropylhydrazine (IP-Hy), a potent toxic metabolite of IPN, were administered as a pretreatment to rats with or without AA. After i.v. injection of AM, the blood concentration of AM was determined by capillary gas chromatography by isotope dilution analysis using deuterium-labeled AM (AM-d9) as the internal standard. The kinetic parameters of AM, Vd, kel and total body clearance, were estimated from the time course of blood concentration. Pretreatment with IPN with AA led to a marked increase in the kel and in the clearance compared with pretreatment using IPN alone. A significant increase in the kel and the clearance was also found in the case of combined pretreatment using IP-Hy with AA. The effects of AA on the hepatic injury induced by IPN were studied according to its histological aspects. In the specimens obtained following the administration of IPN or IP-Hy with AA, the degree of cell necrosis was remarkably lowed both quantitatively and qualitatively. The present results clearly demonstrate that AA was effective in reducing IPN-induced hepatitis.
(Keyword)
Alanine Transaminase / Aminopyrine / Animals / Ascorbic Acid / Aspartate Aminotransferases / Chemical and Drug Induced Liver Injury / Chromatography, Gas / Hydrazines / Iproniazid / Kinetics / Liver / Liver Function Tests / Male / Phenobarbital / Rats / Rats, Wistar
Hideyoshi Harashima, Hiroshi Kiwada, Jirou Kajita and Satoshi Kobayashi : Effects of Benidipine Hydrochloride(Coniel*),a new Calcium Antagonist,on the Cardiac Output,Regional blood Flow and Vascular Resistance in Conscious,Spontaneously Hypertensive Rats, Biopharmaceutics & Drug Disposition, Vol.15, No.5, 431-438, 1994.
(Summary)
Benidipine hydrochloride is a calcium antagonist with a 1,4-dihydropyridine derivative structure, and exhibits long-lasting antihypertensive effects by inhibiting the voltage-dependent Ca2+ channels. This study was undertaken to examine the effect of benidipine on central haemodynamics and regional blood flow (RBF) after intravenous administration of benidipine in conscious, spontaneously hypertensive rats. The microsphere method was used to measure cardiac output and RBF before and after the drug administration, using microspheres labelled with 57Co and 51Cr. Thirty minutes after the intravenous administration of benidipine (3 micrograms kg-1), the mean arterial pressure fell by 15% without significantly increasing the heart rate. The cardiac output increased by 41% and the systemic resistance decreased by 39%. Benidipine significantly increased RBF by 37, 35, and 22% in kidney, heart, and small intestine, respectively, and decreased vascular resistance by 38, 38, and 32%, respectively. We concluded that benidipine reduced blood pressure by increasing RBF in the kidney and heart, while keeping RBF in other organs at a normal level. These results will provide a fundamental basis in support of the clinical benefits of benidipine for hypertensive patients, particularly those with renal failure.
Atsushi Nagayasu, Takashi Shimooka, Yoshihito Kinouchi, Kazuko Uchiyama, Yoh'ichiro Takeichi and Hiroshi Kiwada : Effects of Fluidity and Vesicle Size on Antitumor Activity and Myelosuppresive Activity of Liposomes Loaded with Daunorubicin, Biological & Pharmaceutical Bulletin, Vol.17, No.7, 935-939, 1994.
(Summary)
The effects of fluidity and vesicle size on the antitumor activity and myelosuppressive activity of liposomes loaded with daunorubicin, an anthracycline antitumor drug, were investigated in Yoshida sarcoma-bearing rats. Liposomes composed of egg phosphatidylcholine (EPC) or hydrogenated egg phosphatidylcholine (HEPC), cholesterol and dicetyl phosphate in a molar ratio of 5:4:1 were injected intravenously into rats 5 d after subcutaneous inoculation of Yoshida sarcoma. At non-effect dosage in free drug, HEPC-liposomes with a diameter of 58 or 142 nm showed the greatest inhibitory effect against Yoshida sarcoma among liposomes tested, whereas larger ones (272 nm) had weaker effect. Small EPC-liposomes (57 nm) had no effect. Larger HEPC-liposomes (especially 142 nm) greatly decreased the number of peripheral white blood cell compared with free drug at the same dose, indicating relatively strong myelosuppressive toxicity. However, small EPC- and HEPC-liposomes with a diameter of 57 and 58 nm, respectively, showed toxic effects comparable to that of free drug. Examination of the dose-dependency of therapeutic effects and toxicity indicated encapsulation of daunorubicin in the small HEPC-liposomes to enhance the therapeutic index about 3 times that of free drug. These findings indicate the possibility of using small HEPC-liposome as a drug carrier for targeting solid tumors.
(Keyword)
Animals / Bone Marrow / Daunorubicin / Dose-Response Relationship, Drug / Drug Carriers / liposomes / Male / Membrane Fluidity / Rats / Sarcoma, Yoshida
Sayoko Osaka, Hideki Tsuji and Hiroshi Kiwada : Uptake of Liposoes Surface-Modified with Glycyrrhizin by Primary Cultured Rat Hepatocytes, Biological & Pharmaceutical Bulletin, Vol.17, No.7, 940-943, 1994.
(Summary)
Previously, we synthesized 30-stearyl glycyrrhizin (GLOSt) and reported that small unilamellar liposomes containing GLOSt (GLOSt-SUV) accumulated in the liver several times more than the control liposomes (control-SUV). In the present study, to determine the interaction between GLOSt-SUV and hepatocytes, in vitro uptake experiments were achieved with primary cultured rat hepatocytes. The uptake amount of GLOSt-SUV by rat hepatocytes was considerably higher compared to the control-SUV, while GLOSt-SUV showed about a 10-fold higher uptake level than the control-SUV during 2 h of incubation. It was assumed that GLOSt-SUV not only bind to the surface of the hepatocytes but are internalized and degraded in the cells, because at 37 degrees C, GLOSt-SUV were taken up and the level of the degradable marker was lower than the inert marker, and this did not occur at 4 degrees C. Since the uptake of GLOSt-SUV was inhibited by glycyrrhizin (GL), it was suggested that a binding-site for GL is present on the surface of hepatocytes, and GLOSt-SUV are likely to be internalized via this site by the hepatocytes. Furthermore, it was confirmed that the efficacy of GLOSt on liposomes is not affected by the fluidity of the liposomal membrane.
H Harashima, Y Ochi and Hiroshi Kiwada : Kinetic modeling of liposome degradation in serum:Effect of size and concentration of liposomes in vitro, Biopharmaceutics & Drug Disposition, Vol.15, No.3, 217-225, 1994.
(Summary)
The purpose of this study is to propose a new method for quantitative evaluation of liposome degradation in serum. The time course of liposome degradation in rat serum was monitored continuously, using 6(5)-carboxyfluorescein as an aqueous phase marker. The degradation curves exhibited three characteristic phases: lag time, degradation, and plateau. This curve was described by a kinetic model with three parameters: lag time (tau), first-order degradation rate constant (k), and maximum degradation (alpha). The rate and extent of the degradation of liposomes were evaluated separately in terms of k and alpha, respectively. The effects of size and concentration of liposomes on their degradation kinetics were examined using this method. Both k and alpha increased with increasing liposomal size. The increased affinity of larger liposomes for complement was suggested to increase both k and alpha. On the other hand, alpha decreased with increasing liposomal concentration without altering k. The decreased extent of degradation was considered to result from the depletion of complement components. There was no significant effect of size and concentration of liposomes on tau. Quantitative evaluation of the rate and extent of degradation of liposomes will provide deeper insights into the interaction between liposomes and serum components, and basic information on liposomes as potential drug carriers.
(Keyword)
Animals / Complement Pathway, Alternative / Complement System Proteins / Drug Carriers / In Vitro Techniques / Kinetics / liposomes / Particle Size / Rats
Hideyoshi Harashima, Kazuya Sakata, Kouichi Funato and Hiroshi Kiwada : Enhanced Hepatic Uptake of Liposomes Through Complement Activation Depending on the Size of Liposomes, Pharmaceutical Research, Vol.11, No.3, 402-406, 1994.
(Summary)
The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56 degrees C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.
Hirotami Matsuo, Kouichi Funato, Hideyoshi Harashima and Hiroshi Kiwada : The Complement- but not Mannos Receptor-Mediated Phagocytosis is Involved in the Hepatic Uptake of Cetylmannoside-Modified Liposomes in Situ, Journal of Drug Targeting, Vol.2, No.2, 141-146, 1994.
(Summary)
In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These hepatic uptakes of liposomes were obviously inhibited by preheating the serum at 56 degrees C for thirty minutes or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.
Hideyoshi Harashima, Yoshihiro Kume, Chizu Yamane and Hiroshi Kiwada : Kinetic modeling of liposome degradation in blood circulation, Biopharmaceutics & Drug Disposition, Vol.14, No.3, 265-270, 1993.
(Summary)
The aim of this study is to develop a kinetic model for the quantitative evaluation of, and to examine dose dependency in liposome degradation in blood circulation in vivo. Multilamellar liposomes labeled with 3H-inulin were administered intravenously into rats and the time courses of blood concentration and urinary excretion of 3H-inulin were measured. The dosages of liposomes were fixed at 1, 5, and 100 mumolPCkg-1. Remarkable saturation was found in the time courses of both blood concentration and urinary excretion. Then a kinetic model for the degradation of liposomes in blood was developed, assuming that the degradation follows the first order rate process for each dose. The model fitted the observed time courses of excreted 3H-inulin well, and dose dependency could be observed in the rate constants for liposome degradation, which are more sensitive than urinary excretion of 3H-inulin. The degradation rate constant correlated well with the uptake rate constant, which suggests the same underlying mechanism for both uptake and degradation. These results indicate the usefulness of kinetic modeling in the quantitative evaluation of liposome degradation in blood circulation in vivo.
Hideyoshi Harashima, Kazuya Sakata and Hiroshi Kiwada : Distinction between the depletion of opsonins and the saturation of uptake in the dose-dependent hepatic uptake of liposomes, Pharmaceutical Research, Vol.10, No.4, 606-609, 1993.
