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The abundance of cell surface levels of transferrin receptor 1 (TfR1), which regulates the uptake of iron-bound transferring, correlates with the rate of cell proliferation. Because TfR1 expression is higher in cancer cells than in normal cells, it offers a target for cancer therapy. In this study, we found that the expression of TfR1 in mouse colon cancer cells was affected by the circadian organization of the molecular clock. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop, in which CLOCK and BMAL1 are positive regulators and the Period (Per), Cryptochrome (Cry), and Dec genes act as negative regulators. TfR1 in colon cancer–bearing mice exhibited a 24-hour rhythm in mRNA and protein levels. Luciferase reporter analysis and chromatin immunoprecipitation experiments suggested that the clock-controlled gene c-MYC rhythmically activated the transcription of the TfR1 gene. Platinum incorporation into tumor DNA and the antitumor efficacy of transferrin-conjugated liposome-delivered oxaliplatin could be enhanced by drug administration at times when TfR1 expression increased. Our findings suggest that the 24-hour rhythm of TfR1 expression may form an important aspect of strategies for TfR1-targeted cancer therapy. Cancer Res; 70(15); 6238–46. ©2010 AACR.

Supplementary Figure 5 from Circadian Rhythm of Transferrin Receptor 1 Gene Expression Controlled by c-Myc in Colon Cancer–Bearing Mice

Supplementary Figure 2 from Circadian Rhythm of Transferrin Receptor 1 Gene Expression Controlled by c-Myc in Colon Cancer–Bearing Mice

Supplementary Figure 4 from Circadian Rhythm of Transferrin Receptor 1 Gene Expression Controlled by c-Myc in Colon Cancer–Bearing Mice

Supplementary Figure 1 from Circadian Rhythm of Transferrin Receptor 1 Gene Expression Controlled by c-Myc in Colon Cancer–Bearing Mice

Supplementary Figure 3 from Circadian Rhythm of Transferrin Receptor 1 Gene Expression Controlled by c-Myc in Colon Cancer–Bearing Mice

Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

Introduction Syncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. Methods We generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. Results The human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. Discussion These results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

Tumor blood vessels supply cancer tissues with oxygen and nutrients, and it was therefore believed that inhibition of angiogenesis would induce tumor regression. In fact, the situation is complicated by the presence of normal blood vessels in cancer tissues such as carcinomas and sarcomas as well as abnormal vessels. Here, we describe the development of a dendritic cell (DC)-based immunotherapy which targets tumor endothelial cells (TECs) rather than normal endothelial cells (ECs) or cancer cells themselves. After density gradient centrifugation, the TEC-rich fraction from lungs invaded by B16 melanoma cells was separated from the endothelial cell (EC)-rich fraction on the basis of positivity for angiotensin-converting enzyme (ACE) activity. Prophylactic vaccination with DCs pulsed with lysates of TECs isolated from lungs with metastases significantly suppressed lung metastasis in this B16/BL6 mouse melanoma model. This suggests that DC-based vaccine therapy targeting TECs in cancers tissue could show promise as an effective therapy for distant metastasis.

Human adenoviruses (Ads), common pathogens that cause upper respiratory and gastrointestinal infections, are blocked by neutralizing antibodies (nAbs). However, Ads are not fully eliminated even in hosts with nAbs. In this study, we assessed the infectivity of progeny Ad serotype 5 (Ad5) in the presence of nAb. The infectivity of Ad5 was evaluated according to the expression of the Ad genome and reporter gene. Infection by wild-type Ad5 and Ad5 vector continued to increase until 3 days after infection even in the presence of nAb. We established an assay for determining the infection levels of progeny Ad5 using a sorting system with magnetic beads and observed little difference in progeny Ad5 counts in the presence and absence of nAb 1 day after infection. Moreover, progeny Ad5 in the presence of nAb more effectively infected coxsackievirus and adenovirus receptor (CAR)-positive cells than CAR-negative cells. We investigated the function of fiber proteins, which are the binding partners of CAR, during secondary infection, observing that fibre proteins spread from infected cells to adjacent cells in a CAR-dependent manner. In conclusion, this study revealed that progeny Ad5 could infect cells even in the presence of nAb, differing from the common features of the Ad5 infection cycle. Our findings may be useful for developing new therapeutic agents against Ad infection.

Syncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57We generated p57The human choriocarcinoma cell line, BeWo has undetectable levels of p57These results indicate that the expression of p57

Tumor angiogenesis plays an important role in tumor growth and metastasis, with tumor cells requiring nutrients and oxygen via blood flow for their proliferation. In comparison, angiogenesis also occurs under normal physiological conditions, such as wound healing and in the formation of the corpus luteum. Herein, we report on the development of a novel dendritic cell (DC) vaccine therapy using tumor endothelial cells (TECs) derived from tumor vessels as tumor antigens. After density gradient centrifugation and the detection of angiotensin-converting enzyme activities, a TEC-rich fraction was separated from solid tumor tissues. Prophylactic or therapeutic immunization using DCs pulsed with TECs as vaccine antigens significantly suppressed solid tumor growth in a Colon-26 colorectal adenocarcinoma tumor-bearing mouse model, compared with the use of tumor cells as DC vaccine antigens. Tumor tissues showed reduced angiogenesis. However, vaccination using DCs pulsed with TECs did not inhibit physiological angiogenesis as evidenced by a wound healing assay. Additionally, in a B16/BL6 mouse melanoma lung metastasis model, DC vaccination using TECs derived not only from the same tumor tissue but from a different type of tumor also suppressed metastasis. These results thus show that cancer vaccine therapy targeting TECs is an effective therapy against angiogenesis in several types of cancer, but does not affect normal blood vessel growth.

Cell-penetrating peptides (CPPs) have been highly anticipated as an efficient delivery system due to their ability to cross biological membranes and transport various cargoes into cells. In the present study, we have identified adenovirus-derived CPPs using various capsid-mutant adenovirus (Ad) vectors. First, we examined the endocytosis-inducing ability of these vectors. A fiber-shaft substituted Ad vector, Ad type 5 vector with the fiber shaft domain replaced by that derived from Ad type 35, induced the highest fluorescein isothiocyanate (FITC)-dextran uptake into a human liver cell line, HepG2 cells. In contrast, the FITC-dextran uptake in HepG2 cells was not significantly different between coxsackievirus and adenovirus receptor (CAR)-binding-ablated Ad vector, integrin-binding-ablated Ad vector or conventional Ad vector. Next, we produced a recombinant Ad type 35 shaft protein using the Escherichia coli recombinant system. The recombinant Ad type 35 shaft protein retained the ability for FITC-dextran uptake and efficient gene delivery by plasmid transfection reagent. Furthermore, we identified 26 C-terminal amino acids in the Ad type 35 shaft protein as the cell membrane binding domain. The 26 amino-acid peptides also have the potential to be internalized into cultured cells. The internalization ability of the peptide was dependent on degree and was inhibited by an actin polymerization inhibitor (Latrunculin B) and by a lipid raft formation inhibitor (methyl-β-cyclodextrin). The results of the present study indicate that Ad type 35-derived peptides induce endocytosis in cultured cells and have the ability to cross biological membranes. This report is the first paper to identify Ad-derived CPPs.

Melanoma has an early tendency to metastasize, and the majority of the resulting deaths are caused by metastatic melanoma. It is therefore important to develop effective therapies for metastasis. Dendritic cell (DC)-based cancer immunotherapy has been proposed as an effective therapeutic strategy for metastasis and recurrence due to prime tumor-specific cytotoxic T lymphocytes. In this therapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I molecules. Previously, we developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound. In the present study, we investigated the prevention of melanoma lung metastasis via DC-based immunotherapy. Specifically, antigens were extracted from melanoma cells and used to treat DCs by BL and ultrasound. Delivery into the DCs by this route did not require the endocytic pathway. The delivery efficiency was approximately 74.1%. DCs treated with melanoma-derived antigens were assessed for in vivo efficacy in a mouse model of lung metastasis. Prophylactic immunization with BL/ultrasound-treated DCs provided a four-fold decrease in the frequency of melanoma lung metastases. These in vitro and in vivo results demonstrate that the combination of BLs and ultrasound is a promising method for antigen delivery system into DCs.

Recently, ultrasound-mediated gene delivery with nano- and microbubbles was developed as a novel non-viral vector system. In this gene delivery system, microstreams and microjets, which are induced by disruption of nano/microbubbles exposed to ultrasound, are used as the driving force to transfer genes into cells by opening transient pores in the cell membrane. This system can directly deliver plasmid DNA and siRNA into cytosol without endocytosis pathway. Therefore, these genes are able to escape from degradation in lysosome and result in enhancing the efficiency of gene expression. In addition, it is expected that ultrasound-mediated gene delivery using nano/microbubbles would be a system to establish non-invasive and tissue specific gene expression because ultrasound can transdermally expose to target tissues and organs. This review focuses on the current ultrasound-mediated gene delivery system using nano/microbubbles. We discuss about the feasibility of this gene delivery system as novel non-viral vector system.

