Co-reporter:Tatsunori Hirotsu;Irhan Ibrahim Abu Hashim;Koki Wada;Taishi Higashi;Shogo Misumi;Keiichi Motoyama
Molecular Pharmaceutics February 6, 2017 Volume 14(Issue 2) pp:368-376
Publication Date(Web):December 29, 2016
DOI:10.1021/acs.molpharmaceut.6b00678
Polyethylene glycol (PEG) modification (PEGylation) is one of the best approaches to improve the stabilities and blood half-lives of protein drugs; however, PEGylation dramatically reduces the bioactivities of protein drugs. Here, we present “self-assembly PEGylation retaining activity” (SPRA) technology via a host–guest interaction between PEGylated β-cyclodextrin (PEG-β-CyD) and adamantane-appended (Ad) proteins. PEG-β-CyD formed stable complexes with Ad-insulin and Ad-lysozyme to yield SPRA-insulin and SPRA-lysozyme, respectively. Both SPRA-proteins showed high stability against heat and trypsin digest, comparable with that of covalently PEGylated protein equivalents. Importantly, the SPRA-lysozyme possessed ca. 100% lytic activity, whereas the activity of the covalently PEGylated lysozyme was ca. 23%. Additionally, SPRA-insulin provided a prolonged and peakless blood glucose profile when compared with insulin glargine. It also showed no loss of activity. In contrast, the covalently PEGylated insulin showed a negligible hypoglycemic effect. These findings indicate that SPRA technology has potential as a generic method, surpassing conventional PEGylation methods for proteins.Keywords: adamantine; cyclodextrin; polyethylene glycol; protein; supramolecular host−guest interaction;
Co-reporter:Khaled M. Elamin;Yuki Yamashita
Journal of Inclusion Phenomena and Macrocyclic Chemistry 2017 Volume 89( Issue 3-4) pp:333-342
Publication Date(Web):10 October 2017
DOI:10.1007/s10847-017-0757-x
In recent years, colorectal cancer has gained great attention among various types of cancers. We previously synthesized folate-appended methyl-β-cyclodextrin (FA-M-β-CyD) as a novel autophagic antitumor agent. In this study, to further elucidate the impact of FA-M-β-CyD as an antitumor agent, we evaluated cytotoxic activity and the antiproliferative effect in colon cancer cells and colorectal cancer model mice, respectively. As a result, FA-M-β-CyD showed potent cytotoxic activity in HCT116 cells, a human colon cancer cell line, through folate receptor-α (FR-α)-mediated cellular uptake. In addition, FA-M-β-CyD elicited autophagosome formation and induced mitophagy in HCT116 cells. Importantly, FA-M-β-CyD drastically reduced the number of tumor polyps in colorectal cancer model mice induced by azoxymethane/dextran sodium sulfate after intravenous injections once a week for 4 weeks. These results suggest that FA-M-β-CyD has the antiproliferative effect in the colorectal cancer, due to the FR-α-mediated endocytosis and mitophagy induction.
Co-reporter:Taishi Higashi, Naoko Ohshita, Tatsunori Hirotsu, Yoshihito Yamashita, ... Hidetoshi Arima
Journal of Pharmaceutical Sciences 2017 Volume 106, Issue 5(Volume 106, Issue 5) pp:
Publication Date(Web):1 May 2017
DOI:10.1016/j.xphs.2017.01.002
Antibodies often have poor physicochemical stability during storage and transport, which is a serious drawback for the development of antibody-based drugs. In this study, we prepared polypseudorotaxane (PPRX) hydrogels consisting of cyclodextrins (CyDs) and polyethylene glycol, and evaluated them as stabilizers for commercially available antibody-based drugs. α-CyD and γ-CyD formed PPRX hydrogels with polyethylene glycol (molecular weight 20,000 Da) in the presence of antibody-based drugs such as omalizumab, palivizumab, panitumumab, and ranibizumab. Importantly, both α- and γ-CyD PPRX hydrogel formulations provided high stabilizing effects (ca. 100%) to the all antibody-based drugs used in this study. Furthermore, approximately 100% of the binding activity of omalizumab to the immunoglobulin E receptor was retained after the release from the hydrogels. Plasma levels of omalizumab after subcutaneous injection of the γ-CyD PPRX hydrogel to rats were equivalent to those of omalizumab alone. According to the results of blood chemistry tests, the weights of organs and histological observations α- and γ-CyD PPRX hydrogels induced no serious adverse effects. These results suggest that CyD PPRX hydrogels are useful as safe and promising stabilizing formulations for antibody-based drugs.
Co-reporter:Taishi Higashi, Jun Li, Xia Song, Jingling Zhu, Masatoshi Taniyoshi, Fumitoshi Hirayama, Daisuke Iohara, Keiichi Motoyama, and Hidetoshi Arima
ACS Macro Letters 2016 Volume 5(Issue 2) pp:158
Publication Date(Web):January 11, 2016
DOI:10.1021/acsmacrolett.5b00927
We demonstrated a new strategy for efficient preparation of polypseudorotaxanes (PpRXs) and polyrotaxanes (PRXs) with cyclodextrin derivatives, 2,6-di-O-methyl-cyclodextrins (DM-CyDs), by utilizing the cloud points of DM-CyDs. DM-α-CyD and DM-β-CyD formed PpRXs with polyethylene glycol (PEG) and polypropylene glycol (PPG) in water at >50 °C and >35 °C, respectively, but did not at room temperature. Meanwhile, randomly methylated β-CyD (RM-β-CyD) and 2,3,6-tri-O-methyl-β-CyD (TM-β-CyD) did not form PpRX with PPG at higher temperature. The driving force of thermoresponsive formation of DM-CyD PpRXs was derived from hydrophobic interaction of methyl groups and a hydrogen bond of hydroxyl groups formed by adjacent DM-CyD molecules. Furthermore, in one pot, DM-CyD PRXs were synthesized by capping the PpRXs with bulky ends in high yields.