(Summary)
Opsonins play a role in the hepatic uptake of particles such as bacteria, lipid emulsion, and liposomes. The objective of this study was to distinguish between opsonin depletion and uptake saturation in the dose-dependent hepatic uptake of liposomes. The uptake of opsonized and unopsonized liposomes was determined in the isolated perfused liver. Serum (2.9 mL) was required to opsonize 1 mumol liposomes fully, indicating that a rat (250 g with 10 mL of serum) can opsonize 3.5 mumol liposomes. Next the dose effect on hepatic uptake of opsonized and unopsonized liposomes was examined. Saturation of uptake was found only for the opsonized liposomes. On the other hand, the hepatic uptake clearance decreased dose dependently from 4.31 to 0.79 (mL/min), with increasing doses from 0.075 to 17 mumol/250 g, respectively, after i.v. administration. Thus, the decrease in the hepatic uptake clearance at the medium dose was due to the saturation of uptake alone, and at the high dose it was due to opsonin depletion as well. These results show that the saturation of liposomal uptake in the liver and the depletion of opsonins occurred at different liposome dosage levels.
(Keyword)
Animals / In Vitro Techniques / liposomes / Liver / Male / Opsonin Proteins / Rats / Rats, Wistar
Hideyoshi Harashima, Yukari Midori, Syunji Ohshima, Kiyoto Yachi, Hiroshi Kikuchi and Hiroshi Kiwada : Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats(2), Biopharmaceutics & Drug Disposition, Vol.14, 595-608, 1993.
139.
Hideyoshi Harashima, Chizu Yamane, Yoshihiro Kume and Hiroshi Kiwada : Kinetic analysis of AUC-dependent saturable clearance of liposomes:Mathematical description of AUC dependency, Journal of Pharmacokinetics and Pharmacodynamics, Vol.21, 299-308, 1993.
140.
Hideyoshi Harashima, Yuriko Ohnishi and Hiroshi Kiwada : In vivo evaluation of the effect of the size and opsonization on the hepatic extraction of liposomes in rats:An application of oldendorf method, Biopharmaceutics & Drug Disposition, Vol.13, No.7, 549-553, 1992.
(Summary)
In the hepatic uptake of large particles such as liposomes, a serum component called opsonin plays an important role. In this study, the 'Oldendorf method' is introduced to evaluate the hepatic extraction under the condition of single passage, which enabled examination of the effect of opsonization on liposome uptake by the intact liver. 14C-labelled liposomes and, an internal reference, 3H-H2O were injected as a bolus into portal vein. Liver uptake index (LUI) was calculated from the ratio of the extraction of 14C to that of 3H. The effect of liposome size (mean diameter of 0.8, 0.4, 0.2, and 0.05 micron) and opsonization (preincubation with fresh blood for 5 min) on liposomal hepatic uptake were investigated using this method. LUI increased with size significantly (p < 0.001), and opsonization enhanced LUI only for the large liposomes (0.8 micron). This result suggests that the critical diameter of opsonization for these liposomes lies between 0.4 and 0.8 micron.
Hideyoshi Harashima, Yoshihiro Kume, Chizu Yamane and Hiroshi Kiwada : Non-Michaelis-Menten type hepatic uptake of liposomes in the rat, The Journal of Pharmacy and Pharmacology, Vol.44, No.9, 707-712, 1992.
(Summary)
The objective of this study was to verify the methodology for measuring uptake clearance of liposomes and to characterize kinetically the saturable hepatic uptake of liposomes-through phagocytosis. The correction of vascular space was important in the evaluation of hepatic uptake. The efflux of liposomes from liver was shown to be negligible, by a repeated dose study, and thus, hepatic clearance can be obtained by the hepatic uptake divided by the area under the blood concentration-time curve (AUC). The determinant parameter which describes the saturability of uptake clearance of liposomes, independent of infusion rate, was investigated, using the data of an in-vivo constant infusion study, where infusion rate-dependent saturable hepatic clearance was observed. The mean blood concentration failed to obtain an infusion rate-independent function. On the other hand, the AUC could explain the saturability of hepatic clearance for every infusion rate by a unique relationship. The hepatic uptake amount could also explain this saturability, independent of infusion rate. These kinetic characteristics are inconsistent with Michaelis-Menten type kinetics, therefore a new model is required to describe the saturable hepatic clearance in the disposition of liposomes.
松木 洋子 and Hiroshi Kiwada : シメチジンのpH変化における物性の安定性, Medicine and Drug Journal, Vol.28, No.8, 1683-1685, 1992.
143.
Hideyoshi Harashima, Syunji Ohshima, Yukari Midori, Kiyoto Yachi, Hiroshi Kikuchi and Hiroshi Kiwada : Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats:I, Biopharmaceutics & Drug Disposition, Vol.13, No.3, 155-170, 1992.
(Summary)
The purpose of this study was to perform a kinetic analysis of the tissue distribution of doxorubicin (DXR) and liposomes separately after intravenous administration of DXR entrapped in liposomes in rats. Liposomes were double labeled with 14C-DXR (L-DXR) and 3H-inulin (L-INU). Blood and tissues were sampled at specified times until 120 min. Blood clearance of L-DXR was similar to that of L-INU. Distribution of both L-DXR and L-INU into the liver was parallel and extensive, while in the heart, the pattern of distribution differed between L-DXR and L-INU after peak concentration. Time courses of tissue concentration were explained well by dividing tissue into a shallow compartment with efflux and a deep compartment without efflux. In the liver, pharmacokinetic parameters of L-DXR and L-INU were similar, and the two kinetically different compartments may correspond to different uptake processes in hepatic endocytosis. In the heart, the shallow compartment was considered to correspond to the cardiac vascular space, and the intercompartmental rate constant (k3) for L-DXR was much larger than that for L-INU. The estimated half-life for this process was 20 min. The half-life for the degradation of liposomes in blood circulation was also estimated at 20 min from data on the urinary excretion of released 3H-inulin. These results suggest that the release of DXR from liposomes may be the rate-limiting process in the tissue distribution of DXR to the heart.
Kouichi Funato, Ritsuko Yoda and Hiroshi Kiwada : Contribution of complement system on destabilization of liposomes composed of hydrogenated egg phosphatidylcholine in rat fresh plasma, Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1103, No.2, 198-204, 1992.
(Summary)
Large multilamellar vesicles (MLV) composed of hydrogenated egg phosphatidylcholine (HEPC), cholesterol (CH), and dicetyl phosphate (DCP) rapidly release part of an entrapped aqueous marker when incubated with fresh rat plasma and thus have severely limited usefulness as drug carriers. The mechanisms causing the instability of liposomes in plasma were investigated in this study. The leakage of liposomal constituents was completely inhibited by pre-heating at 56 degrees C for 30 min with plasma or by treating with EDTA, K-76COOH, or anti-C3 antiserum but was not inhibited with EGTA/MgCl2. These results indicated that the destabilization of liposomes in fresh rat plasma was induced by activation of the alternative complement pathway (ACP). Furthermore, the complement third component (C3) was detected from the liposomes incubated with fresh plasma by SDS-PAGE followed by Western blotting and immune detection. The C3b deposited on the liposomal surface via ACP was rapidly cleaved to iC3b. The results obtained in the present study suggest a possibility that the liposomes composed of HEPC (without any surface modification) may be effective carriers for macrophages because C3b and its degradative products, iC3b are related to the opsonic function on phagocytosis of foreign particles by macrophages.
(Keyword)
liposomes / Phosphatidylcholine,hydrogenated / Drug carrier / stability / Plasma / Alternative complement pathway / Opsonin / iC3b / (Rat)
松木 洋子, 赤沢 美保, Koichiro Tsuchiya, 桜井 弘, Hiroshi Kiwada and 五郎丸 毅 : [Effects of ascorbic acid on the free radical formations of isoniazid and its metabolites]., Journal of the Pharmaceutical Society of Japan, Vol.111, No.10, 600-605, 1991.
(Summary)
By the use of electron spin resonance (ESR) spectroscopy and of spin-trapping technique, the effects of ascorbic acid on the formation of the free radical intermediates due to isoniazid (INAH) and its metabolites were investigated with a microsomal system. When alpha-(4-pyridyl 1-oxide)-N-tert butylnitrone (4-POBN) was used as a spin trapping agent, the ESR signal due to hydrazine (Hy) was formed to be most intensive among others. Therefore, it was presumed that Hy is a potent intermediate to cause an INAH-induced hepatic injury. In the presence of ascorbic acid (AA), the free radical formation of Hy, INAH and acetyl hydrazine was significantly inhibited, suggesting that AA may affect the INAH-hepatitis. By the addition of inhibitors of cytochrome P-450 like metyrapone and CO, the generation of the radical from Hy decreased, confirming that the radical is formed by the cytochrome P-450 dependent microsome systems. The 4-POBN-trapped radical species generated from Hy was presumed to be the hydrazyl radical by the results of mass spectrometry.
(Keyword)
Animals / Ascorbic Acid / Chemical and Drug Induced Liver Injury / Free Radical Scavengers / Free Radicals / Hydrazines / In Vitro Techniques / Isoniazid / Microsomes, Liver / Rats / Rats, Inbred Strains
Chikamasa Yamashita, Saburo Sone, Takeshi Ogura and Hiroshi Kiwada : Potential Value of Cetylmannoside-modified Liposomes as Carriers of Macrophage Activators to Human Blood Monocytes, Japanese Journal of Cancer Research, Vol.82, No.5, 569-576, 1991.
(Summary)
The present study was undertaken to examine the potential value of cetylmannoside-modified multilamellar liposomes (Man-MLV) as carriers for transfer of macrophage activators to blood monocytes. Highly purified blood monocytes were isolated by centrifugal elutriation from healthy donors under endotoxin-free conditions. Freshly prepared monocytes phagocytosed Man-MLV to a lesser extent than monocyte-derived macrophages, but they took up Man-MLV much more effectively than control liposomes without cetylmannoside (control MLV). Phagocytosis of Man-MLV, but not control MLV by monocytes was inhibited by addition of D-mannose, but not of D-galactose. Desmethyl-muramyl dipeptide (norMDP) entrapped in Man-MLV was far more effective than norMDP entrapped in MLV in activating monocytes to the tumoricidal state. The effect of encapsulation of recombinant human macrophage colony-stimulating factor (M-CSF) in Man-MLV on prolongation of survival of monocytes was examined. Blood monocytes that had been incubated for up to 21 days with Man-MLV containing 5-20 U of M-CSF per ml were effective in prolonging monocyte survival, but monocytes that had been incubated in medium with less than 50 U/ml of M-CSF or with control MLV containing 5-10 U of M-CSF showed no increase of monocyte survival over that in medium alone. Addition of rabbit anti-M-CSF antiserum did not affect survival prolongation of monocytes by M-CSF encapsulated in Man-MLV. We conclude that liposomes modified with cetylmannoside are far more effective than unmodified liposomes as a carrier to deliver biological response modifiers to human blood monocytes.