The abundance of cell surface levels of transferrin receptor 1 (TfR1), which regulates the uptake of iron-bound transferring, correlates with the rate of cell proliferation. Because TfR1 expression is higher in cancer cells than in normal cells, it offers a target for cancer therapy. In this study, we found that the expression of TfR1 in mouse colon cancer cells was affected by the circadian organization of the molecular clock. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop, in which CLOCK and BMAL1 are positive regulators and the Period (Per), Cryptochrome (Cry), and Dec genes act as negative regulators. TfR1 in colon cancer–bearing mice exhibited a 24-hour rhythm in mRNA and protein levels. Luciferase reporter analysis and chromatin immunoprecipitation experiments suggested that the clock-controlled gene c-MYC rhythmically activated the transcription of the TfR1 gene. Platinum incorporation into tumor DNA and the antitumor efficacy of transferrin-conjugated liposome-delivered oxaliplatin could be enhanced by drug administration at times when TfR1 expression increased. Our findings suggest that the 24-hour rhythm of TfR1 expression may form an important aspect of strategies for TfR1-targeted cancer therapy. Cancer Res; 70(15); 6238–46. ©2010 AACR.

Interleukin-12 (IL-12) gene therapy is expected to be effective against cancers because it primes the immune system for cancer cells. In this therapy, it is important to induce IL-12 gene expression in the tumor tissue. Sonoporation is an attractive technique for developing non-invasive and non-viral gene delivery systems, but simple sonoporation using only ultrasound is not an effective cancer gene therapy because of the low efficiency of gene delivery. We addressed this problem by combining ultrasound and novel ultrasound-sensitive liposomes (Bubble liposomes) which contain the ultrasound imaging gas perfluoropropane. Our previous work showed that this is an effective gene delivery system, and that Bubble liposome collapse (cavitation) is induced by ultrasound exposure. In this study, we assessed the utility of this system in cancer gene therapy using IL-12 corded plasmid DNA. The combination of Bubble liposomes and ultrasound dramatically suppressed tumor growth. This therapeutic effect was T-cell dependent, requiring mainly CD8 + T lymphocytes in the effector phase, as confirmed by a mouse in vivo depletion assay. In addition, migration of CD8 + T cells was observed in the mice, indicating that the combination of Bubble liposomes and ultrasound is a good non-viral vector system in IL-12 cancer gene therapy.

近年，樹状細胞（DCs）の強力な抗原提示能を利用したがん免疫療法が注目されている．この療法の中核をなす細胞傷害性T細胞（CTL）を活性化するためには，DC上のMHCクラスIを介してがん関連抗原を提示させる必要がある．そこで筆者らは，DCsへの抗原送達キャリアとしてIgG表面修飾した抗原封入リポソームを開発した．このIgGリポソームはDCs上のFcγレセプターを介して取り込まれ，内封した抗原のMHCクラスIへの抗原提示を誘導することで抗原特異的なCTL誘導を可能とした．本稿では，がん免疫療法における抗原デリバリーキャリアとしてのIgGリポソームの可能性について紹介する．

Bubble liposomes (liposomes which entrap an ultrasound imaging gas) may constitute a unique system for delivering various molecules efficiently into mammalian cells in vitro. In this study, Bubble liposomes were compared with cationic lipid (CL)-DNA complexes as potential gene delivery carriers into tumor in vivo. The delivery of genes by Bubble liposomes depended on the intensity of the applied ultrasound. Transfection efficiency plateaued at 0.7 W/cm(2) ultrasound intensity. Bubble liposomes efficiently transferred genes into cultured cells even when the cells were exposed to ultrasound for only 1 s. In addition, Bubble liposomes could introduce the luciferase gene more effectively than CL-DNA complexes into mouse ascites tumor cells and solid tumor tissue. We conclude that the combination of Bubble liposomes and ultrasound is a minimally-invasive and tumor specific gene transfer method in vivo.