Co-reporter:Ayumu Ohyama, Taishi Higashi, Keiichi Motoyama, and Hidetoshi Arima
Bioconjugate Chemistry 2016 Volume 27(Issue 3) pp:521
Publication Date(Web):December 30, 2015
DOI:10.1021/acs.bioconjchem.5b00545
We previously reported that folate-polyethylene glycol (PEG)-appended dendrimer (generation 3)/α-cyclodextrin conjugate (Fol-PαC (G3)) shows folate receptor-α (FR-α)-overexpressing tumor cell-selective in vitro siRNA transfer activity. However, Fol-PαC (G3)/siRNA complex did not induce a significant in vivo RNAi effect after intravenous administration to tumor-bearing mice, possibly resulting from immediate dissociation of the complex in blood. Herein, to develop the novel siRNA carrier having high blood circulating ability, high in vivo siRNA transfer activity, and high safety profile, we newly prepared Fol-PαCs with higher generation (G4) and evaluated their potential as tumor-targeting siRNA carriers in vitro and in vivo. Fol-PαC (G4, average degree of substitution of α-cyclodextrin (DSC) 2.9, average degree of substitution of folate-PEG (DSF) 2)/siRNA complex had the prominent RNAi effect through adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to cytoplasm with negligible cytotoxicity. Importantly, Fol-PαC (G4, DSC2.9, DSF2) improved the serum stability, blood circulating ability, and in vivo RNAi effects of siRNA, compared to Fol-PαC (G3). Furthermore, Fol-PαC (G4, DSC2.9, DSF2) complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed the tumor growth compared to control siRNA complex. These results suggest that Fol-PαC (G4, DSC2.9, DSF2) has the potential as a novel tumor-targeting siRNA carrier in vitro and in vivo.
Co-reporter:Keiichi Motoyama; Ryosuke Mitsuyasu; Chiho Akao; Irhan Ibrahim Abu Hashim; Nana Sato; Takahiro Tanaka; Taishi Higashi
Molecular Pharmaceutics 2015 Volume 12(Issue 9) pp:3129-3136
Publication Date(Web):August 13, 2015
DOI:10.1021/mp500814f
NF-κB and its associated pathways are complicatedly concerned about hepatic homeostasis. Discriminating inhibition of NF-κB signaling has been expected to treat various liver diseases including fulminant hepatitis. To clarify the potential use of thioalkylated mannose-appended dendrimer (generation 3; G3) conjugates with α-cyclodextrin with average degree of substitution of mannose (DSM4) (Man-S-α-CDE (G3, DSM4)) as a novel antigen presenting cell (APC)-specific siRNA carrier, we evaluated the RNAi effect of NF-κB p65 siRNA (sip65) complex with Man-S-α-CDE (G3, DSM4) both in vitro and in vivo. Man-S-α-CDE (G3, DSM4)/sip65 complex significantly suppressed NF-κB p65 mRNA expression and nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated NR8383 cells, a rat alveolar macrophage cell line, by adequate physicochemical properties and mannose receptor-mediated cellular uptake. Intravenous injection of Man-S-α-CDE (G3, DSM4)/sip65 complex extended the survival rate of LPS-induced fulminant hepatitis model mice. In addition, intravenous administration of Man-S-α-CDE (G3, DSM4)/sip65 complex had the potential to induce the in vivo RNAi effect by significant suppression of mRNA expression of NF-κB p65 and inflammatory cytokines in the liver of fulminant hepatitis model mice induced by LPS/d-galactosamine (d-Gal) without any significant side effects. Also, the serum levels of enzymes were significantly attenuated by injection of Man-S-α-CDE (G3, DSM4)/sip65 complex in fulminant hepatitis model mice. Collectively, these results suggest that Man-S-α-CDE (G3, DSM4) has the potential as a novel APC-selective sip65 carrier for the treatment of LPS/d-Gal-induced fulminant hepatitis in mice.
Co-reporter:Taishi Higashi;Anna Tajima;Naoko Ohshita;Tatsunori Hirotsu
AAPS PharmSciTech 2015 Volume 16( Issue 6) pp:1290-1298
Publication Date(Web):2015 December
DOI:10.1208/s12249-015-0309-x
To achieve the potent therapeutic effects of human immunoglobulin G (IgG), highly concentrated formulations are required. However, the stabilization for highly concentrated human IgG is laborious work. In the present study, to investigate the potentials of polypseudorotaxane (PPRX) hydrogels consisting of polyethylene glycol (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as pharmaceutical materials for highly concentrated human IgG, we designed the PPRX hydrogels including human IgG and evaluated their pharmaceutical properties. The α- and γ-CyDs formed PPRX hydrogels with PEG (M.W. 20,000) even in the presence of highly concentrated human IgG (>100 mg/mL). According to the results of 1H-NMR, powder X-ray diffraction, and Raman microscopy, the formation of human IgG/CyD PPRX hydrogels was based on physical cross-linking arising from their columnar structures. The release profiles of human IgG from the hydrogels were in accordance with the non-Fickian diffusion model. Importantly, the stabilities of human IgG included into the hydrogels against thermal and shaking stresses were markedly improved. These findings suggest that PEG/CyD PPRX hydrogels are useful to prepare the formulation for highly concentrated human IgG.
