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Recently, we demonstrated that asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined 2’-O-methyl RNA and DNA, termed MED-siRNAs, show high RNase resistance, efficient cleavage of target mRNA, and the subsequent reduction of target protein expression. The 5’-terminal phosphate group and the 3’-overhang of the guide strand were required to fully activate the RNAi activity of MED-siRNAs. Here, we evaluated MED-siRNAs modified with aryl phosphate groups at the 5’-end of the guide strand. The 5’-aryl phosphorylated MED-siRNAs showed highly efficient reduction of target protein expression comparable to 5’-phosphorylated MED-siRNAs. Moreover, 5’-aryl phosphorylated MED-siRNAs linked between the aryl phosphate group at the 5’-end of the guide strand and the hydroxyl group at the 3’-end of the passenger strand with alkyl amide linkers or peptides (e.g., DL-Ser-L-Ala-L-Tyr), resulted in single-stranded MED-siRNAs with a highly efficient cleavage activity of target mRNA with binding to Argonaute 2 via an RNA interference mechanism. These linker techniques could also be used to create siRNAs composed of naturally-occurring molecules such as amino acids. These findings suggest the possibility of using these single-stranded MED-siRNAs as siRNA reagents. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1927077 .

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2′-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'

Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [(3)H]-labeled dihydroS1P ([(3)H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [(3)H]dhS1P rather than [(3)H]dihydrosphingosine (the non-phosphorylated form of [(3)H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.

Summary Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI.

We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P₁ receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P₁ and S1P₃ agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P₁/S1P₃ selectivity. These changes of the S1P₁ and S1P₃ agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P₁ X-ray crystal structure and S1P₃ homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P₁/S1P₃ selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ∼10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.

We have proposed continuous and discrete time resolved acceleration control methods for underwater vehicle-manipulator systems and the effectiveness of the control methods have been shown by experiments. In this paper, we propose a digital type disturbance compensation control method based on the RAC method considering singular configuration of manipulator. Experimental results show the effectiveness of the proposed method.

Object Malignant gliomas are often highly invasive and can migrate along blood vessels. The purpose of the current study was to identify the substance in human serum and/or cerebrospinal fluid (CSF) that promotes glioma cell migration. Methods The authors used a Boyden chamber cell migration assay to study the effect of serum from patients with glioma and healthy volunteers on chemotaxis of A172 human glioma cells. Heat inactivation, trypsinization, and ultra-filtration of serum were used to establish the nature of the active factor. Vitronectin and fibronectin were chosen for further investigations; chemotactic effects were studied in both serum and CSF. Results Serum from both patients with glioma and healthy volunteers was found to promote chemotaxis of human glioma cells. This activity was greatly reduced by heat inactivation or trypsinization. Fractionation of the serum by ultrafiltration through membranes with various pore sizes showed that the active molecule was larger than 50 kD. Antibodies against integrin αv or αvβ5 or arginine-glycine-aspartic acid–containing peptides, both of which block the vitronectin–glioma cell interactions, significantly reduced serum-induced cell migration, whereas blocking the interaction of glioma cells with fibronectin had no effect. Furthermore, the ability of serum to promote the migration of A172 or T98G glioma cells was suppressed by immunodepletion of vitronectin and restored by the addition of exogenous vitronectin. The migration of glioma cells induced by CSF collected from the postoperative cavity of a malignant glioma patient was also reduced by blocking the interaction of glioma cells with vitronectin. Conclusions These results suggest that vitronectin is one of the major factors in serum- and CSF-induced glioma cell migration.

We have proposed continuous-time and discrete-time resolved acceleration control methods for underwater vehicle-manipulator systems and the effectiveness of the control methods has been shown by experiments. In this article, we propose a digital control method considering the singular configuration of manipulator. Experimental results show the effectiveness of the proposed method.