Gene therapy has a potentiality for treatment of cancer and diseases from genomic defects. It is important to select a vector which has good potency in terms of gene transduction efficiency, and is safe and easy to apply. Many researchers have attempted to develop an effective gene delivery carrier. Recently, it was reported that microbubbles, which are ultrasound (US) contrast agents, improved the transfection efficiency by cavitation with US exposure. However, microbubbles had problems with stability and targeting ability. To solve these problems, we focused on liposomes that had many advantages such as being stable and safe in vivo and easily modifying targeting ligand. We succeeded in preparing the liposomes ("Bubble liposomes" (BLs)) entrapping perfluoropropane gas which was utilized for contrast enhancement in ultrasonography. In this study, we assessed the feasibility of BLs as gene delivery carrier utilized cavitation by US exposure. BLs could deliver plasmid DNA to various cell types in vitro by combining with US without cytotoxicity. To evaluate the ability of BLs to in vivo gene delivery, we attempted to deliver plasmid DNA into the femoral artery. The gene expression at this artery treated with BLs and US combination was higher than with US only, BLs without US or Lipofectamine 2000. This result suggested that Bubble liposomes could quickly deliver plasmid DNA into the artery even under conditions of short contact time between BLs and the endothelial cells and the existence of the bloodstream and serum. These results suggested that BLs might be a non-invasive and effective carrier for gene delivery.

In cancer immunotherapy with dendritic cells (DCs), which are the most potent antigen-presenting cells, it is important that DCs present peptides derived from tumor-associated antigens on major histocompatibility complex (MHC) class I molecules and activate tumor-specific cytotoxic T lymphocytes. However, exogenous antigens are generally presented on MHC class II but not class I molecules. To develop effective immunotherapy for cancer, an antigen delivery carrier that can induce MHC class I presentation of exogenous antigens is necessary. Several strategies to induce DCs to present exogenous antigens on MHC class I molecules have been reported. First, DCs that phagocytosed a particulate form of antigens present peptides derived from the antigens on MHC class I molecules. Second, DCs that incorporated antigens via certain endocytic receptors such as Fc receptors efficiently present peptides on MHC class I molecules. We combined these two strategies and prepared antigen-containing IgG-conjugated liposomes (IgG-liposomes). In this study, we investigated the feasibility of IgG-liposomes as antigen delivery carriers in cancer immunotherapy with DCs. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG-liposomes, but not OVA-containing bare liposomes or soluble OVA, completely prevented the growth of OVA-expressing lymphoma cells. These results suggest that IgG-liposomes represent an efficient antigen delivery carrier for DCs in cancer immunotherapy.

Liposomes represent a promising vehicle to deliver exogenous antigens to dendritic cells (DCs) for tumor immunotherapy. Targeting exogenous antigens to Fcgamma receptors on DCs has been shown to result in efficient presentation of antigen-derived peptides on major histocompatibility complex (MHC) class I and class II molecules. In this study, it was investigated whether DCs that endocytosed physicochemically optimized antigen-containing liposomes conjugated with IgG efficiently present antigens on MHC class I and class II molecules, and consequently induce strong antitumor immune responses. IgG-conjugated liposomes that were 200 nm in diameter without attaching polyethylene glycol were most efficiently endocytosed by DCs. Human monocyte-derived DCs that endocytosed tetanus toxoid (TT)-containing IgG liposomes via CD32 stimulated CD4(+) T cells more strongly than DCs pulsed with TT-containing bare liposomes or with soluble TT. Immunization of mice with DCs that endocytosed ovalbumin (OVA)-containing IgG liposomes but not OVA-containing bare liposomes or soluble OVA completely prevented the growth of OVA-expressing lymphoma cells. Importantly, administration of DCs that endocytosed OVA-containing IgG liposomes to the mice with established OVA-expressing tumors strongly suppressed tumor growth. This study demonstrates an IgG liposome with physicochemical properties suitable for delivering antigens to DCs and paves the way to the application of IgG liposomes for tumor immunotherapy using DCs.

ポリエンマクロライド系抗真菌剤であるアムホテリシンB (AmB) は, 深在性真菌症の治療に用いられているが, 腎障害などの重篤な副作用を示すため, その使用に制限がある. したがって, DDSによる副作用の軽減が望まれている. AmBは水やほとんどの有機溶媒に不溶なため, そのリポソーム化は困難であったが, ポリエチレングリコールのリン脂質誘導体 (DSPE-PEG) と9% sucroseを用いることで, AmBを容易かつ多量にリポソームに封入できることを見いだした. AmB-PEGリポソームは, アスペルギルス感染マウスにおいて高い血中滞留性を示し, 肺内皮細胞に対する抗体 (34A) の付与で, AmBisomeにくらべて高い治療効果を示した.