Co-reporter:Chiho Akao, Takahiro Tanaka, Risako Onodera, Ayumu Ohyama, Nana Sato, Keiichi Motoyama, Taishi Higashi, Hidetoshi Arima
Journal of Controlled Release 2014 Volume 193() pp:35-41
Publication Date(Web):10 November 2014
DOI:10.1016/j.jconrel.2014.07.004
The purpose of the present study is to treat lipopolysaccharide (LPS)-induced fulminant hepatitis by NF-κB decoy complex with fucose-appended dendrimer (generation 2; G2) conjugate with α-cyclodextrin (Fuc-S-α-CDE (G2)). Fuc-S-α-CDE (G2, average degree of substitution of fucose (DSF2))/NF-κB decoy complex significantly suppressed nitric oxide and tumor necrosis factor-α (TNF-α) production from LPS-stimulated NR8383 cells, a rat alveolar macrophage cell line, by adequate physicochemical properties and fucose receptor-mediated cellular uptake. Intravenous injection of Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex extended the survival of LPS-induced fulminant hepatitis model mice. In addition, Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex administered intravenously highly accumulated in the liver, compared to naked NF-κB decoy alone. Furthermore, the liver accumulation of Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex was inhibited by the pretreatment with GdCl3, a specific inhibitor of Kupffer cell uptake. Also, the serum aspartate aminotransferase, alanine aminotransferase and TNF-α levels in LPS-induced fulminant hepatitis model mice were significantly attenuated by the treatment with Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex, compared with naked NF-κB decoy alone. Taken together, these results suggest that Fuc-S-α-CDE (G2, DSF2) has the potential for a novel Kupffer cell-selective NF-κB decoy carrier for the treatment of LPS-induced fulminant hepatitis in mice.
Co-reporter:Tatsunori Hirotsu;Taishi Higashi
Journal of Inclusion Phenomena and Macrocyclic Chemistry 2014 Volume 80( Issue 1-2) pp:107-112
Publication Date(Web):2014 October
DOI:10.1007/s10847-014-0407-5
Recently, a large number of peptides and proteins have been utilized as active pharmaceutical ingredients in the clinical field. However, the stability of peptide and protein drugs is often low. In addition, some peptides and proteins adsorb onto glass or polypropylene tube. In the present study, to improve these pharmaceutical properties of peptides and proteins, we newly prepared glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with insulin, a model protein drug, and evaluated its enzymatic or thermal stability and adsorption onto glass or polypropylene tube. The insulin conjugate with GUG-β-CyD was successfully prepared by condensation of amine group of insulin and carboxyl group of GUG-β-CyD. Circular dichroism spectra showed that the secondary structure of insulin in this conjugate was retained. Adsorption of insulin onto glass or polypropylene tube was decreased by the conjugation with GUG-β-CyD. Moreover, enzymatic and thermal stabilities of the conjugate were higher than those of insulin and the mixture of insulin and GUG-β-CyD. These results suggest that insulin conjugation with GUG-β-CyD could improve the pharmaceutical properties of insulin.
Co-reporter:Ayaka Okamatsu, Keiichi Motoyama, Risako Onodera, Taishi Higashi, Takahiro Koshigoe, Yasutaka Shimada, Kenjiro Hattori, Tomoko Takeuchi, and Hidetoshi Arima
Bioconjugate Chemistry 2013 Volume 24(Issue 4) pp:724
Publication Date(Web):March 4, 2013
DOI:10.1021/bc400015r
A large number of antitumor drug delivery carriers based on passive targeting and/or active targeting have been developed. However, encapsulation of antitumor drugs into these drug carriers is often complicated, and antitumor activities of these targeting systems are not satisfactory. In the present study, we first prepared heptakis-6-folic acid (FA)-appended β-cyclodextrin (β-CyD) possessing two caproic acids between FA and a β-CyD molecule as a spacer (Fol-c2-β-CyD) and evaluated the potential as a novel tumor targeting carrier for antitumor drugs through a complexation. Fol-c2-β-CyD formed an inclusion complex with doxorubicin (DOX) at a 1:1 molar ratio with a markedly high stability constant (>106 M–1). Cellular uptake of DOX was increased by the addition of Fol-c2-β-CyD in KB cells, a folate receptor-α (FR-α)-positive cell line. Additionally, Fol-c2-β-CyD increased in vitro antitumor activities of antitumor drugs such as DOX, vinblastine (VBL), and paclitaxel (PTX) in KB cells, but not in A549 cells, a FR-α-negative cell line. The complex of DOX with Fol-c2-β-CyD markedly increased antitumor activity of DOX, not only after intratumoral administration but also after intravenous administration to mice subcutaneously inoculated Colon-26 cells, a FR-α-positive cell line. These findings suggest that Fol-c2-β-CyD could be useful as a promising antitumor drug carrier.
