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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.

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.

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.

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.

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．

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.

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.

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)

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.

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.

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.

The interaction between a sodium-type synthetic mica (Na-TSM), a material of reactive layered compounds which exhibit characteristic light scattering, and some aminobenzoic acid derivatives which act as UV absorbing agents (UV absorber), was investigated. Due to the observed elongation of interlayer spacing (increase in the d-value) by X-ray diffraction analysis, it was found that the powdered form of 4-aminobenzoic acid ethyl ester (4-ABE) interacts with Na-TSM, and can be immobilized in the interlayer space of Na-TSM. Consequently, an intercalation compound of 4-ABE and Na-TSM was obtained. The formation of the intercalation compound of 4-ABE and Na-TSM was accelerated by heat processing, particularly at temperatures exceeding the melting point of 4-ABE. When 2-aminobenzoic acid methyl ester (2-ABM) in liquid form at room temperature was used instead of 4-ABE, the intercalation compound of 2-ABM and Na-TSM was formed in a similar manner to that of 4-ABE and Na-TSM. The heat processing again accelerated the formation of the intercalation compound of 2-ABM and Na-TSM. Interestingly, a difference in the formation behavior of intercalation compounds between 4-ABE and 2-ABM was observed. The intercalation compound of Na-TSM and 4-ABE or 2-ABM showed efficient UV absorption in the UV B and UV A wavelength regions. Therefore, application of a layered compound of Na-TSM and 4-ABE or 2-ABM as a novel UV screening system is promising.

Rat KMT-17 fibrosarcoma-derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching-speed separation technique, and their properties in culture were examined. The primary cultured tumor-derived endothelial cells (TEC) showed angiotensin-converting enzyme activity, positivity for Factor VIII-related antigen staining, and typical capillary-like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissue-derived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC-dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat-derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first-passage cell cultures, but not in the fourth-passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.

The purpose of the present study was to understand the permeability of tumor-derived endothelial cell monolayer using tumor-conditioned medium and primary cultured tumor-derived endothelial cells. B16 melanoma-conditioned medium increased permeabilities of bovine aortic, bovine vein and human umbilical vein-derived endothelial monolayers. This characteristic resemble that of tumor vascular endothelium in vivo. Ascorbic acid, which stimulated endothelial collagen synthesis, decreased endothelial permeability. 3, 4-Dihydroxybenzoic acid ethyl ester, a collagen synthesis inhibitor, increased the permeability. The conditioned medium decreased endothelial collagen content. These findings suggested that the hyperpermeability of the endothelial monolayer which were cultured with tumor-conditioned medium was due to low collagen content. The isolation of rat KMT-17 fibrosarcoma-derived endothelial cells by Percoll gradient centrifugation and an attaching speed separation technique is described, together with their properties in culture. Primary cultured tumor-derived endothelial monolayer was more permeable than normal tissue-derived endothelial monolayer. Leukocyte adhesion to tumor-derived endothelial cells was reduced compared to normal tissue-derived endothelial cells. These characteristics resemble those of tumor vascular endothelium. Our data indicate that tumor-conditioned medium or primary cultured tumor-derived endothelial cells are applicable for studies on physiological characteristics of tumor endothelial cells.

The aim of the present study was to evaluate the bioavailability of a drug from rapidly disintegrating tablets prepared using fine spherical crystalline cellulose (PH-M-06) and spherical sugar granules (Nonpareil, NP). Rapidly disintegrating tablets containing acetaminophen as the model drug in combination with a mixture of NP-108 (purified n-mannitol) and PH-M-06 were prepared. Plasma concentration profiles and pharmacokinetic parameters of acetaminophen in rabbits were investigated after oral administration of the prepared tablets. No significant difference in Cmax and AUC(0-infinity) of acetaminophen between rapidly disintegrating tablets and conventional tablets was observed after direct administration of these tablets into the stomach of rabbits. However, tmax (15 min) of acetaminophen from rapidly disintegrating tablets was significantly (p