To characterize the influence of nitric oxide (NO) donors on the intestinal absorption of macromolecules, the relationship between the release rate of NO from NO donors and their absorption-enhancing effects and the effects of several scavengers and generators on the absorption-enhancing effects of NO donor were investigated. The t1/2 values of the NO release rate from 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-1-propanamine (NOC5), 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12) are 25, 5 and 100min, respectively. The absorption-enhancing effects of NO donors on the absorption of fluorescein isothiocyanate dextrans with an average molecular weight of 4400 (FD-4) are NOC5 > NOC7 > NOC12 in the colon. The lowest enhancing effect of NOC12 may be due to the slow rate of NO release. The enhancing effect of NOC7 rapidly disappeared compared with the effect of NOC5. The results raise the possibility that the difference between NOC5 and NOC7 on enhancing effect is related to the t1/2 of the NO release. The NOC7-induced enhancing effect was prevented by the co-administration of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide sodium salt (C-PTIO), an NO scavenger; tiron, an O2(-) scavenger; mannitol, an OH* scavenger, and deferoxamine, peroxynitrate scavenger. Pyrogallol, an O2(-) generator, potentiated the NOC7-induced enhancing effect. These results support a role for peroxynitrate, and possibly OH*, in the NO donor-induced intestinal enhancing effect.

The successful treatment of cancer by boron neutron-capture therapy (BNCT) requires the selective delivery of relatively high concentration of 10B compounds to malignant tumor tissue. This study focuses on a new tumor-targeting drug delivery system for BNCT that uses small (less than 200 nm in diameter), unilamellar mercaptoundecahydrododecaborate (BSH)-encapsulating, transferrin (TF)-conjugated polyethyleneglycol liposomes (TF-PEG liposomes). When TF-PEG liposomes were injected at a dose of 35 mg 10B/kg, we observed a prolonged residence time in the circulation and low uptake by the reticuloendothelial system (RES) in Colon 26 tumor-bearing mice, resulting in enhanced accumulation of 10B into the solid tumor tissue (e.g., 35.5 microg/g). TF-PEG liposomes maintained a high 10B level in the tumor, with concentrations over 30 microg/g for at least 72 h after injection. This high retention of 10B in tumor tissue indicates that binding and concomitant cellular uptake of the extravasated TF-PEG liposomes occurs by TF receptor and receptor-mediated endocytosis, respectively. On the other hand, the plasma level of 10B decreased, resulting in a tumor/plasma ratio of 6.0 at 72 h after injection. Therefore, 72 h after injection of TF-PEG liposomes was selected as the time point of BNCT treatment. Administration of BSH encapsulated in TF-PEG liposomes at a dose of 5 or 20 mg 10B/kg and irradiation with 2 x 10(12) neutrons/cm2 for 37 min produced tumor growth suppression and improved long-term survival compared with PEG liposomes, bare liposomes and free BSH. Thus, intravenous injection of TF-PEG liposomes can increase the tumor retention of 10B atoms, which were introduced by receptor-mediated endocytosis of liposomes after binding, causing tumor growth suppression in vivo upon thermal neutron irradiation. These results suggest that BSH-encapsulating TF-PEG liposomes may be useful as a new intracellular targeting carrier in BNCT therapy for cancer.

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and alphav integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and alphav integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while alphav integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as alphav integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with alphav integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with alphav integrin is likely to be crucial to the development of targeted Ad vectors.

Human choriocarcinoma cell lines have been used as placental models for the study of endocrine function, including aromatase (CYP19) activity and the secretion of human CG (hCG). In the present study, we investigated the effects of trialkyltin compounds, which are suspected endocrine disrupters, on aromatase activity and hCG secretion in human choriocarcinoma JAR, JEG-3, and BeWo cells. Protein synthesis as measured by 35 S-methionine incorporation in all cell lines was markedly decreased by treatment with both tributyltin (TBT) and triphenyltin (TPT) at concentrations above 3 10 7 M, due to cytotoxicity. In JAR cells, 35 S-methionine uptake was decreased by 50% at 3 10 7 M of TBT. At a TPT concentration of 1 10 7 M, protein synthesis in JAR cells was not affected, whereas JEG-3 and BeWo cells demonstrated slightly decreases. In all cell lines, both TBT and TPT increased levels of hCG secretion and aromatase activity in a dose- and timedependent fashion following exposure to nontoxic concentration ranges. In addition, these trialkyltin compounds enhanced 8-bromo-cAMP-induced hCG secretion and aromatase activity in JAR cells. TBT caused dose-related increases in steady-state mRNA levels of both hCG and CYP19 in JAR cells following 24- or 48-h exposure to nontoxic concentrations of TBT. However, these mRNA changes in JAR cells were not comparable to the changes in both hCG secretion and aromatase activity. These results indicate that the observed trialkyltin-induced alterations in human choriocarcinoma cells are due to other mechanism in addition to a regulation of hCG and CYP19 mRNA levels. Our studies suggest that trialkyltin compounds are potent stimulators of human placental hCG production and aromatase activity in vitro; and the placenta represents a potential target organ for trialkyltin compounds, whose endocrine-disrupting effects might be the result of local changes in hCG and estrogen concentrations in pregnant women. (J Clin Endocrinol Metab 87: 2830 –2837, 2002)