Co-reporter:Ayaka Okamatsu, Keiichi Motoyama, Risako Onodera, Taishi Higashi, Takahiro Koshigoe, Yasutaka Shimada, Kenjiro Hattori, Tomoko Takeuchi, and Hidetoshi Arima
Biomacromolecules 2013 Volume 14(Issue 12) pp:
Publication Date(Web):October 31, 2013
DOI:10.1021/bm401340g
We reported that per-6-folic acid (FA)-appended β-cyclodextrin (β-CyD) possessing two caproic acids between FA and a β-CyD molecule as a spacer (Fol-c2-β-CyD) could be useful as a promising antitumor drug carrier. However, the effects of the cavity size and the spacer length on the carrier ability are not still known. In this study, we designed and evaluated the FA-appended three kinds of CyDs possessing a caproic acid as a spacer between FA and a CyD molecule (Fol-c1-CyDs) as a tumor targeting carrier for antitumor drugs. The stability constant of the Fol-c1-β-CyD/doxorubicin (DOX) complex was much higher than those of Fol-c1-α-CyD and Fol-c1-γ-CyD at pH 7.3. Antitumor activity of DOX was increased by the complexation with Fol-c1-β-CyD, but not with Fol-c1-α-CyD or Fol-c1-γ-CyD in KB cells, a folate receptor-α-positive cell line. Also, Fol-c1-β-CyD increased antitumor activities of paclitaxel and vinblastine, but not 5-fluorouracil. Furthermore, Fol-c1-β-CyD accelerated cellular uptake of DOX and inhibited its efflux from KB cells. The Fol-c1-β-CyD/DOX complex showed much higher antitumor activity than DOX alone after intratumoral and intravenous administrations to tumor-bearing mice with a negligible change of the blood chemistry values. These findings suggest that Fol-c1-β-CyD could be useful as a tumor-selective carrier for antitumor drugs.
Co-reporter:Hidetoshi Arima, Ayumi Yoshimatsu, Haruna Ikeda, Ayumu Ohyama, Keiichi Motoyama, Taishi Higashi, Akira Tsuchiya, Takuro Niidome, Yoshiki Katayama, Kenjiro Hattori, and Tomoko Takeuchi
Molecular Pharmaceutics 2012 Volume 9(Issue 9) pp:2591-2604
Publication Date(Web):July 26, 2012
DOI:10.1021/mp300188f
We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo.Keywords: conjugate; folate; PAMAM dendrimer; PEG; siRNA; α-cyclodextrin;
Co-reporter:Yuya Hayashi, Yoshimasa Mori, Shogo Yamashita, Keiichi Motoyama, Taishi Higashi, Hirofumi Jono, Yukio Ando, and Hidetoshi Arima
Molecular Pharmaceutics 2012 Volume 9(Issue 6) pp:1645-1653
Publication Date(Web):April 17, 2012
DOI:10.1021/mp200654g
To reveal the potential use of lactosylated-dendrimer (G3) conjugates with α-cyclodextrin (Lac-α-CDE (G3)) as novel hepatocyte-specific siRNA carriers in order to treat transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), we evaluated the RNAi effect of siRNA complexes with Lac-α-CDE (G3) both in vitro and in vivo. Herein, we targeted TTR gene expression because TTR-related FAP was often caused by amyloidogenic TTR (ATTR), which mainly expresses in hepatocytes. Lac-α-CDE (G3, average degree of substitution of lactose (DSL) 1.2)/siRNA complex had a potent RNAi effect against TTR gene expression through adequate physicochemical properties, asialoglycoprotein receptor (ASGP-R)-mediated cellular uptake, efficient endosomal escape and the delivery of the siRNA complex to cytoplasm, but not nucleus, with negligible cytotoxicity. Lac-α-CDE (G3, DSL 1.2)/siRNA complex had the potential to induce the in vivo RNAi effect after intravenous administration in the liver of mice. The blood chemistry values in the α-CDE (G3) and Lac-α-CDE (G3, DSL 1.2) systems were almost equivalent to those in the control system (5% mannitol solution). Taken together, these results suggest that Lac-α-CDE (G3, DSL 1.2) has the potential for a novel hepatocyte-selective siRNA carrier in vitro and in vivo, and has a possibility as a therapeutic tool for FAP to the liver transplantation.Keywords: cyclodextrin; dendrimer; familial amyloidotic polyneuropathy; hepatocyte-specific delivery; siRNA;
Co-reporter:Keiichi Motoyama, Kayoko Hayashida, Taishi Higashi, Hidetoshi Arima
Bioorganic & Medicinal Chemistry 2012 Volume 20(Issue 4) pp:1425-1433
Publication Date(Web):15 February 2012
DOI:10.1016/j.bmc.2011.12.060
Nonviral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. In this study, we examined whether polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2,000)-grafted α-cyclodextrin (α-CyD)/polyamidoamine dendrimer conjugate (PEG-α-CDE) with CyDs have the potential for the novel sustained release systems for plasmid DNA (pDNA). The PEG-α-CDE/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-α-CDE/CyDs PPRX systems, 20.6 mol of α-CyD and 11.8 mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively, consistent with in the PEG-dendrimer/CyDs systems. PEG-α-CDE/pDNA/α-CyD PPRX and PEG-α-CDE/pDNA/γ-CyD PPRX formed hexagonal and tetragonal columnar channels in the crystalline phase, respectively. In addition, the CyDs PPRX provided the sustained release of pDNA from PEG-α-CDE complex with pDNA at least 72 h in vitro. The release of pDNA from CyDs PPRX retarded as the volume of dissolution medium decreased. Furthermore, the PEG-α-CDE/γ-CyD PPRX system showed sustained transfection efficiency after intramuscular injection to mice at least for 14 days. These results suggest that the PEG-α-CDE/CyD PPRX systems are useful for novel sustained DNA release systems.Proposed scheme for gene transfer mechanism of PEG-α-CDE/γ-CyD PPRX suspension after intramuscular injection to mice.