Ultrasound (US) has been utilized as a useful tool for diagnosis and therapy. US mediated drug and gene delivery is paid to attention as a non-invasive system. The combination of US and microbubbles generated microjet stream by inducing disruption of bubbles and resulted in enhancing permeability of cell membrane. This phenomenon has been utilized as driving force for drug and gene delivery. Recently, we developed ultrasound sensitive liposome [Bubble liposome (BL)] containing perfluoropropane gas. US combined with BL could effectively transfer gene in vivo compared to conventional cationic liposomes. Using this method, we succeeded to obtain a therapeutic effect in cancer gene therapy with Interleukin-12 corded plasmid DNA. Therefore, it is expected that US combined with BL might be a useful non-viral vector system. From this result, the fusion of liposomal and ultrasound technologies would be important for establishment of advanced cancer therapy.

In gene therapy, it is important to develop an effective and safe gene delivery system. Especially, from the viewpoint of reducing side effects, gene delivery into a specific site is essential. We previously, developed liposomal bubbles (Bubble liposomes) containing perfluoropropane. Bubble liposomes were useful as ultrasound enhanced gene delivery tools in vitro and in vivo. In this review, we introduced the characteristics of Bubble liposomes as ultrasound imaging agents and ultrasound enhanced gene delivery tools. Bubble liposomes worked as ultrasound imaging agents in cardiosonography. In addition, their combination with ultrasound exposure was able to deliver plasmid DNA in the femoral artery. The gene expression was only observed at the site of ultrasound exposure. Moreover, the gene delivery by Bubble liposomes and ultrasound exposure was more efficient than that by conventional lipofection method using Lipofectamine 2000. Therefore, it was suggested that Bubble liposomes might be a new class of tools for site specific gene delivery.

Previously we reported fiber-modified adenovirus (Ad) vectors containing the Arg-Gly-Asp (RGD) motif on the HI loop of the fiber knob (Ad-RGD vectors) have high gene transfer efficacy into some human trophoblast cell lines. In the current study, we investigate transgene activity of Ad-RGD during differentiation of human cytotrophoblast BeWo cells into syncytiotrophoblast-like cells. Although cellular differentiation into syncytiotrophoblast cells was followed by a decrease in the coxsackievirus and adenovirus receptor levels on the cell membrane, the alphaVbeta3 and alphaVbeta5 integrin levels did not change. Conventional adenovirus vector had lower transduction activity in the differentiated cells than non-differentiated cells. In contrast, Ad-RGD vector had no influence on differentiation and had a ca. 2-5 fold higher transduction activity than that of the conventional Ad vector. Thus, Ad-RGD vector can be a powerful tool for gene transfer experiments in syncytiotrophoblast cells.

Microbubbles and ultrasound have recently been investigated with a view to improving the transfection efficiency of non-viral gene delivery systems. However, microbubbles are unstable and their targeting ability is insufficient for clinical use. To circumvent these problems, we developed novel polyethyleneglycol (PEG) modified liposomes (Bubble liposomes) containing perfluoropropane, which is an ultrasound imaging gas. Here, we used ultrasound to induce cavitation in Bubble liposomes and then investigated their ability to deliver genes in vitro and in vivo. Bubble liposomes could deliver plasmid DNA to many cell types without cytotoxicity. Additionally, in vivo gene delivery, Bubble liposomes were more effective delivery into femoral artery than lipofection method. Thus, Bubble liposomes might be efficient and novel non-viral tools for gene delivery.

Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

The aim of this study was to prepare, using taste-masked granules, tablets which can rapidly disintegrate in saliva (rapidly disintegrating tablet), of drugs with bitter taste (pirenzepine HCl or oxybutynin HCl). The taste-masked granules were prepared using aminoalkyl methacrylate copolymers (Eudragit E-100) by the extrusion method. None of the drugs dissolved from the granules (% of dissolved, < 5%) even at 480 min at pH 6.8 in the dissolution test. However, the drugs dissolved rapidly in the medium at pH 1.2 in the dissolution test. Rapidly disintegrating tablets were prepared using the prepared taste-masked granules, and a mixture of excipients consisting of crystalline cellulose (Avicel PH-102) and low-substituted hydroxypropylcellulose (L-HPC, LH-11). The granules and excipients were mixed well (mixing ratio by weight, crystalline cellulose: L-HPC = 8:2) with 1% magnesium stearate, and subsequently compressed at 500-1500 kgf in a single-punch tableting machine. The prepared tablets (compressed at 500 kgf) containing the taste-masked granules have sufficient strength (the crushing strength: oxybutynin tablet, 3.5 kg; pirenzepine tablet, 2.2 kg), and a rapid disintegration time (within 20 s) was observed in the saliva of healthy volunteers. None of the volunteers felt any bitter taste after the disintegration of the tablet which contained the taste-masked granules. We confirmed that the rapidly disintegrating tablets can be prepared using these taste-masked granules and excipients which are commonly used in tablet preparation.

The tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of 125I-labelled TES-23 were also up to 50 times higher than those of control antibody with little distribution to normal tissues. The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer. © 1999 Cancer Research Campaign

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of 125 I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis andlor necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.

In this study, the effects of a protease inhibitor, endocytosis inhibitors and an absorption-enhancing agent on the absorption of ((Asu1,7)-eel calcitonin, ECT) from the nasal mucous membrane in rabbits were examined, and the results were compared with those obtained following the rectal absorption of ECT reported in our previous paper. ECT was efficiently absorbed from the nasal mucous membrane and effectively decreased serum calcium (Ca) concentrations. The increase in the area under the percent decrease in serum Ca concentration (deltaCa%)-time curve (deltaCa%-AUC) value, assumed to be an index of the pharmacodynamics (hypocalcemic effect) of ECT, depended on the dose of ECT administered intranasally. When nafamostat mesilate, a protease inhibitor, was coadministered with ECT, the deltaCa%-AUC markedly increased. It is presumed that the influence (enzymatic barrier function) of protease on the nasal absorption of ECT is significant. However, no significant difference in the deltaCa%-AUC value was observed when an endocytosis inhibitor (cytochalasin B or monensin) was coadministered with ECT. ECT administration in combination with sodium decanoate, the sodium salt of a medium-chain fatty acid, effectively increased the deltaCa%-AUC value due to the enhancing effect of sodium decanoate on the nasal absorption of ECT. We conclude that the nasal application offers a promising approach for the administration of pharmaceutical preparations containing ECT with additives such as nafamostat mesilate and sodium decanoate.

In this study, we developed a new hollow-type suppository containing elcatonin ((Asu1, 7)-eel calcitonin, ECT), a synthetic derivative of eel calcitonin, which produces hypocalcemia, as a pharmaceutical preparation for self administration, to be used instead of parenteral injections for patients with osteoporosis. The absorption of ECT from the rectal mucous membrane was evaluated by observation of the decrease in serum calcium (Ca) concentrations following rectal administration in rabbits. ECT was efficiently absorbed from the rectum and effectively decreased serum Ca concentrations. The data of the area under the percent decrease in serum Ca concentration (ΔCa%)-time curve (ΔCa%-AUC), assumed to be an index of the pharmacodynamics (pharmacological effect) of ECT, indicated that similar hypocalcemic effects were obtained following rectal and intravenous administrations of ECT. In regard to the effect of coadministration of other compounds on rectal absorption of ECT, no significant difference in the ΔCa%-AUC between rectal ECT administration with or without nafamostat mesilate (a protease inhibitor) was observed. However, the coadministration of ECT with cytochalasin B or monensin (endocytosis inhibitors) significantly decreased the ΔCa%-AUC, indicating that rectal ECT absorption is probably inhibited by endocytosis inhibitors by endocytosis inhibitors. On the other hand, it was found that sodium decanoate, a mediumchain fatty acid (sodium salt), significantly enhanced the rectal absorption of ECT. We conclude that this ECT hollow-type suppository offers promise as a new method for the administartion of ECT.