Chikamasa Yamashita, Hirotami Matsuo, Kazue Akiyama and Hiroshi Kiwada : Enhancing effect of cetylmannoside on targeting of liposomes to Kupffer cells in rats, International Journal of Pharmaceutics, Vol.70, No.3, 225-233, 1991.
Hideki Tsuji, Sayoko Osaka and Hiroshi Kiwada : Targeting of lipsomes surface-modified with glycyrrhizin to the liver I Preparation and biological disposition, Chemical & Pharmaceutical Bulletin, Vol.39, No.4, 1004-1008, 1991.
(Summary)
We consider glycyrrhizin to be a new ligand for liposomes to the liver because it is known that about 80% of glycyrrhizin is excreted into the bile after intravenous administration in rats. In order to modify the liposomal surface with glycyrrhizin, 30-stearyl glycyrrhizin (GLOSt), one of the lipophilic glycyrrhizin derivatives, was synthesized. The structure of this new compound was identified by nuclear magnetic resonance (NMR), infrared (IR) and mass spectra (MS). Sonicated liposomes were prepared from hydrogenated egg phosphatidylcholine-cholesterol-GLOSt or dicetyl phosphate (DCP) (4:4:1) and were labelled with [3H]inulin as an aqueous marker. It was confirmed by measuring the encapsulation efficiencies and the mean diameters that GLOSt-containing sonicated liposomes (GLOSt-SUV) were SUV-type as well as DCP-containing control liposomes (control-SUV). Four hours after intravenous injection into rats at a dose of 90 mumol as total lipid per kg of rat body weight, GLOSt-SUV showed 4-fold more accumulation (42.4%) in the liver than control-SUV. Therefore, glycyrrhizin is considered to be a useful new ligand on liposomes for targeting to the liver.
(Keyword)
Animals / Drug Carriers / Glycyrrhetinic Acid / Glycyrrhizic Acid / liposomes / Liver / Male / Rats / Rats, Inbred Strains / Tissue Distribution
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 1893485
Yoshihiro Kume, Fumiyo Maeda, Hideyoshi Harashima and Hiroshi Kiwada : Saturable,non-Michaelis-Menten uptake of liposomes by the reticuloendothelial system, The Journal of Pharmacy and Pharmacology, Vol.43, No.3, 162-166, 1991.
(Summary)
Multilamellar vesicles (300-350 nm) were infused into the rat femoral vein at the rate of 4, 40 and 400 nmol phosphatidycholine min-1 for 6 h using [3H]inulin as an aqueous marker. The time courses of blood concentration of vesicles, normalized for infusion rate, were not superimposable, showing the non-linearity of liposome disposition in the blood circulation. These time courses of blood concentration were well fitted by a single Michaelis-Menten equation. On the other hand, the time courses of tissue content could not be so accommodated. Additionally, the observed relationship between the uptake of liposomes by the liver and their clearance from it and other organs differed essentially from a simulation based on Michaelis-Menten type saturable kinetics. Therefore, it is suggested that there is a time-dependent non-Michaelis-Menten type process in the phagocytosis of macrophages in the reticuloendothelial system.
(Keyword)
Animals / In Vitro Techniques / insulin / liposomes / Liver / Male / Mononuclear Phagocyte System / Phagocytosis / Rats / Rats, Inbred Strains / Spleen
久米 義博, 山根 千鶴, 原島 秀吉 and Hiroshi Kiwada : In vivo実験系における血中でのリポソームの安定性の定量的評価法, 薬物動態, Vol.6, 201-208, 1991.
153.
Yoko Matsuki, Yoshimitu Katakuse, Hiroshi Matsuura, Hiroshi Kiwada and Tsuyoshi Goromaru : Effects of glucose and ascorbic acid on absorption and first pass metabolism of isoniazid in rats, Chemical & Pharmaceutical Bulletin, Vol.39, 445-448, 1991.
154.
山下 親正, Saburo Sone, 小倉 剛 and Hiroshi Kiwada : マクロファージコロニー刺激因子含有マンノース修飾リポソームによるヒト単球の生存率増強効果, Drug Delivery System, Vol.6, No.1, 19-24, 1991.
Koichiro Tahara, Masahiro Hayashi, Shoji Awazu, Yuriko Kato and Hiroshi Kiwada : Effect of serum on dose- and temperature-dependent hepatic uptake of multilamellar vesicles(MLV), Journal of Pharmacobio-Dynamics, Vol.12, No.10, 596-601, 1989.
(Summary)
Effects of the addition of serum to the perfusate on hepatic uptake of multilamellar vesicles (MLV) were examined in recirculating perfused rat liver. MLV was labelled with both membrane lipid marker ([14C]cholesteryl oleate) and aqueous phase marker ([3H]inulin or 5(6)-carboxyfluorescein (CF)). The uptake rates of [14C]cholesteryl oleate and CF at 37 degrees C coincided well, indicating both markers to be taken up as MLV. Inulin was released from MLV at 37 degrees C but its uptake rate tended to approach that of [14C]cholesteryl oleate at 4 degrees C or at high MLV dose. Some factor in serum promoted MLV uptake at 37 degrees C, but its effect was inhibited at 4 degrees C. The promoting effect was indicated to be possibly due to activation of adsorption of MLV to the hepatic Kupffer cell surface and/or that of phagocytosis by the cells. The saturation of MLV uptake was observed with increase in MLV dose, suggesting the saturation of MLV adsorption to the hepatic cell or the consumption of serum factor which promotes MLV uptake.
Hiroshi Kiwada, Masami Akimoto, Michiyo Araki, Mitsuko Tsuji and Yuriko Kato : Application of synthetic liposomes based on acyl amino acids or acyl peptides as drug carriers I.Their preparation and transport of glutathione into the liver, Chemical & Pharmaceutical Bulletin, Vol.35, No.7, 2935-2942, 1987.
Hiroshi Kiwada, Takaaki YiYajima and Yuriko Kato : Studies on the uptake mechanism of liposomes by perfused rat liver 2.An indispensable factor for liver uptake in serum, Chemical & Pharmaceutical Bulletin, Vol.35, No.3, 1189-1195, 1987.
(Keyword)
Animals / In Vitro Techniques / liposomes / Liver / Male / Perfusion / Rats / Rats, Inbred Strains
Hiroshi Kiwada, Junko Sato, Sunju Yamada and Yuriko Kato : Feasibility of magnetic liposomes as a Targeting device for drugs, Chemical & Pharmaceutical Bulletin, Vol.34, No.10, 4253-4258, 1986.
Hiroshi Kiwada, Sakae Obara, Hiroko Nishiwaki and Yuriko Kato : Studies on the uptake mechanism of liposomes by perfused rat liver 1.An investigation of effuluent profiles with perfusate containing no blood component, Chemical & Pharmaceutical Bulletin, Vol.34, No.3, 1249-1256, 1986.
(Keyword)
Animals / Antimetabolites / In Vitro Techniques / liposomes / Liver / Male / Perfusion / Rats / Rats, Inbred Strains / temperature
Yasushi Sato, Hiroshi Kiwada and Yuriko Kato : Effects of dose and vesicle size on the pharmacokinetics of liposomes, Chemical & Pharmaceutical Bulletin, Vol.34, 4244-4252, 1986.
162.
C Hunt Anthony, R R Burnette, R D MacGregor, A E Strubbe, D T Lau, N Taylor and Hiroshi Kiwada : Synthesis and evaluation of a prototypal artificial red cell, Science, Vol.230, No.4730, 1165-1168, 1985.
(Summary)
A new process allows microencapsulation of purified human hemoglobin and 2,3-diphosphoglycerate to form neohemocytes. The microcapsule membrane is composed of phospholipids and cholesterol. Neohemocytes are substantially smaller than erythrocytes, contain 15.1 grams per decaliter of hemoglobin, and have a P50 value (the partial pressure of oxygen at which the hemoglobin is half-saturated) of 24.0 torr. All rats given 50-percent exchange transfusions survived with only limited evidence of reversible toxicity. Normal serum glutamate-pyruvate-transaminase values at 1, 7, and 30 days after transfusion were consistent with minimal hepatotoxicity. The concentration of blood urea-nitrogen was elevated by 35 percent after 1 day but returned to normal by day 7. However, histopathology revealed normal kidneys on day 1 as well as on days 7 and 30. Neohemocytes cleared from the circulation of transfused rats with an apparent half-life of 5.8 hours.
Hiroshi Kiwada, Hitoshi Niimura and Yuriko Kato : Tissue distribution and pharmacokinetic evaluation of targeting efficiency of synthetic alkyl glycoside vesicles, Chemical & Pharmaceutical Bulletin, Vol.33, No.6, 2475-2482, 1985.
Hiroshi Kiwada, Hiroyuki Kojima and Yuriko Kato : Correlation between in vivo and in vitro dissolution rates of(a-bromoisovaleryl)urea polymorphs, Chemical & Pharmaceutical Bulletin, Vol.32, 3164-3172, 1984.
166.
Hiroshi Kiwada, Hiroyuki Kojima and Yuriko Kato : Pharmacokinetic study on the polymorphs of(a-bromoisovaleryl)urea in rat, Chemical & Pharmaceutical Bulletin, Vol.31, 1345-1349, 1983.
167.
Hiroyuki Kojima, Hitoshi Niimura, Hiroshi Kiwada and Yuriko Kato : The determination of(a-bromoisovaleryl)urea in plasma and the bioavailability of its polymorphic forms in rat, Chemical & Pharmaceutical Bulletin, Vol.30, 1831-1836, 1982.
168.
Hiroyuki Kojima, Hiroshi Kiwada and Yuriko Kato : The dissolution properties and physico-chemical properties of polymorphic forms of(a-bromoisovaleryl)urea, Chemical & Pharmaceutical Bulletin, Vol.30, 1824-1830, 1982.
169.
Hiroshi Kiwada, Shoji Awazu and Manabu Hanano : The study on the biological fate of pasraben at the dose of practical usage in rat 3.The effect of salicylic acid on the fate of paraben., Journal of Pharmacobio-Dynamics, Vol.4, No.8, 643-648, 1981.
170.
Hiroshi Kiwada, Shoji Awazu and Manabu Hanano : The study on the biological fate of paraben at the dose of practical usage in rat 2.The pharmacokinetic study on the blood concentration after the administration of ethyl paraben or p-hydroxybenzoic acid., Journal of Pharmacobio-Dynamics, Vol.3, No.7, 353-363, 1980.