Co-reporter:Taishi Higashi;Anna Tajima;Keiichi Motoyama
Journal of Pharmaceutical Sciences 2012 Volume 101( Issue 8) pp:2891-2899
Publication Date(Web):
DOI:10.1002/jps.23232
Abstract
In this study, to clarify the utility of polypseudorotaxane (PPRX) hydrogels composed of poly(ethylene glycol) (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as a sustained-release system for protein drugs, we prepared CyD PPRX hydrogels including lysozyme, and then the release profiles of lysozyme from these hydrogels and the release mechanisms were investigated. The α- and γ-CyD formed PPRX hydrogels by threading onto one PEG chain and two PEG chains, respectively. The formation of α- and γ-CyD PPRX hydrogels including lysozyme was based on physical cross-linking arisen from their columnar structures. The in vitro release rates of lysozyme were markedly decreased by the encapsulation into CyD PPRX hydrogels. In addition, when release data were plotted according to Korsmeyer–Peppas model, the exponent values (n) in the α- and γ-CyD systems had no statistically significant difference, suggesting that these release mechanisms were almost same. In conclusion, these results suggest that α- and γ-CyD PPRX hydrogels possess the potential as a sustained-release system for lysozyme. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2891–2899, 2012
Co-reporter:H Arima, M Arizono, T Higashi, A Yoshimatsu, H Ikeda, K Motoyama, K Hattori, T Takeuchi, F Hirayama and K Uekama
Cancer Gene Therapy 2012 19(5) pp:358-366
Publication Date(Web):March 9, 2012
DOI:10.1038/cgt.2012.9
We previously reported that polyamidoamine STARBURST dendrimer (generation 3, G3) (dendrimer) conjugate with α-cyclodextrin (α-CyD) having an average degree of substitution of 2.4 of α-CyD (α-CDE) provided remarkable aspects as novel carriers for DNA and small-interfering RNA. To develop novel α-CDE derivatives with tumor cell specificity, we prepared folate-appended α-CDEs (Fol-α-CDEs) and folate-polyethylene glycol (PEG)-appended α-CDEs (Fol-PαCs) with the various degrees of substitution of folate (DSF), and evaluated in vitro and in vivo gene transfer activity, cytotoxicity, cellular association and physicochemical properties. In vitro gene transfer activity of Fol-α-CDEs (G3, DSF 2, 5 or 7) was lower than that of α-CDE (G3) in KB cells, folate receptor (FR)-overexpressing cancer cells. Of the three Fol-PαCs (G3, DSF 2, 5 or 7), Fol-PαC (G3, DSF 5) had the highest gene transfer activity in KB cells. The activity of Fol-PαC (G3, DSF 5) was significantly higher than that of α-CDE (G3) in KB cells, but not in A549 cells, FR-negative cells. Negligible cytotoxicity of the plasmid DNA (pDNA) complex with Fol-PαC (G3, DSF 5) was observed in KB cells or A549 cells up to a charge ratio of 100/1 (carrier/pDNA). The cellular association of the pDNA complex with Fol-PαC (G3, DSF 5) could be mediated by FR on KB cells, resulting in its efficient cellular uptake. Fol-PαC (G3, DSF 5) had a higher binding affinity with folate-binding protein than α-CDE (G3), although the physicochemical properties of pDNA complex with Fol-PαC (G3, DSF 5) were almost comparable to that with α-CDE (G3), although the onset charge ratio and the compaction ability of Fol-PαC (G3, DSF 5) were slightly different. Fol-PαC (G3, DSF 5) tended to show a higher gene transfer activity than α-CDE (G3) 12 h after intratumoral administration in mice. These results suggest that Fol-PαC (G3, DSF 5), not Fol-α-CDEs, could be potentially used as a FR-overexpressing cancer cell-selective DNA carrier.