The effects of ethanol on the simultaneous transport and metabolism of methyl p-hydroxybenzoate (HBM) were investigated in the skin of Yucatan micropig in vitro. It was found that transesterification occurred in the permeation studies involving ethanol. This was confirmed by monitoring the flux of ethyl p-hydroxybenzoate (HBE) into the receptor phase, as well as by monitoring the fluxes of HBM and p-hydroxybenzoic acid (HBA). The apparent flux of total HBM was decreased. The solubility of HBM increased with ethanol concentration, thus, the activity of HBM in ethanol solution became low because we used 10 mM HBM solution for permeation studies. The enhancement factor (E) was calculated to correct the activity. E increased with increasing the flux of ethanol, thus, ethanol may function as an enhancer of HBM transport. The hydrolysis of HBM to HBA was inhibited, whereas transesterification of HBM to HBE was induced at all concentrations of ethanol used (10-40%). The formation of HBE occurred much more readily than that of HBA at all concentrations of ethanol used.

In an attempt to achieve chronopharmacotherapy for asthma, press-coated tablets (250 mg), which contained aminophylline in the core tablet in the form of low-substituted hydroxypropylcellulose (L-HPC) and coated with crystalline cellulose (PH-102) and polyethylene glycol (PEG) at various molecular weights and mixing ratios in the amounts of PH-102 and PEG as the outer shell (press-coating material), were prepared (chronopharmaceutics). Their applicability as timed-release (delayed-release) tablets with a lag time of disintegration and a subsequent rapid drug release phase was investigated. Various types of press-coated tablets were prepared using a tableting machine, and their aminophylline dissolution profiles were evaluated by the JP paddle method. Tablets with the timed-release characteristics could be prepared, and the lag time of disintegration was prolonged as the molecular weight and the amount of PEG, for example PEG 500,000, in the outer shell were increased. The lag time of disintegration could be controlled by the above-mentioned method, however, the pH of the medium had no effect on disintegration of the tablet and dissolution behavior of theophylline. The press-coated tablet (core tablet:aminophylline 50 mg, L-HPC and PEG 6000; outer shell:PH-102:PEG = 8:2 200 mg) with the timed-release characteristics was administered orally to rabbits for an in vivo test. Theophylline was first detected in plasma more than 2 h after administration; thus, this tablet showed a timed-release characteristics in the gastrointestinal tract. The time (tmax) required to reach the maximum plasma theophylline concentration (Cmax) observed after administration of the press-coated tablet was significantly (p0.05) delayed compared with that observed after administration of aminophylline solution in the control experiment. However, there was no difference in Cmax and area under the plasma theophylline concentration-time curve (AUC0--24) between the press-coated tablet and aminophylline solution. These results suggest that the press-coated aminophylline tablet (with the timed-release characteristic) offers a promising forms of theophylline chronotherapy for asthma.

To determine the effectiveness of human chorionic gonadotropin (hCG) administered rectally, we studied the pharmacokinetics and pharmacodynamics of hCG using a hollow-type suppository. HCG was not detected in plasma when only hCG was administered rectally, even at a higher dose (4,000 IU/kg body weight) than intravenous injection, because of its low bioavailability due to high molecular weight or degradation by proteolytic activity. To enhance the rectal absorption of hCG, the effectiveness of its coadministration with alpha-cyclodextrin (alpha-CyD), an absorption-enhancing agent, was investigated in male rabbits. HCG was detected in plasma following coadministration of hCG and alpha-CyD (10 mg/kg body weight) into the rectum. The plasma hCG concentration increased with increasing dose of alpha-CyD. The AUC(0-48) observed after coadministration of hCG and alpha-CyD at 30 mg/kg body weight was approximately four times higher than that of hCG and alpha-CyD at 10mg/kg body weight. HCG at a high concentration induced a rapid increase in the plasma testosterone concentration (74.2 +/- 3.4 ng/ml) 2 h after intravenous administration. However, the testosterone concentration 24 h after intravenous administration decreased to the physiological level (approximately 20 ng/ml) which had been observed before such administration. On the other hand, the maximum level of testosterone concentration (40.0 +/- 12.6 ng/ml) was observed 24 h after rectal administration of hCG (400 IU/kg body weight) in combination with alpha-CyD (30 mg/kg body weight). Moreover, the plasma testosterone concentration (31.0 +/- 11.4 ng/ml) obtained 72 h after rectal administration tended to be maintained at a higher level than that (14.4 +/- 0.9ng/ml) observed before the administration. These results suggest that the hollow-type suppository as a rectal delivery system of hCG is promising as a new mode of hCG therapy.