(Summary)
The biological fates of ethyl paraben and p-hydroxybenzoic acid in rat were investigated after intravenous and intraduodenal administrations at the dose of 2 mg/kg. The blood concentrations were measured in detail from 3 min after the administration at appropriate time intervals until 90 min. Areas under the blood concentration curves and clearances were calculated from these time course data. Ethyl paraben was little detected in blood after intraduodenal administration. It is suggested that the intestinal metabolism and the first pass effect in liver greatly contribute to the hydrolysis of ethyl paraben. Total radio activity after intraduodenal administration did not show the maximum peak and decreased rapidly. The maximum peak was not observed also in the time course of p-hydroxyhippuric acid after intravenous administration. It shows that not only the rate of hydrolysis but also absorption and conjugation are very rapid. The differences of the areas under the blood concentration curves of p-hydroxybenzoic acid or p-hydroxyhippuric acid was found between the routes or chemical forms of administration. The complex kinetic mechanisms were assumed in the biological fates of these compounds as follows: Conjugation to p-hydroxyhippuric acid is excellent in ethyl paraben administration than p-hydroxybenzoic acid, and in intraduodenal administration than intravenous administration. These phenomenon can not be explained by the conventional kinetic model which is constructed with the connected blood compartments in series. The kinetic models including the assumed routes conjugating directly ethyl paraben in blood or intestine to p-hydroxyhippuric acid were presented, and the least square curve fitting analyses were carried out on these kinetic models.
Hiroshi Kiwada, Kazuo Takami and Yuriko Kato : The preparation and properties of a new polymorphic form of(a-bromoisovaleryl)urea, Chemical & Pharmaceutical Bulletin, Vol.28, 1351-1356, 1980.
172.
Hiroshi Kiwada, Shoji Awazu and Manabu Hanano : The study on the biological fate of parabens at the dose of practical usage in rat 1.The metabolism and excretion of etyl p-hydroxybenzoate(ethyl paraben)and p-hydroxybenzoic acid, Journal of Pharmacobio-Dynamics, Vol.2, No.6, 356-364, 1979.
173.
Hiroshi Kiwada, Masahiro Hayashi, Tohru Fuwa, Shoji Awazu and Manabu Hanano : The pharmacokinetic study on the fate of 8-hydroxyquinoline in rat, Chemical & Pharmaceutical Bulletin, Vol.25, No.7, 1566-1573, 1977.
Hiroshi Kiwada, Kaname Morita, Masahiro Hayashi, Shoji Awazu and Manabu Hanano : A New numerical calculation method for deconvolution in linear compartment analysis of pharmacokinetics, Chemical & Pharmaceutical Bulletin, Vol.25, No.6, 1312-1318, 1977.
Lila Selim Ahmed Ali Abu Amr, Hiroshi Kiwada and Tatsuhiro Ishida : Liposomal delivery systems: design optimization and current applications, Biological & Pharmaceutical Bulletin, Vol.40, No.1, 1-10, Jan. 2017.
(Summary)
The liposome, a closed phospholipid bilayered vesicular system, has received considerable attention as a pharmaceutical carrier of great potential over the past 30 years. The ability of liposomes to encapsulate both hydrophilic and hydrophobic drugs, coupled with their biocompatibility and biodegradability, make liposomes attractive vehicles in the field of drug delivery. In addition, great technical advances such as remote drug loading, triggered release liposomes, ligand-targeted liposomes, liposomes containing combinations of drugs, and so on, have led to the widespread use of liposomes in diverse areas as delivery vehicles for anti-cancer, bio-active molecules, diagnostics, and therapeutic agents. In this review, we summarize design optimization of liposomal systems and invaluable applications of liposomes as effective delivery systems.
(Keyword)
Animals / Drug Delivery Systems / Humans / Liposomes
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Selective delivery of oxaliplatin to tumor tissue by nanocarrier system enhances overall therapeutic efficacy of the encapsulated oxaliplatin, Biological & Pharmaceutical Bulletin, Vol.37, No.2, 206-211, Feb. 2014.
(Summary)
Oxaliplatin (trans-l-diaminocyclohexane oxalatoplatinum; l-OHP), a third-generation platinum antitumor drug, is currently approved in combination with 5-flurouracil (5-FU)/leucovorin (FOLFOX) for standard first- and second-line treatment of metastatic or advanced-stage colorectal cancer. Despite l-OHP's better tolerability in comparison with other platinum compounds such as cisplatin and carboplatin, its clinical efficiency is limited by the dose-limiting side effects including cumulative neurotoxicity and acute dysesthesias. In addition, like other platinum chemotherapeutic agents, l-OHP therapy is limited by reduced accumulation levels in tumor tissues, nonselective accumulation in healthy organs and/or tissues, inactivation by conjugation with glutathione, and the development of drug resistance. Accordingly, successful outcome of cancer treatment using l-OHP requires selective delivery of a relatively high concentration of the drug to tumor tissues. In this review we focus on utilization of different drug-delivery vehicles such as liposomes, polymeric nanocarriers, and carbon nanotubes in enhancing selective delivery of l-OHP to tumor tissues and consequently improving overall efficacy of l-OHP-containing drug-delivery systems.
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : The accelerated blood clearance (ABC) phenomenon: Clinical challenge and approaches to manage, Journal of Controlled Release, Vol.172, No.1, 38-47, Aug. 2013.
(Summary)
Despite the clinical introduction of an increasing number of polyethylene glycol (PEG)-conjugated substances, PEG has been named as the cause of an unexpected immunogenic response known as the "accelerated blood clearance (ABC) phenomenon." This phenomenon has been extensively observed during the repeated administration of PEG-conjugated substances and PEGylated nanocarriers including PEGylated liposomes, PEGylated nanoparticles, PEGylated micelles, etc., resulting in the increased clearance and reduced efficacy of PEG-conjugated substances/PEGylated nanocarriers. In this review, therefore, we focused on the possible mechanisms underlying the induction of such a phenomenon and emphasized the factors affecting its magnitude. In addition, the clinical implications of the ABC phenomenon on the therapeutic efficacy of PEG-conjugated substances/PEGylated nanocarriers, along with the new approaches that can be applied to manage and/or abrogate the induction of the ABC phenomenon, are also discussed.
In contrast to the general assumption that polyethyleneglycol (PEG)-conjugated substances lack immunogenicity and antigenic, it has been reported that they can elicit antibodies against PEG (mainly anti-PEG immunoglobulin M (IgM)). In patients, the presence of anti-PEG antibodies may limit therapeutic efficacy of PEGylated substances as a consequence of inducing rapid clearance of and neutralizing biological activity of the substances. Here, we introduce specific examples of PEGylated substances including several PEGylated proteins and PEGylated particles (PEGylated nanocarriers) which induce anti-PEG antibody responses. Finally, we emphasize that the immunogenicity of PEGylated substances should be tested in the development stage and that the titer of anti-PEG antibodies in patients should be pre-screened and monitored prior to and throughout a course of treatment with a PEGylated substance.
(Keyword)
Animals / Drug Carriers / Humans / Immunoglobulin M / Nanoparticles / Pharmaceutical Preparations / Polyethylene Glycols / Proteins
Tatsuhiro Ishida and Hiroshi Kiwada : Alteration of tumor microenvironment for improved delivery and intratumor distribution of nanocarriers., Biological & Pharmaceutical Bulletin, Vol.36, No.5, 692-697, May 2013.
(Summary)
Nanocarrier-based cancer chemotherapeutics are thought to increase therapeutic efficiency and reduce the side effects of associated chemotherapeutic agents by altering the agents' pharmacokinetics and tissue distribution following intravenous administration. In spite of these favorable properties, nanocarrier-based cancer chemotherapeutics are not always effective because of their heterogeneous intratumoral localization. Homogeneous distribution of nanocarriers in a tumor would improve the efficacy of nanocarrier-based cancer chemotherapeutics. In this article, we describe and discuss some trials that attempt to manipulate the barriers in the tumor microenvironment that hinder extravasation through the tumor vasculature and penetration of nanocarriers in solid tumors. Alterations of the tumor microenvironment that relate directly to the intratumoral distribution of nanocarriers may be potential strategies to improve the delivery of nanocarrier-based cancer chemotherapeutics.
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Targeting anticancer drugs to tumor vasculature using cationic liposomes., Pharmaceutical Research, Vol.27, No.7, 1171-1183, Mar. 2010.
(Summary)
Liposomal drug delivery systems improve the therapeutic index of chemotherapeutic agents, and the use of cationic liposomes to deliver anticancer drugs to solid tumors has recently been recognized as a promising therapeutic strategy to improve the effectiveness of conventional chemotherapeutics. This review summarizes the selective targeting of cationic liposomes to tumor vasculature, the merits of incorporating the polymer polyethylene-glycol (PEG), and the impact of the molar percent of the cationic lipid included in cationic liposomes on liposomal targeting efficacy. In addition, the discussion herein includes the therapeutic benefit of a dual targeting approach, using PEG-coated cationic liposomes in vascular targeting (of tumor endothelial cells), and tumor targeting (of tumor cells) of anticancer drugs. Cationic liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development.
(Keyword)
Antineoplastic Agents / Cations / Drug Delivery Systems / Humans / Liposomes / Neoplasms / Neovascularization, Pathologic
小出 裕之, 浅井 知浩, 畑中 剣太朗, 清水 広介, 横山 昌幸, Tatsuhiro Ishida, Hiroshi Kiwada and 奥 直人 : Elucidation of Accelerated Blood Clearance Phenomenon Caused by Repeat Injection of PEGylated Nanocarriers, Journal of the Pharmaceutical Society of Japan, Vol.129, No.12, 1445-1451, Dec. 2009.
(Summary)
Liposomes modified with polyethylene glycol (PEG) can stably exist in the bloodstream because the PEG on the liposomes attracts a water shell to the liposomal surface. Since these liposomes are long circulating nanocarriers, they are used as drug and gene delivery tools. Repeat injection of PEGylated liposomes, however, is known to induce the accelerated blood clearance (ABC) phenomenon. In the ABC phenomenon, PEGylated liposomes that are injected subsequent to the first injection are cleared rapidly from the bloodstream and accumulate in the liver, resulting in loss of their long-circulating characteristics. The induction of ABC phenomenon is related to the production of anti-PEG IgM from splenic B cells. To elucidate the mechanism of the phenomenon, we firstly examined the relationship between the induction of ABC phenomenon and the concentration of PEGylated liposomes, and observed that the high dose of those did not induce the phenomenon. Next, we investigated whether polymeric micelles trigger ABC phenomenon or not. Finally, the size-dependency of ABC phenomenon was investigated by use of variously sized PEGylated liposomes and polymeric micelles having PEG chains. Our data suggest that the initiation of ABC phenomenon would be size-dependent, and particles smaller than 30 nm did not induce ABC phenomenon. We anticipate that the elucidation of the ABC phenomenon will be helpful for the development of DDS formulations.