Co-reporter:Takayuki Anno;Keiichi Motoyama
Journal of Inclusion Phenomena and Macrocyclic Chemistry 2011 Volume 70( Issue 3-4) pp:339-344
Publication Date(Web):2011 August
DOI:10.1007/s10847-010-9868-3
The purpose of this study is to evaluate the potential use of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugates with 6-O-α-(4-O-α-d-glucuronyl)-d-glucosyl-β-cyclodextrin (GUG-β-CDE (G2)) having glucose as a spacer between dendrimer and cyclodextrin (CyD) as a novel gene transfer carrier. GUG-β-CDE (G2) was found to have lower hemolytic activity than dendrimer (G2), suggesting that GUG-β-CDE (G2) had lower local irritation than dendrimer (G2). Of GUG-β-CDEs (G2) having the various average degree of substitution (DS) of a glucuronylglycoside group, GUG-β-CDE (G2, DS 1.8) possessed much higher gene transfer activity than α-CDE (G2, DS 1.2) and β-CDE (G2, DS 1.3) in A549 and RAW264.7 cells, suggesting the crucial role of a spacer between dendrimer and CyD for high gene transfer activity of GUG-β-CDE (G2, DS 1.8). In sharp contrast to linear polyethyleneimine (10 kDa, PEI), GUG-β-CDE (G2, DS 1.8) had negligible cytotoxicity. These results suggest that GUG-β-CDE (G2, DS 1.8) could have the potential for a novel gene transfer carrier, compared to α-CDE (G2, DS 1.2), β-CDE (G2, DS 1.3) and PEI.
Co-reporter:Keiichi Motoyama;Yoshimasa Mori
Journal of Inclusion Phenomena and Macrocyclic Chemistry 2011 Volume 70( Issue 3-4) pp:333-338
Publication Date(Web):2011 August
DOI:10.1007/s10847-010-9842-0
The purpose of this study is to evaluate in vitro gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 3, G3) conjugates with α-cyclodextrin (α-CDE (G3)) bearing lactose (Lac-α-CDE) with various degrees of substitution of the lactose moiety (DSL) as a novel hepatocyte-selective carrier. Lac-α-CDE (G3, DSL 1.2) was found to have much higher gene transfer activity than α-CDE (G3), Lac-α-CDE (G2, DSL 2.6) and Lac-α-CDEs (G3, DSL 2.6, 4.1 and 6.1) in HepG2 cells, which are dependent on the expression of cell-surface asialoglycoprotein receptor (ASGP-R). Lac-α-CDE (G3, DSL 1.2) provided negligible cytotoxicity up to a charge ratio of 100 (carrier/pDNA) in HepG2 cells. These results suggest the potential use of Lac-α-CDE (G3, DSL 1.2) as a non-viral vector for gene delivery toward hepatocytes.
Co-reporter:Risako Onodera;Keiichi Motoyama
Journal of Inclusion Phenomena and Macrocyclic Chemistry 2011 Volume 70( Issue 3-4) pp:321-326
Publication Date(Web):2011 August
DOI:10.1007/s10847-010-9843-z
Methyl-β-cyclodextrin (M-β-CyD) is widely used as a raft disrupting agent through extraction of cholesterol from lipid rafts which are highly expressed in cell membranes of tumor cells, but it does not have tumor cell-selective action. Meanwhile, the widespread use of folic acid (FA) as a tumor-targeting ligand has been known, because folate receptor (FR) overexpresses in various kinds of epithelial tumor cells. In the present study, in order to obtain more tumor cell-selectivity and antitumor activity of M-β-CyD, we designed folate-appended M-β-CyD (FA-M-β-CyD), and evaluated its physicochemical properties and antitumor activity. The 1H-NMR study demonstrated that FA-M-β-CyD having average degree of substitution of FA (DSF) of 1.0 was prepared. In addition, FA-M-β-CyD (DSF 1.0) was found to be amorphous in a solid state and surface-active. Importantly, FA-M-β-CyD (DSF 1.0) had potent cytotoxicity, compared to M-β-CyD in KB cells, but not in A549 cells. These results suggest that FA-M-β-CyD (DSF 1.0) has the potential as a novel antitumor agent.
Co-reporter:Hidetoshi Arima, Shogo Yamashita, Yoshimasa Mori, Yuya Hayashi, Keiichi Motoyama, Kenjiro Hattori, Tomoko Takeuchi, Hirofumi Jono, Yukio Ando, Fumitoshi Hirayama, Kaneto Uekama
Journal of Controlled Release 2010 Volume 146(Issue 1) pp:106-117
Publication Date(Web):17 August 2010
DOI:10.1016/j.jconrel.2010.05.030
The purpose of this study is to evaluate in vitro and in vivo gene delivery efficiency of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugates with α-cyclodextrin (α-CDE (G2)) bearing lactose (Lac-α-CDE) with various degrees of substitution of the lactose moiety (DSL) as a novel hepatocyte-selective carrier in hepatocytes. Lac-α-CDE (DSL 2.6) was found to have much higher gene transfer activity than dendrimer, α-CDE, Lac-α-CDE (DSL 1.2, 4.6, 6.2 and 10.2) and lactosylated dendrimer (Lac-dendrimer, DSL 2.4) in HepG2 cells, which are dependent on the expression of cell-surface asialoglycoprotein receptor (ASGP-R), reflecting the cellular association of the plasmid DNA (pDNA) complexes. The physicochemical properties of pDNA complex with Lac-α-CDE (DSL 2.6) were almost comparable to that with α-CDE. Lac-α-CDE (DSL 2.6) provided negligible cytotoxicity up to a charge ratio of 150 in HepG2 cells. Lac-α-CDE (DSL 2.6) provided gene transfer activity higher than jetPEITM-Hepatocyte to hepatocytes with much less changes of blood chemistry values 12 h after intravenous administration in mice. These results suggest the potential use of Lac-α-CDE (DSL 2.6) as a non-viral vector for gene delivery toward hepatocytes.