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

To decrease the sensation of roughness when a tablet, which is rapidly disintegrated by saliva (rapidly disintegrating tablet), is orally taken, we prepared rapidly disintegrating tablets using microcrystalline cellulose (Avicel PH-M series), a new type of pharmaceutical excipient that is spherical and has a very small particle size (particle size, 7-32 microm), instead of conventional microcrystalline cellulose (PH-102) used in the formulation of tablets containing acetaminophen or ascorbic acid as model drugs for tableting study. Tablets (200 mg) prepared using spherical microcrystalline cellulose, PH-M-06, with the smallest particle size (mean value, 7 microm) had sufficient crushing tolerance (approximately, 8 kg) and were very rapidly, disintegrated (within 15 s) when the mixing ratio of PH-M-06 to low-substituted hydroxypropylcellulose (L-HPC) was 9:1. Sensory evaluation by volunteers showed that PH-M-06 was superior to PH-102 in terms of the feeling of roughness in the mouth. Consequently, it was found that particle size is an important factor for tablet preparation using microcrystalline cellulose. It is possible to prepare drugs such as acetaminophen and ascorbic acid (concentration of approximately 50%) in the tablet form using PH-NM-06 in combination with L-HPC as a good disintegrant at a low compression force (1-6 kN). To solve the problem of poor fluidity in the preparation of these tablets, we investigated the use of spherical sugar granules (Nonpareil, NP-101 (sucrose and starch, composition ratio of 7:3), NP-103 (purified sucrose), NP-107 (purified lactose) and NP-108 (purified D-mannitol)). Rapidly disintegrating tablets can be prepared by the direct compression method when suitable excipients such as fine microcrystalline cellulose (PH-M-06) and spherical sugar granules (NP) are used.

Immunoconjugate targeting of solid tumors has not been routinely successful because the endo-thelial cells of blood vessels act as a physical barrier against the transport of macromolecules, such as antibodies. In the present study, we attempted to achieve tumor vascular targeting with an anti-tumor tissue endothelium-specific monoclonal antibody (TES-23). TES-23, an IgG1 monoclonal antibody raised against rat KMT-17 fibrosarcoma-derived endothelial cells, was covalently conjugated with neocarzinostatin (NCS) in a previous study. The TES-23-NCS conjugate induced tumor hemorrhagic necrosis, and showed marked anti-tumor effects against rat KMT-17 fibrosarcoma. This result prompted us to investigate whether this approach would be applicable to various other types of solid tumors. One hour after injection of (125)I-labeled TES-23 into BALB / c mice bearing Meth-A fibrosarcoma and Colon 26 adenocarcinoma, the tumor accumulation of TES-23 was greater than that of the control IgG. In the present study, we report the anti-tumor effects of this monoclonal antibody in mice bearing Meth-A fibrosarcoma. Mice treated with the immunoconjugate showed improved survival with no side effects. This result indicates that common antigens may be found in different kinds of tumor endothelial cells, and that TES-23 might recognize these antigens.

Nitric oxide (NO) is one of the most versatile mediators in mammalian biology. In the present study, we investigated the absorption-enhancing effects of an NO donor, 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propa namine (NOC7), on drugs that are poorly absorbed from the gastrointestinal tract. NOC7 significantly increased the jejunal absorption of fluorescein isothiocyanate dextrans (FDs) of different average molecular weights (4000-20,000). This enhancing effect decreased as the FD molecular weight increased. Another NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), also increased the absorption of FD-4 from the jejunum. The absorption enhancement effect of NOC7 significantly decreased after coadministration with an NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazole-1-oxyl-3-oxide, sodium salt. Furthermore, the enhancement effect of NOC7 was reversed shortly after cessation of the enhancer treatment. Little damage by NOC7 to the intestinal mucosa was observed in terms of release of lactose dehydrogenase and protein from the intestinal mucosa. NOC7 also increased the absorption of FD-4 by the colon and rectum. The findings suggest that an NO donor can improve the absorption of macromolecules from all regions of the rat intestine with very little mucosal damage and that an NO donor can act as a potent absorption enhancer.