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Recent advances in tumor vasculature targeting using liposomal drug delivery systems., Expert Opinion on Drug Delivery, Vol.6, No.12, 1297-1309, Dec. 2009.
(Summary)
Tumor vessels possess unique physiological features that might be exploited for improved drug delivery. The targeting of liposomal anticancer drugs to tumor vasculature is increasingly recognized as an effective strategy to obtain superior therapeutic efficacy with limited host toxicity compared with conventional treatments. This review introduces recent advances in the field of liposomal targeting of tumor vasculature, along with new approaches that can be used in the design and optimization of liposomal delivery systems. In addition, cationic liposome is focused on as a promising carrier for achieving efficient vascular targeting. The clinical implications are discussed of several approaches using a single liposomal anticancer drug formulation: dual targeting, vascular targeting (targeting tumor endothelial cells) and tumor targeting (targeting tumor cells).
(Keyword)
Angiogenesis Inhibitors / Animals / Antineoplastic Agents / Cations / Drug Delivery Systems / Drug Design / Humans / Liposomes / Neoplasms / Neovascularization, Pathologic
Tatsuhiro Ishida and Hiroshi Kiwada : Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes., International Journal of Pharmaceutics, Vol.354, No.1-2, 56-62, Apr. 2008.
(Summary)
We and a Dutch group reported that "empty" PEGylated liposomes (approximately 100 nm) lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). Very recently, we showed that anti-PEG IgM, induced by the first dose of "empty" PEGylated liposomes, is responsible for inducing the phenomenon, based on the observation that IgM thus produced selectively binds to the surface of subsequently injected PEGylated liposomes, leading to substantial complement activation. It is generally believed that nanocarriers coated with a polymer, such as PEG, have no or lower immunogenicity. However, the results indicated evidence that unexpected immune responses occur even to such polymer-coated liposomes. Such immunogenicity of "empty" liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. In addition, through series of our studies, it was demonstrated that the magnitude of the ABC phenomenon depends on the physicochemical property of injected liposomes as a first dose, time interval between injection, lipid dose and drug-encapsulation.
(Keyword)
Animals / Complement Activation / Drug Administration Schedule / Drug Carriers / Immunoglobulin M / Injections, Intravenous / Liposomes / Polyethylene Glycols
Tatsuhiro Ishida and Hiroshi Kiwada : Accelerated Blood Clearance (ABC) Phenomenon Induced by Administration of PEGylated Liposome(Symposium Reviews), Journal of the Pharmaceutical Society of Japan, Vol.128, No.2, 233-243, Feb. 2008.
(Summary)
PEGylated liposomes (approximately 100nm in diameter) lose their long-circulating characteristic upon repeated injection at certain intervals in the same animal (referred to as the "accelerated blood clearance (ABC) phenomenon"), as described by our group and by researchers in the Netherlands. Recently, it was demonstrated by our group that anti-PEG IgM, induced by the first dose of PEGylated liposomes, is responsible for the ABC phenomenon. The IgM produced in this manner then selectively bound to the surface of subsequently injected PEGylated liposomes, leading to substantial complement activation. It is generally believed that nanocarriers coated with a polymer, such as PEG, have no immunogenicity. However, unexpected immune responses occurred even in response to polymer-coated liposomes. This immunogenicity to PEGylated liposomes presents a serious concern in the development and clinical use of liposomal formulations. In this review, we demonstrate our recent observations regarding with the ABC phenomenon against liposomes.
Tatsuhiro Ishida, Yoshihiro Takanashi and Hiroshi Kiwada : Safe and efficient drug delivery system with liposomes for intrathecal application of an antivasospastic drug, fasudil., Biological & Pharmaceutical Bulletin, Vol.29, No.3, 397-402, Mar. 2006.
(Summary)
Pharmacological treatment for cerebral ischemia and cerebral vasospasm following subarachnoid hemorrhage (SAH) cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. We recently developed a liposomal drug delivery system for intrathecal application that can maintain effective concentrations of cerebral vasodilator, fasudil, in the CSF. A single intrathecal injection of liposomal fasudil could maintain a therapeutic drug concentration in the CSF over a period time due to their sustained-release property, significantly decreasing infarct size in a rat model of acute ischemia and reducing vasoconstriction of the rat and dog basilar artery in a model of SAH. In this review, we are introducing our new less-invasive intrathecal drug delivery system that provides an alternative and safe method to deliver therapeutic agents.
(Keyword)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Animals / Brain Ischemia / Chemistry, Pharmaceutical / Cholesterol / Chromatography, High Pressure Liquid / Drug Carriers / Drug Delivery Systems / Half-Life / Humans / Injections, Spinal / Liposomes / Neuroprotective Agents / Solubility / Vasodilator Agents
Tatsuhiro Ishida and Hiroshi Kiwada : がん治療を目指した機能性リポソームの開発研究, Chemical Industry, Vol.57, 39-43, 2006.
19.
Tatsuhiro Ishida and Hiroshi Kiwada : Accelerated blood clearance of PEGylated liposomes after repeated injection, Drug Delivery System, Vol.19, No.6, 495-510, Nov. 2004.
(Summary)
We and a Dutch group have recently reported the accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes, that is the enhanced accumulation of PEGylated liposomes in liver and consequently rapidly cleared the liposomes from blood circulation. The phenomenon is a general characteristic of liposomes and the induction of the phenomenon depends on the physicochemical property and dose of liposomes and animal species. In addition, our study indicated that the IgM secreted in response to first dose is involved in the occurrence of the phenomenon. In this article, our and a Dutch group's recent results related to the ABC phenomenon were described.
Tatsuhiro Ishida, H Harashima and Hiroshi Kiwada : Interactions of Liposomes with Cells In Vitro and In Vivo:Opsonins and Receptors, Current drug metabolism, Vol.2, No.4, 397-409, Dec. 2001.
Rieko Tachibana, Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviarl vectors, Advanced Drug Delivery Reviews, Vol.52, No.3, 219-226, Nov. 2001.
27.
Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Intracellular control of gene trafficking using liposomes as drug carriers, European Journal of Pharmaceutical Sciences, Vol.13, No.1, 85-89, Apr. 2001.
(Summary)
The objective of this review is to summarize some of the critical barriers in gene delivery and recent progress in overcoming such barriers using non-viral carrier systems. Receptor-mediated endocytosis is generally considered to be a principal entering pathway. Therefore, endosomal escape is an essential step for achieving efficient transfection. The nuclear membrane is also a critical barrier in gene delivery and the application of the nuclear localization signal is discussed, based on recent strategies. It is essential to optimize the carrier system, in order to enhance the transfection ability equivalent to a viral system. The importance of developing an intracellular pharmacokinetic model of genes is emphasized in the optimization of non-viral carrier systems.
(Keyword)
Liposomes / Drug delivery system / Gene delivery / Carrier system / Nuclear localization signal
Rieko Tachibana, Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Intracellular control of macromolecules by use of liposomes as drug carriers, Journal of Biological Macromolecules, Vol.1, No.3, 60-67, 2001.
29.
原島 秀吉, 土井 久子, 土橋 麻理 and Hiroshi Kiwada : 血中滞留性リポソームによる抗癌剤の処方設計における最適化, Drug Delivery System, Vol.15, 442-467, 2000.
30.
A Nagayasu, K Uchiyama and Hiroshi Kiwada : The size of liposomes:Factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs, Advanced Drug Delivery Reviews, Vol.40, No.1-2, 75-87, Nov. 1999.
(Summary)
The size of liposomes has been shown to be an important factor in the efficient delivery of an antitumor agent to a tumor. In this paper, the effects of the size of liposomes on the pharmacokinetics of liposomes and liposome-encapsulated drugs are discussed with reference to: (1) the circulation amount and residence time of liposomes in the blood, (2) the accumulation of liposomes in the tumor, and (3) in vivo drug release from liposomes. In addition, the effect of size on therapeutic activity (antitumor efficacy and toxicity) of a liposomal anticancer preparation is discussed. Finally we discuss the importance of liposome size in the design of a more effective liposomal antitumor preparation.
H Harashima, M Tsuchihashi, S Iida, H Doi and Hiroshi Kiwada : Pharmacokinetic/Pharmacodynamic modeling of antitumor agents encapsulated into Liposomes, Advanced Drug Delivery Reviews, Vol.40, No.1-2, 39-61, Nov. 1999.
(Summary)
Pharmacokinetic/pharmacodynamic (PK/PD) modeling of antitumor agents has been developed for doxorubicin (DOX) in order to predict the optimum conditions for a drug carrier to maximize the antitumor effect. A PK model was constructed for free and liposomal doxorubicin using a hybrid model wherein the disposition in the whole body is described by compartment models, which were linked to the tumor compartment via the blood flow rate. The PD model for doxorubicin was described by a cell-kill kinetic model, which represents the number of tumor cells quantitatively, as a function of the free concentration of doxorubicin in the tumor compartment. The influence of each parameter on the antitumor effects was examined by sensitivity analysis based on the PK/PD model, which clearly showed the importance of optimizing the release rate of DOX from liposomes. The validity of the model has been tested using animal experiments. Preliminary simulations were also performed for humans after scaling up the PK/PD model from rodents to humans. The optimum conditions in the rate of drug release from liposomes were different for rodents vis-a-vis humans, which indicates the limitations involved in extrapolating optimum conditions for experimental animals to those for humans.
Hideyoshi Harashima and Hiroshi Kiwada : The pharmacokinetics of liposomes in tumor targeting, Advanced Drug Delivery Reviews, Vol.40, No.1-2, 1-2, Nov. 1999.
原島 秀吉, Yasuo Shinohara and Hiroshi Kiwada : キャリアによる細胞内デリバリー戦略と今後の課題, Drug Delivery System, Vol.14, 157-164, 1999.
35.
H Harashima, H Matsuo and Hiroshi Kiwada : Identification of proteins mediating clearance of liposomes using a liver perfusion system, Advanced Drug Delivery Reviews, Vol.32, No.1-2, 61-79, Jun. 1998.