Co-reporter:Toshihito Tsutsumi;Fumitoshi Hirayama;Kaneto Uekama
Journal of Pharmaceutical Sciences 2008 Volume 97( Issue 8) pp:3022-3034
Publication Date(Web):
DOI:10.1002/jps.21206
Abstract
The potential of starburst® polyamidoamine dendrimer (dendrimer, generation 3, G3) conjugate with α-cyclodextrin (α-CyD) having an average degree of substitution of 2.4 (α-CDE) as a novel carrier of short hairpin RNA (shRNA) expressing plasmid DNA (shpDNA) was evaluated and the shpDNA transfer activity of α-CDE was compared with that of dendrimer (G3). α-CDE formed a stable and condensed complex with shpDNA and induced a conformational transition of shpDNA in solution even in the low charge ratios. In addition, α-CDE markedly inhibited the enzymatic degradation of shpDNA by DNase I. The shpDNA complex with α-CDE at the charge ratio of 20/1 (α-CDE/shpDNA) elicited the most potent RNAi effects in cells transiently and stably expressing the GL3 and GL2 luciferase genes without cytotoxicity among the complexes with the various charge ratios. Besides, the RNAi effects were strikingly enhanced by the further addition of the adequate amounts of siRNA to the shpDNA complex with α-CDE. Taken together, the prominent RNAi effects of the shpDNA complex with α-CDE could be attributed to the stabilizing effect of α-CDE on enzymatic degradation of shpDNA and negligible cytotoxicity. These results suggest that α-CDE possesses the potential to be a novel carrier for shpDNA and siRNA. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 3022–3034, 2008
Co-reporter:Toshihito Tsutsumi, Fumitoshi Hirayama, Kaneto Uekama, Hidetoshi Arima
Journal of Controlled Release 2007 Volume 119(Issue 3) pp:349-359
Publication Date(Web):22 June 2007
DOI:10.1016/j.jconrel.2007.03.013
As the first step toward an evaluation of the potential use of the PAMAM dendrimer (G3) conjugate with α-cyclodextrin (α-CDE) for a small interfering RNA (siRNA) carrier, the ternary complexes of α-CDE or the transfection reagents such as Lipofactamine™ 2000 (L2), TransFast™ (TF) and Lipofectin™ (LF) with plasmid DNA (pDNA) and siRNA were prepared, and their RNAi effects, cytotoxicity, physicochemical properties and intracellular distribution were compared. Here the pGL2 control vector (pGL2) and pGL3 control vector (pGL3) encoding the firefly luciferase gene and the two corresponding siRNAs (siGL2 and siGL3) were used. The ternary complexes of pGL3/siGL3/α-CDE showed the potent RNAi effects with negligible cytotoxicity compared to those of the transfection reagents in various cells. α-CDE strongly interacted with both pDNA and siRNA, and suppressed siRNA degradation by serum, compared to those of the transfection reagents. α-CDE allowed fluorescent labeled siRNA to distribute in cytoplasm, whereas the transfection reagents resided in both nucleus and cytoplasm in NIH3T3 cells. Furthermore, the binary complex of siRNA/α-CDE provided the significant RNAi effect in NIH3T3 cells transiently and stably expressing luciferase gene. These results suggest that α-CDE may be utilized as a novel carrier for siRNA.
Co-reporter:Yuki Maeda, Keiichi Motoyama, Taishi Higashi, Yuka Horikoshi, Toru Takeo, Naomi Nakagata, Yuki Kurauchi, Hiroshi Katsuki, Yuki Kondo, Yoichi Ishitsuka, Tetsumi Irie, Takumi Era, Hidetoshi Arima
Asian Journal of Pharmaceutical Sciences (February 2016) Volume 11(Issue 1) pp:183-184
Publication Date(Web):February 2016
DOI:10.1016/j.ajps.2015.11.046
Co-reporter:Keiichi Motoyama, Chiho Akao, Ryosuke Mitsuyasu, Taishi Higashi, Hidetoshi Arima
Asian Journal of Pharmaceutical Sciences (February 2016) Volume 11(Issue 1) pp:152-153
Publication Date(Web):February 2016
DOI:10.1016/j.ajps.2015.11.080
Co-reporter:Mari Sugio, Tatsunori Hirotsu, Taishi Higashi, Keiichi Motoyama, Fumitoshi Hirayama, Kaneto Uekama, Hidetoshi Arima
Asian Journal of Pharmaceutical Sciences (February 2016) Volume 11(Issue 1) pp:120-121
Publication Date(Web):February 2016
DOI:10.1016/j.ajps.2015.11.097
Co-reporter:Hidetoshi Arima, Keiichi Motoyama, Taishi Higashi
Advanced Drug Delivery Reviews (August 2013) Volume 65(Issue 9) pp:1204-1214
Publication Date(Web):1 August 2013
DOI:10.1016/j.addr.2013.04.001
The widespread use of various cyclodextrin (CyD)-appended polymers and polyrotaxanes as gene carriers has been reported. Among the various polyamidoamine dendrimer (dendrimer) conjugates with CyDs (CDE), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution (DS) of 2.4 (α-CDE (G3, DS 2)) displayed remarkable properties as DNA carriers. In an attempt to develop cell-specific gene transfer carriers, we prepared some sugar-appended α-CDEs, e.g. mannosylated, galactosylated, and lactosylated α-CDEs. In addition, PEGylated Lac-α-CDEs (G3) were prepared and evaluated as a hepatocyte-selective and serum-resistant gene transfer carrier. Moreover, PEGylated-α-CDE/CyD polypseudorotaxane systems for novel sustained DNA release system have been developed. Interestingly, glucronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugates with dendrimer (G2) (GUG-β-CDE (G2)) had superior gene transfer activity to α-CDE (G2), expecting a development of new series of sugar-appended CDEs over α-CDEs (G2). Collectively, sugar-appended α-CDEs have the potential as novel cell-specific and safe carriers for DNA.Download high-res image (128KB)Download full-size image