The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but not anion exchange inhibitor. Selected monocarboxylic acids inhibited the uptake of [14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver-Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [14C]valproic acid from the apical-to-basal side of the monolayers was significantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model.

The monolayer-forming, human choriocarcinoma cell line, BeWo, was used to study the mechanisms of monocarboxylic acid transport across the human trophoblast. Benzoic acid, acetic acid, and lactic acid were used as markers for monocarboxylic acid carrier-mediated transport. The uptake of benzoic acid by BeWo cells was saturable (K(t) = 0.6 +/- 0.3 mM) at higher concentrations and significantly inhibited by typical metabolic inhibitors, sodium azide and 2, 4-dinitrophenol. A selection of different monocarboxylic acids, including a natural substrate lactic acid, also substantially inhibited the uptake of benzoic acid and acetic acid by BeWo cells, whereas dicarboxylic acids did not affect the uptake of either marker. Monocarboxylic acid uptake was pH-dependent and inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore. Kinetic analysis using Lineweaver-Burk plots revealed that monocarboxylic acids competitively inhibited the uptake of benzoic, lactic, and acetic acid by BeWo cells. In transport experiments, the permeation of benzoic acid from apical-to-basolateral side was greater than the permeation from the basolateral-to-apical side, and the transport of benzoic acid from apical-to-basolateral side was inhibited by monocarboxylic acids. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for monocarboxylic acids across the BeWo cell, a representative of the human trophoblast.

In this study, we attempted to develop tumor vascular targeting with a tumor tissue endothelium-specific monoclonal antibody. TES-23, which strongly and selectively recognizes tumor tissue endothelial cells, was chemically conjugated with Neocarzinostatin (NCS), and the anti-tumor effect was examined. The immunoconjugate, TES-23-NCS, showed, through the use of tumor hemorrhagic necrosis, a marked anti-tumor effect on KMT-17 tumors in rats at a dosage of 17 micrograms/kg (NCS equivalent) without any side effects, probably due to specific tumor vascular injury. By contrast, TES-23 alone (107 micrograms/kg), NCS alone (17 micrograms/kg), and Mopc-NCS (Mopc, 107 micrograms/kg; NCS, 17 micrograms/kg), the immunoconjugate of control antibody, did not have any anti-tumor activities. By tissue distribution analysis, TES-23 and TES-23-NCS showed high accumulation in KMT-17 tumors 1 h after intravenous administration. Moreover TES-23 also accumulated in Sarcoma-180 tumors in mice 1 h after intravenous administration. These results suggest that TES-23 may be a candidate for a potential tumor vascular targeting agent that is applicable to a wide variety of tumor types.

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca2+]i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10−7−10−5 M), permeability of FITC-dextran (molecular weight 70,000) and [Ca2+]i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca2+]i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca2+]i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca2+]i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.

Previously, we found that monocarboxylic acids undergo carrier-mediated transport in primary cultures of oral mucosal epithelial cells.1 In this study, we investigated whether carrier-mediated absorption of a monocarboxylic acid from the oral mucosa occurs in vivo. Salicylic acid was administered to hamster cheek pouch. At predetermined intervals, the concentration of salicylic acid in the fluid remaining in the cheek pouch lumen and the blood salicylic acid concentration were determined. The absorption of salicylic acid was saturable at high salicylic acid concentrations. Sodium azide, a metabolic inhibitor, and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, significantly inhibited the absorption of salicylic acid but not the absorption of salicylamide from the oral mucosa. Various monocarboxylic acids inhibited the absorption of salicylic acid, whereas dicarboxylic acids had no such effect. Transfer of [14C]salicylic acid from the cheek pouch mucosa to the systemic circulation was observed, and the blood [14C]salicylic acid concentration in the case of coadministration with propionic acid was significantly lower than that in the case of no propionic acid coadministration. These results show that monocarboxylic acids undergo carrier-mediated absorption from the hamster cheek pouch mucosa.

Purpose. The objective of this investigation is to evaluate the potential of nitric oxide (NO) donors as a new class of absorption enhancers which may act on intestinal epithelial cells through epithelial actions of the chemical mediator, NO.