(Summary)
The objective of this paper is to identify the principal blood components governing the fate of liposomes in blood circulation. Information based on an isolated perfused liver system in rats has revealed the central role of the complement system in enhancing the uptake of liposomes by the liver. A species difference was an important factor in determining the uptake mechanisms of liposomes by the liver. Limited evidence revealed the tendency that opsonin-dependent hepatic uptake is principal in rats, while opsonin-independent or dysopsonin-dependent uptake governs in mice, although there are some exceptions. These studies provide us with important information for understanding the uptake mechanisms of liposomes by the liver, and useful insights in predicting the in vivo disposition of liposomes in humans.
H Harashima and Hiroshi Kiwada : Liposomal Targeting and Drug Delivery:Kinetic Consideration, Advanced Drug Delivery Reviews, Vol.19, No.3, 425-444, Jun. 1996.
H Harashima and Hiroshi Kiwada : Studies on the Mechanism of Uptake of Liposomes Using an Isolated Perfused Liver System, Journal of Liposome Research, Vol.6, 61-75, 1996.
Hiroshi Kiwada : リポソームを利用した薬物投与ーより有効なキャリアーシステムへの可能性, SUT Bulletin, Vol.2, 56-57, 1985.
47.
C A Hunt, Hiroshi Kiwada, M Reader-Shikorr, K Hirano, J Nakamura, R Abra and H Schreier : Liposomes-Are they effective drug carriers?, Pharm Int, Vol.4, 181, 1983.
Taro Shimizu, Yuki Watanabe, Yu Mima, Tatsuhiro Ishida and Hiroshi Kiwada : Antigen delivery with PEGylated liposome and presentation via MZ-B cells enhanced induction of antigen-specific cytotoxic T lymphocyte, Liposome Research Days 2014, Copenhagen, Aug. 2014.
2.
Munehira Kawanishi, Yosuke Hashimoto, Taro Shimizu, I Sagawa, Tatsuhiro Ishida and Hiroshi Kiwada : Comprehensive analysis of proteins bound on PEGylated liposome relating to the accelerated blood clearance (ABC) phenomenon by shotgun analysis, Liposome Research Days 2014, Copenhagen, Aug. 2014.
3.
Yukako Fujiwara, Hiroyuki Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : Increased PEG-coated liposomal l-OHP accumulation in the liver relates antitumor effect against M5076 ovarian sarcoma liver metastasis, Liposome Research Days 2014, Copenhagen, Aug. 2014.
4.
Yu Mima, Taro Shimizu, Hiroshi Kiwada and Tatsuhiro Ishida : Inclusion of ganglioside into PEGylated liposome suppresses the anti-PEG immunity against PEGylated liposome, Liposome Research Days 2014, Copenhagen, Aug. 2014.
5.
Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Characterization of optimally designed liposomal formulation of Oxaliplatin, The 41st Annual Meeting & Exposition of the Controlled Release Society, Chicago, Jul. 2014.
6.
Noriko Saito-Tarashima, Kojima Takamitsu, Hashimoto Yosuke, Kazuhiro Furukawa, Naoshi Yamazaki, Hiroshi Kiwada, Tatsuhiro Ishida, Noriaki Minakawa and Yoshiharu Takiguchi : A novel approach of gene suppression using an intelligent shRNA expressing device (iRed), 9th Annual Meeting of the Oligonucleotide Therapeutics Society, Naples(Italy), Oct. 2013.
7.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Potentiation of specific antibody response against antigen encapsulated in PEGylated liposomes via active transport of the liposomes from marginal zone into follicle, 5th Asian Arden Conference, Nagoya, Aug. 2013.
8.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Transport of PEGylated liposomes to follicle induced by pre-stimulation of empty liposomes: Potentiating specific antibody responses against antigen encapsulated in the liposomes, 40th Annual Meeting & Exposition of the Controlled Release Society, Hawaii, Jul. 2013.
9.
Yosuke Hashimoto, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Serum anti-PEG IgM concentration is a determinant factor on hepatic accumulation of PEGylated liposome in the accelerated blood clearance phenomenon, 40th Annual Meeting & Exposition of the Controlled Release Society, Hawaii, Jul. 2013.
10.
Yu Mima, Yosuke Hashimoto, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Accelerated blood clearance phenomenon upon PEGylated protein, 40th Annual Meeting & Exposition of the Controlled Release Society, Hawaii, Jul. 2013.
11.
Miho Nishio, Hiroyuki Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : PEGylated liposomes injected sequentially distribute in different region of solid tumor, 40th Annual Meeting & Exposition of the Controlled Release Society, Hawaii, Jul. 2013.
12.
Risako Fujita, Yosuke Hashimoto, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of terminal group of PEG on induction of ABC phenomenon to PEGylated liposome, 40th Annual Meeting & Exposition of the Controlled Release Society, Hawaii, Jul. 2013.
13.
Tatsuhiro Ishida and Hiroshi Kiwada : Improvement of tumor-targeting therapy with nanocarrier system by changing the tumor microenvironment., 10th DDS Symposium France-Japan, France, Oct. 2012.
14.
Tatsuhiro Ishida and Hiroshi Kiwada : Improved intratumoral delivery of PEG-coated siRNA-lipoplexes by combination with metronomic S-1 dosing in a murine solid tumor model., Oligonucleotide Delivery: Biology, Engineering and Development, Austria, Oct. 2012.
15.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Activation of splenic marginal zone B cell by PEGylated liposome with lower dose: triggering transport of antigen-containing second dose PEGylated liposome from marginal zone to follicle, 39th Annual Meeting & Exposition of the Controlled Release Society, Québec City, Jul. 2012.
16.
Hiroyuki Nakamura, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Sequential dose of l-OHP containing PEGylated liposome improve the intratumor delivery and efficacy of PEGylated liposomal anticancer agents., 39th Annual Meeting & Exposition of the Controlled Release Society, Québec City, Jul. 2012.
17.
Yosuke Hashimoto, Tatsuhiro Ishida and Hiroshi Kiwada : Anti-PEG IgM response against PEG-coated liposome is further increased by immune stimulation of siRNA via TLR7,, 39th Annual Meeting & Exposition of the Controlled Release Society, Québec City, Jul. 2012.
18.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Contribution of spleen marginal zone B cells on anti-PEG IgM responses against PEGylated liposomes and transport of PEGylated liposomes., International Liposome Society 2011 meeting, University of London,, London, Dec. 2011.
19.
Naoto Moriyoshi, Mariko Matsunaga, Kazuya Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : Development of new cancer treatment strategy that combines PEG-coated siRNA-lipoplex with metronomic S-1 dosing., International Liposome Society 2011 meeting, University of London,, London, Dec. 2011.
20.
Tatsuhiro Ishida and Hiroshi Kiwada : Biological responses against lipid membrane containing PEG., 3rd International Symposium on Surface and Interface of Biomaterials (SIB2011), Sapporo, Jul. 2011.
21.
Tatsuhiro Ishida and Hiroshi Kiwada : Improvement of tumor-targeting therapy with nanocarrier system by changing the tumor microenviroment, 2011 RCAA International Symposium Endogenous Ligands Modulating Anticancer Agents, Seoul, Feb. 2011.
22.
Yuki Hashiguchi, Taro Shimizu, Masako Ichihara, Tatsuhiro Ishida and Hiroshi Kiwada : PEG on nanocarriers induces anti PEG IgM production as a result of activation of immune system., Nano in Cancer: Linking Chemistry, Biology and Clinical Applications in vivo, Miami, Jan. 2011.
23.
Tatsuhiro Ishida, Kazuya Nakamura, Yusuke Doi, Naoto Moriyoshi and Hiroshi Kiwada : A novel double modulation strategy in cancer treatment with chemotherapeutic agent and siRNA: S-1 improves siRNA accumulation and then Bcl-2 knockdown by the siRNA increases chemosensitivity of 5-FU., Nano in Cancer: Linking Chemistry, Biology and Clinical Applications in vivo, Miami, Jan. 2011.
24.
Yuki Hashiguchi, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Sequential treatment, pre-dose with empty PEGylated liposome (SL) and second dose antigen-encapsulating SL, promotes activation in a primary immune response., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
25.
Haruna Matsumoto, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Tumor localization and therapeutic effect of PEG-coated liposomal anticancer agent: Tumor type-dependency., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
26.
Tomoko Okada, Yusuke Doi, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Metronomic oral S-1, a fluoropyrimidine anticancer agent, dosing affects tumor accumulation of PEG-coated liposome., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
27.
Mariko Matsunaga, Kazuya Nakamura, Yusuke Doi, Naoto Moriyoshi, Tatsuhiro Ishida and Hiroshi Kiwada : DEVELOPMENT OF NEW CANCER TREATMENT STRATEGY THAT COMBINES siRNA-LIPOPLEX WITH ORAL TEGAFUR ANTICANCER DRUG S-1., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
28.
Takahiro Iwaki, Tatsuaki Tagami, Kazuya Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : THE EFFECT OF PEGYLATION OF siRNA-LIPOPLEX ON INTRACELLULAR UPTAKE QUANTITY OF SIRNA AND INTRACELLULAR BEHAVIOR OF siRNA., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
29.
Masako Ichihara, Ami Imoto, Yuki Hashiguchi, Yumi Uehara, Tatsuhiro Ishida and Hiroshi Kiwada : Spleen cells secret anti-PEG IgM following intravenous injection of PEGylated liposome., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
30.
Tatsuhiro Ishida and Hiroshi Kiwada : Immunostimulatory properties of liposomes containing PEG., International Liposome Research Days & Lipids, Liposomes & Membrane Biophysics, Vancouver, Aug. 2010.
31.
Yusuke Doi, Tomoko Okada, Haruna Matsumoto, Tatsuhiro Ishida and Hiroshi Kiwada : Alteration of tumor microenvironment by metronomic S-1 dosing results in augmentation of EPR effect against PEG-coated liposomes., 4th International Liposome Society Conference. Liposome advances: Progress in Drug and Vaccine Delivery, London, Dec. 2009.
32.
Tatsuhiro Ishida, Kazuya Nakamura, Mariko Matsunaga, Yusuke Doi and Hiroshi Kiwada : A novel double modulation strategy in cancer treatment with chemotherapeutic agent and siRNA: siRNA-induced Bcl-2 knockdown increases chemosensitivity against 5-FU and S-1 improves siRNA accumulation in human colorectal tumor xenograft model., 4th International Liposome Society Conference. Liposome advances: Progress in Drug and Vaccine Delivery, London, Dec. 2009.
33.