Co-reporter:Taishi Higashi, Anna Tajima, Keiichi Motoyama, Hidetoshi Arima
Journal of Pharmaceutical Sciences (August 2012) Volume 101(Issue 8) pp:2891-2899
Publication Date(Web):1 August 2012
DOI:10.1002/jps.23232
In this study, to clarify the utility of polypseudorotaxane (PPRX) hydrogels composed of poly(ethylene glycol) (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as a sustained-release system for protein drugs, we prepared CyD PPRX hydrogels including lysozyme, and then the release profiles of lysozyme from these hydrogels and the release mechanisms were investigated. The α- and γ-CyD formed PPRX hydrogels by threading onto one PEG chain and two PEG chains, respectively. The formation of α- and γ-CyD PPRX hydrogels including lysozyme was based on physical cross-linking arisen from their columnar structures. The in vitro release rates of lysozyme were markedly decreased by the encapsulation into CyD PPRX hydrogels. In addition, when release data were plotted according to Korsmeyer–Peppas model, the exponent values (n) in the α- and γ-CyD systems had no statistically significant difference, suggesting that these release mechanisms were almost same. In conclusion, these results suggest that α- and γ-CyD PPRX hydrogels possess the potential as a sustained-release system for lysozyme. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
Co-reporter:Hidetoshi Arima, Toshihito Tsutsumi, Ayumi Yoshimatsu, Haruna Ikeda, Keiichi Motoyama, Taishi Higashi, Fumitoshi Hirayama, Kaneto Uekama
European Journal of Pharmaceutical Sciences (9 October 2011) Volume 44(Issue 3) pp:375-384
Publication Date(Web):9 October 2011
DOI:10.1016/j.ejps.2011.08.019
In the present study, we prepared the small interfering RNA (siRNA) complexes with polyamidoamine (PAMAM) dendrimer (G3) conjugate with α-cyclodextrin (α-CDE (G3)), and examined the physicochemical properties, serum resistance, in vitro RNAi effects on endogenous gene expression, cytotoxicity, interferon response, hemolytic activity, cellular association and intracellular distribution. In addition, these results were compared to the siRNA complexes with the commercial transfection reagents such as linear polyethyleneimine (PEI), Lipofectamine™2000 (L2) and RNAiFect™ (RF). α-CDE (G3) interacted with siRNA, and suppressed siRNA degradation by serum. The siRNA complex with α-CDE (G3) showed the potent RNAi effects against Lamin A/C and Fas expression with negligible cytotoxicity and hemolytic activity, compared to those of the transfection reagents in Colon-26-luc cells and NIH3T3-luc cells. Cell-death patterns induced by siRNA polyplexes with α-CDE (G3) and PEI were different from siRNA lipoplexes with L2 and RF. α-CDE (G3) delivered fluorescent-labeled siRNA to cytoplasm, not nucleus, after transfection in NIH3T3-luc cells. Taken together, α-CDE (G3) could be potentially used as a siRNA carrier to provide the RNAi effect on endogenous gene expression with negligible cytotoxicity.Download full-size image
Co-reporter:Tatsunori Hirotsu, Taishi Higashi, Keiichi Motoyama, Hidetoshi Arima
Carbohydrate Polymers (15 May 2017) Volume 164() pp:
Publication Date(Web):15 May 2017
DOI:10.1016/j.carbpol.2017.01.074
•Combination system of cyclodextrin-based supramolecular drug delivery technologies.•Sustained and controllable release system for insulin.•Evaluation of cyclodextrin-based supramolecular drug carriers in vitro and in vivo.Sustained and controllable release of insulin is strongly required to achieve the ideal treatment of diabetes. We previously developed “self-assembly PEGylation retaining activity (SPRA) technology” via a host–guest interaction between PEGylated β-cyclodextrin and adamantane-appended insulin, and resulting PEGylated insulin was termed SPRA-insulin. So far, we also demonstrated that covalently PEGylated insulin forms polypseudorotaxanes (PPRXs) with cyclodextrins (PPRX technology). In the present study, we designed and evaluated the combination system of SPRA technology and PPRX technology to achieve a sustained and controllable release system of insulin. SPRA-insulin formed PPRXs with α-cyclodextrin and γ-cyclodextrin. In addition, SPRA-insulin/cyclodextrin PPRXs provided sustained and controllable release of insulin beyond the each single technology both in vitro and in vivo. These results suggest that the combination system of SPRA technology and PPRX technology is useful for design of a sustained and controllable release system of insulin.
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