H Koide, T Asai, M Yokoyama, Tatsuhiro Ishida, Hiroshi Kiwada and N Oku : Elucidation of ABC phenomenon caused by repeat injection of PEGylated nanocarrier., 4th International Liposome Society Conference. Liposome advances: Progress in Drug and Vaccine Delivery, London, Dec. 2009.
34.
Tatsuhiro Ishida and Hiroshi Kiwada : Improvement of tumor-targeting therapy with nanocarriers by changing the tumor microenvironment., 2009 International Symposium of the Intelligent Drug Delivery System, Seoul, Apr. 2009.
35.
Tatsuhiro Ishida, Lila Selim Ahmed Ali Abu Amr, Takuya Suzuki, Yusuke Doi and Hiroshi Kiwada : Oxaliplatin encapsulated in PEG-coated cationic liposome induces significant tumor growth suppression via dual-targeting approach in murine solid tumor model., AACR 100th Annual Meeting 2009, USA, Apr. 2009.
36.
Yusuke Doi, Yurie Hira, Tatsuhiro Ishida and Hiroshi Kiwada : Synergistic antitumor activity of metronomic S-1 dosing in combination with oxaliplatin (1-OHP) containing PEGylated liposome in a murine solid tumor model., AACR 100th Annual Meeting 2009, USA, Apr. 2009.
37.
Tatsuhiro Ishida and Hiroshi Kiwada : Improvement of tumor targeting therapy with nanocarriers by changing the tumor microenvironment., 6th international workshop on Drug Delivery Systems for Nanomedicine, Prague (the Czech Republic), Oct. 2008.
38.
Tatsuaki Tagami, Kazuya Nakamura, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of siRNA on anti-PEG IgM production in the accelerated blood clearance (ABC) phenomenon when formulated with PEGylated cationic liposome., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
39.
Takuya Suzuki, Kazuya Nakamura, Tatsuaki Tagami, Jose Mario Barichello, T Asai, Tatsuhiro Ishida, N Oku and Hiroshi Kiwada : Anticancer activity of Argonaute2 (Ago2) gene silencing induced by siRNA in mouse tumor xenograft model., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
40.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Interaction of PEGylated liposomes with splenic marginal zone B cells may trigger production of anti-PEG IgM in the ABC phenomenon., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
41.
Shinji Kizuki, Jose Mario Barichello, Tatsuaki Tagami, H Kikuchi, Tatsuhiro Ishida and Hiroshi Kiwada : The impact of novel siRNA-lipoplex preparation method on cellular uptake pathway and gene knockdown efficiency., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
42.
Yusuke Doi, Yurie Hira, Tatsuhiro Ishida and Hiroshi Kiwada : Metronomic dosing of oral anticancer agent affects microenvironment in solid tumor, resulting in enhanced intratumor accumulation and distribution of nanocarrior., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
43.
Lila Selim Ahmed Ali Abu Amr, Takuya Suzuki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin (l-OHP) targeting to angiogenic vessels by PEGylated cationic liposomes suppresses the angiogenesis in a dorsal air sac mouse model., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
44.
Jose Mario Barichello, Shinji Kizuki, Tatsuaki Tagami, T Asai, Tatsuhiro Ishida, K Hiroshi, N Oku and Hiroshi Kiwada : Application of vortex-mixing during lipoplex formation affects siRNA-liposome association, siRNA internalization in cells and the RNAi effect., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
45.
M Matsushita, (名) Asai, Y Suzuki, Tatsuhiro Ishida, N Maeda, T Dewa, Hiroshi Kiwada, (名) Nango and N Oku : Antiangiogenic effect by Argonaute2 knockdown used polycation liposome., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
46.
Tatsuhiro Ishida and Hiroshi Kiwada : Improvement of tumor targeting therapy with nanocarriers by changing the tumor microenvironment., 11th Liposome Research Days conference, Yokohama, Jul. 2008.
47.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : Accelerated blood clearance (ABC) phenomenon on PEGylated nanocarrier: Unexpected immune-reaction against PEGylated liposome., International Symposium on ``Nanotoxicology Assessment and Biomedical, Environmental Application of Fine Particles and Nanotubes'' (ISNT2008), Sapporo, Jun. 2008.
48.
Tatsuhiro Ishida and Hiroshi Kiwada : Unexpected immunological response against sterically stabilized, PEGylated, liposome after intravenous injection., International Symposium on ``Nanotoxicology Assessment and Biomedical, Environmental Application of Fine Particles and Nanotubes'' (ISNT2008), Sapporo, Jun. 2008.
49.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Design, preparation and directional insertion of peptides into lipid bilayer membrane and heir application for the preparation of liposome of which surface could be coated by externally added antibody, International symposium on system cell engineering by multi-scale manipulation, Nagoya, Nov. 2007.
50.
H Miyauchi, K Ichikawa, T Urakami, S Yonezawa, K Shimizu, Tatsuhiro Ishida, Hiroshi Kiwada, T Asai and N Oku : Antigen-conjugated liposomes enhanced hyposensitization immune therapy with extra-low doses., 34th Annual Meeting & Exposition of the Controlled Release Society, USA, Jul. 2007.
51.
T Asai, Y Suzuki, S Matsushita, J Yokota, Tatsuhiro Ishida, Hiroshi Kiwada and N Oku : Development of antineovascular RNAi cancer therapy targeting to Argonaute 2., American Society of Gene Therapy's 10th annual Meeting, Seattle USA, May 2007.
52.
Barichello M. Jose, Tatsuhiro Ishida, Soares A. L. Luiz, Shinji Kizuki, Tatsuaki Tagami, Kiyomi Hirose, Hideo Kobayashi, Kouichi Hashimoto, Hiroshi Kikuchi and Hiroshi Kiwada : Agitation during siRNA-cationic liposomes (CL) complex formation is a key step to improve in vitro RNA interference by lipofection., American Society of Gene Therapy's 10th annual Meeting, Seattle USA, May 2007.
53.
Tatsuhiro Ishida, Tatsuaki Tagami, Barichello M. Jose, Kiyomi Hirose, Naoshi Yamazaki, Tomohiro Asai, Naoto Oku and Hiroshi Kiwada : Argonaute2 (Ago2) gene silencing by liposomal transfection with siRNA for Ago2 induces apoptosis on HT1080 cells and HUVECs., American Society of Gene Therapy's 10th annual Meeting, Seattle USA, May 2007.
54.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Mutant coat proteins of Pf3 bacteriophage as models of membrane proteins and their interactions with lipid bilayer membrane, Pre-Satellite Meeting of the 3rd Pharmaceutical Sciences World Congress (PSWC 2007) for and by Ph.D. students and postdoctoral fellows, Amsterdam, Apr. 2007.
55.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Mutant coat proteins of Pf3 bacteriophage as models of membrane proteins and their interactions with lipid bilayer membrane, International symposium on system cell engineering by multi-scale manipulation, Nagoya, Nov. 2006.
56.
Tatsuhiro Ishida and Hiroshi Kiwada : Immunological responses to liposome carriers, The 1st FIP-APSTJ Joint Work Shop on Gene Delivery, Sapporo, Jul. 2006.
57.
Tatsuhiro Ishida, Kazutaka Atobe, Wang Xinyu and Hiroshi Kiwada : The influence of administration of PEGylated liposomal doxorubicin as a first dose on induction of accelerated blood clearance (ABC) phenomenon., 8th US-Japan symposium on drug delivery systems, Hawaii, Dec. 2005.
58.
Tatsuhiro Ishida, Masako Ichihara, Masae Harada, Shuntarou Kashima, Wang Xinyu and Hiroshi Kiwada : IgM secreted in response to the first injection of PEGylated liposomes involves in induction of the ABC phenomenon., 31st Annual Meeting and Exposition of the Controlled Release Society., Hawaii, Jun. 2004.
59.
Hiroshi Kiwada and Tatsuhiro Ishida : Immunological responses to nano-drug carriers., The 2nd Japan-Korea Joint Symposium on Drug Delivery and Therapy, Kyoto, May 2004.
60.
Tatsuhiro Ishida, Masako Ichihara, Shuntarou Kashima, Wang Xinyu and Hiroshi Kiwada : IgM secreted in response to injection of PEGylated liposomes causes accelerated blood clearance of a subsequent dose., 9th Liposome Research Days Conference, Hsinchu, Taiwan, May 2004.
Chihiro Katoh, Lila Selim Ahmed Ali Abu Amr, Hidenori ANDO, Tatsuhiro Ishida and Hiroshi Kiwada : 悪性胸膜中皮腫治療のための核酸搭載カチオニックリポソームの胸腔内投与とその動態評価, 第36回生体膜と薬物の相互作用シンポジウム, Nov. 2014.
Eri Hondoh, Yu Mima, Yosuke Hashimoto, Tatsuhiro Ishida and Hiroshi Kiwada : PEGASYS投与により誘導されるanti-PEG IgMのPEG修飾タンパクに対する反応性, 第52回日本薬学会・日本薬剤師会・日本病院薬剤師会中国四国支部学術大会, Oct. 2013.
24.
Chihiro Katoh, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : In vivo imaging systemを用いたリポソーム胸腔内投与後の体内動態の評価, 第52回日本薬学会・日本薬剤師会・日本病院薬剤師会中国四国支部学術大会, Oct. 2013.
Eri Hondoh, Yu Mima, Yosuke Hashimoto, Tatsuhiro Ishida and Hiroshi Kiwada : PEG修飾タンパク投与時に分泌されるanti-PEG IgMによるABC現象の誘導, ナノライフサイエンス・オープンセミナー2013, Sep. 2013.
32.
Chihiro Katoh, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : 脂質組成の違いが与える胸腔内投与後のリポソームの体内動態への影響, ナノライフサイエンス・オープンセミナー2013, Sep. 2013.
33.
Taichi Hasui, Miho Nishio, Tatsuhiro Ishida and Hiroshi Kiwada : マイクロアレイ解析によるOxaliplatin封入PEG修飾リポソーム製剤治療後の腫瘍内遺伝子発現変化の検討, ナノライフサイエンス・オープンセミナー2013, Aug. 2013.
34.
Kohsuke Takahashi, Hiroyuki Nakamura, Miho Nishio, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin封入PEG修飾リポソーム頻回投与による抗腫瘍効果の検討, ナノライフサイエンス・オープンセミナー2013, Aug. 2013.
Ami Imoto, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : 抗PEG-IgMを分泌する脾臓B細胞に関する検討, 創剤フォーラム第18回若手研究会, Nov. 2012.
55.
Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : Gene knockdown of thymidylate synthase via RNA interference modulates the antitumor efficacy of pemetrexed in malignant mesothelioma xenograft model, 創剤フォーラム第18回若手研究会, Nov. 2012.
56.
E Alaaeldin, Lila Selim Ahmed Ali Abu Amr, Hiroyuki Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : Effect of oxaliplatin on anti-PEG IgM and cytokine induced by IV injection of siRNA/PEG-cationic liposome complex., 創剤フォーラム第18回若手研究会, Nov. 2012.
Masako Ichihara, Taro Shimizu, Ami Imoto, Tatsuhiro Ishida and Hiroshi Kiwada : 脾臓辺縁帯B細胞がABC現象における抗PEG IgM分泌細胞である, 日本薬剤学会第27年会, May 2012.
85.
Yosuke Hashimoto, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : PEG修飾リポソームの体内動態変動因子anti-PEG IgMの定量評価系の構築, 日本薬剤学会第27年会, May 2012.
86.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : 脾臓辺縁帯B細胞によるPEG修飾リポソーム輸送現象を利用した免疫誘導の試み, 日本薬剤学会第27年会, May 2012.
87.
Hirotaka Kira, Naoto Moriyoshi, Tatsuhiro Ishida, Hiroshi Kiwada and Noriaki Minakawa : ケミカルツールを用いたRNA干渉発現の分子認識機構の解明, 日本薬学会第132年会, Mar. 2012.
88.
Tomoko Okada, Yusuke Doi, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : フッ化ピリミジン系経口抗癌剤S-1がPEG修飾カチオニックリポソームの腫瘍移行性に与える影響, 日本薬学会第132年会, Mar. 2012.
89.
Hiroyuki Nakamura, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : S-1とオキサリプラチン封入PEG修飾リポソームの併用療法におけるリポソームの体内動態の変化に関する検討, 日本薬学会第132年会, Mar. 2012.
90.
Tatsuhiro Ishida and Hiroshi Kiwada : 腫瘍内微小環境の能動的制御に基づくsiRNAデリバリー技術の開発とがん治療への展開, 日本薬学会第132年会, Mar. 2012.
91.
Takamitsu Kojima, Naoto Moriyoshi, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada and Noriaki Minakawa : 4'-チオDNAを利用した遺伝子発現抑制法の開発, 日本薬学会第132年会, Mar. 2012.
92.
Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : PEG修飾リポソームと脾臓辺縁帯B 細胞との相互作用に関する検討, 日本薬学会第132年会, Mar. 2012.
Tomoko Okada, Yusuke Doi, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : フッ化ピリミジン系経口抗癌剤がPEG修飾カチオニックリポソームの腫瘍移行性に与える影響, 第20回DDSカンファランス, Sep. 2011.
Masako Ichihara, Ami Imoto, Yuki Hashiguchi, Yumi Uehara, Tatsuhiro Ishida and Hiroshi Kiwada : PEG修飾リポソーム投与マウス脾臓の抗PEG IgM分泌における役割, 日本薬剤学会第25年会, May 2010.
134.
Lila Selim Ahmed Ali Abu Amr, Yusuke Doi, Kazuya Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor, 日本薬剤学会第25年会, May 2010.
135.
Yusuke Doi, Haruna Matsumoto, Tomoko Okada, Tatsuhiro Ishida and Hiroshi Kiwada : ナノキャリアに対するEPR効果に寄与する腫瘍内微小環境変化に関する研究:S-1の低用量頻回投与(metronomic chemotherapy)から得られた知見, 日本薬学会第130年会, Mar. 2010.
136.
Takahiro Iwaki, Tatsuaki Tagami, Kazuya Nakamura, Tatsuhiro Ishida and Hiroshi Kiwada : RNAi効果発現に対するLipoplexへのPEG修飾の影響に関する検討, 日本薬学会第130年会, Mar. 2010.
137.
Yuki Hashiguchi, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : ABC現象における抗PEG-IgM分泌とMZ-B細胞の活性化について, 日本薬学会第130年会, Mar. 2010.
138.
Yumi Uehara, Tatsuaki Tagami, Tatsuhiro Ishida and Hiroshi Kiwada : ABC現象回避を目的としたlipoplexに対するpolymer修飾剤polyglycerolの有効性評価, 日本薬学会第130年会, Mar. 2010.
139.
Kazuya Nakamura, Mariko Matsunaga, Takahiro Iwaki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : テガフール製剤S-1とBcl-2標的siRNA封入リポソームの併用によるdouble modulation therapyの有用性, 日本薬学会第130年会, Mar. 2010.
140.
Haruna Matsumoto, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : がん細胞種が及ぼす抗がん剤封入リポソームの腫瘍移行性とその治療効果への影響, 日本薬学会第130年会, Mar. 2010.
Tomoko Okada, 土井 祐輔, Lila Selim Ahmed Ali Abu Amr, Tatsuhiro Ishida and Hiroshi Kiwada : フッ化ピリミジン系経口抗癌剤とOxaliplatin封入PEG修飾カチオニックリポソーム併用による抗腫瘍効果の検討, 第48回日本薬学会・日本薬剤師会・日本病院薬剤師会 中国四国支部学術大会, Nov. 2009.
Lila Selim Ahmed Ali Abu Amr, Shinji Kizuki, Yusuke Doi, Takuya Suzuki, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin encapsulated in PEG-coated cationic liposomes induces significant tumor growth suppression via dual-targeting approach in a murine solid tumor model, 第25回日本DDS学会, Jul. 2009.
Tatsuhiro Ishida, 浅井 知浩, 奥 直人 and Hiroshi Kiwada : Argonaute2標的siRNAリポプレックスを用いたがん治療戦略, 日本薬剤学会第24年会 学術シンポジウム4 RNAi医薬の展望 –From Bench to Bedside -, May 2009.
161.
Mariko Matsunaga, Kazuya Nakamura, Takuya Suzuki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : フッ化ピリミジン系経口抗がん剤併用によるsiRNA-lipoplexの腫瘍移行向上の試み, 日本薬剤学会第24年会, May 2009.
162.
Lila Selim Ahmed Ali Abu Amr, Takuya Suzuki, Yusuke Doi, Tatsuhiro Ishida and Hiroshi Kiwada : Oxaliplatin targeting to angiogenic vessels by PEG-coated cationic liposomes suppresses the angiogenesis in a dorsal air sac mouse model., 日本薬剤学会第24年会, May 2009.
163.
Shinji Kizuki, Tatsuaki Tagami, Michi Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : siRNAリポプレックスのエンドソーム脱出ならびにキャリアからのsiRNA解離過程の評価, 日本薬学会第129年会, Mar. 2009.
Takuya Suzuki, Kazuya Nakamura, Tatsuaki Tagami, Kiyomi Hirose, Jose Mario Barichello, 浅井 知浩, Tatsuhiro Ishida, 奥 直人 and Hiroshi Kiwada : Argonaute2 (Ago2)標的siRNA-lipoplexを用いたin vivo抗腫瘍効果, 第24回日本DDS学会, Jun. 2008.
187.
Kazuya Nakamura, Takuya Suzuki, Tatsuaki Tagami, Jose Mario Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : siRNA―カチオニックリポソーム複合体の表面荷電が新生血管親和性に及ぼす影響, 第24回日本DDS学会, Jun. 2008.
188.
Kazuya Nakamura, Takuya Suzuki, Tatsuaki Tagami, Jose Mario Barichello, Tatsuhiro Ishida and Hiroshi Kiwada : siRNA-カチオニックリポソーム複合体の新生血管親和性向上の要因の解明に関する研究, 日本薬剤学会第23年会, May 2008.
189.
Taro Shimizu, Kousuke Nawata, Kazushige Naruke, Tatsuhiro Ishida and Hiroshi Kiwada : ポリエチレングリコール修飾タンパク投与による抗PEG IgM誘導に関する研究, 日本薬剤学会第23年会, May 2008.
190.
Shinji Kizuki, Jose Mario Barichello, Tatsuaki Tagami, 菊池 寛, Tatsuhiro Ishida and Hiroshi Kiwada : siRNAリポプレックスの新規調製法がもたらす細胞内取り込み機構の変化及びRNAi効果に与える影響に関する研究, 日本薬剤学会第23年会, May 2008.
191.
Yusuke Doi, Yurie Hira, Tatsuhiro Ishida and Hiroshi Kiwada : S-1を用いたlow dose chemotherapyによって生ずる腫瘍内微少環境の変化が与えるリポソームの腫瘍内動態変化に関する検討, 日本薬剤学会第23年会, May 2008.
192.
Ayumi Hagi, Tatsuaki Tagami, Tatsuhiro Ishida and Hiroshi Kiwada : がん細胞およびがん新生血管に親和性を有するPEG修飾Cationic liposomeの開発に関する検討, 日本薬剤学会第23年会, May 2008.
193.
Kaori Shimoda, Kazutaka Atobe, 浅井 知浩, Tatsuhiro Ishida, 菊池 寛, 奥 直人 and Hiroshi Kiwada : 抗がん剤リポソーム製剤化検討」資料収集および「腫瘍細胞および腫瘍新生血管を標的とした抗MT1-MMP抗体修飾リポソームの有用性に関する検討, 日本薬剤学会第23年会, May 2008.
194.
Takuya Suzuki, Kazuya Nakamura, Tatsuaki Tagami, Kiyomi Hirose, M J Barichello, 浅井 知浩, Tatsuhiro Ishida, 菊池 寛, 奥 直人 and Hiroshi Kiwada : Argonaute2(Ago2)標的siRNA腫瘍内直接投与による抗がん効果の検討, 日本薬剤学会第23年会, May 2008.
195.
Tatsuaki Tagami, Kazuya Nakamura, Taro Shimizu, Tatsuhiro Ishida and Hiroshi Kiwada : PEG修飾siRNAリポプレックス投与時におけるanti-PEG IgM分泌に関する検討, 日本薬剤学会第23年会, May 2008.
Improvement of tumor-targeting therapy with nanocarrier system by changing the tumor microenvironment. (Project/Area Number: 24390010 )
Development of novel vaccine system with nanocarrier (Project/Area Number: 23659082 )
Anti-PEG Immunity upon nucleic acid delivery (Project/Area Number: 23390012 )
Evaluation on EPR effect and active control of EPR effect for achieving efficient tumor accumulation of nanocarrier delivery system (Project/Area Number: 21689002 )
Study for Interaction of Biological Milieu with Nano-drug Carrier system (Project/Area Number: 20390013 )