The small GTP-binding protein Rho and its target Rho-associated kinase trigger an intracellular signaling cascade that controls actin cytoskeleton and plays an essential role in cell motility and adhesion. A specific Rho-associated kinase inhibitor, Y-27632, has been reported to inhibit cancer invasion. Clinically, disseminated tumor cells in the cerebrospinal fluid invade the intraparenchymal region, damaging the brain and nerves, resulting in fatal brain stem dysfunction, despite intrathecal chemotherapy. To expand therapeutic options for this devastating neoplastic meningitis, we evaluated the potential use of intrathecal Y-27632 administration by employing Walker 256 cells, a rat mammary cancer cell line. Y-27632 dose-dependently inhibited chemotactic and invasive activity of Walker 256 cells. Y-27632 also inhibited the phosphorylation level of regulatory myosin light chain in vitro, but the effect was temporary and was considerably diminished within 16 hours. Y-27632 induced striking morphologic changes in Walker 256 cells, as evidenced by decreased cell-matrix adhesion in culture dishes and three-dimensional collagen I gels, and slightly inhibited colony formation in soft agar. Nevertheless, this drug treatment did not affect Walker 256 cell growth rate. We were able to administer continuous delivery of this inhibitor using an osmotic pump and maintaining drug concentration of 10 μmol/L within the brain. Importantly, this concentration of Y-27632 showed minimal neurotoxicity both in vitro and in vivo. We found that an intrathecal therapy, combining 5-fluoro-2′-deoxyuridine with Y-27632, significantly increased the survival time of rats bearing meningeal carcinomatosis in comparison with animals treated with 5-fluoro-2′-deoxyuridine alone. Taken together, our findings indicate that continuous intrathecal administration of Y-27632 could be a promising therapeutic method when combined with chemotherapy for treating human neoplastic meningitis.

Our laboratory has employed replication-defective herpes simplex virus type 1 gene transfer vectors for treatment of animal models of human malignant glioblastoma. The base vectors were defective for the immediate early (IE) genes ICP4, ICP27, and ICP22 but expressed the IE gene ICP0, which can arrest tumor cell division, and an IE thymidine kinase (alpha-tk) gene construct that mediates suicide gene therapy (SGT) in the presence of ganciclovir (GCV). Previously, we reported that SGT using ICP0/alpha-tk vectors in nude mouse models of glioblastoma was improved by coexpression of the gap-junction-forming protein connexin43 (Cx43) or human tumor necrosis factor alpha (TNF alpha). We also showed that further gains in therapeutic outcome could be achieved by combining TNF alpha-enhanced SGT with gamma-knife radiosurgery (GKR). To expand these observations, we have first repeated these studies in immunocompetent rats with brain tumors derived from implanted 9L gliosarcoma cells and second compared the most efficient vector from this study with a new recombinant vector, NUREL-C2, which expressed both TNF alpha and Cx43 along with ICP0 and alpha-tk. Results from the first part indicated that our ICP0/alpha-tk/TNF alpha vector in combination with GKR provides an effective therapy although this treatment was not statistically better than GKR combined with the ICP0/alpha-tk/Cx43 vector. Our observations in the second part suggested that NUREL-C2 may be more effective than the ICP0/alpha-tk/TNF alpha vector in combination treatments with GCV (P = 0.08) or GCV plus GKR (P = 0.10). GKR significantly enhanced the efficacy of NUREL-C2/GCV treatment (P = 0.02) as well as other virus/GCV treatments (P < or = 0.05). Conversely, the efficacy of GKR was significantly improved by both the ICP0/alpha-tk/TNF alpha vector and NUREL-C2 in combination with GCV (P = 0.02 and P < 0.01, respectively). Together these results indicate that NUREL-C2 may be an attractive candidate for Phase I gene-therapy safety studies in patients with recurrent malignant glioblastoma.

Herpes simplex virus type 1 (HSV-1) deleted for the immediate-early gene was applied for treatment of hepatoma cells of SKHep 1 and Huh-7. Hepatoma cells were cultured in medium containing HSV1 expressing GFP gene (QOZ/HG) to determine its transfection rate, and both cell lines infected by MOI 1 of QOZ/HG were found to have high expression of GFP without cytotoxicity. Subcutaneous growth of SKHep 1 cell tumor in nude mice was significantly reduced by injection of replicative-deficient herpes virus (TOZ.1) containing Tk-gene with administration of GCV, in comparison with that of noninjected tumor. SCID mice of peritonitis carcinomatosis due to Huh-7 hepatoma cells infected with TOZ.1 could survive longer under administration of GCV than those without TOZ.1. Therefore replicative-deficient HSV1 is a useful vector for treatment of human hepatoma cells, and TOZ.1 with GCV may be applied to suicide gene therapy for hepatoma and peritonitis carcinomatosis of hepatoma cells.

We have previously reported that repeated intravenous administration of cis-diamminedichloroplatinum(CDDP)prevented local recurrence of metastatic brain tumors after surgical total excision; however, data on CDDP distribution in the postoperative cavity after intravenous CDDP administration are not available. In the present study, we evaluated the penetration of total platinum(Pt)into the cerebrospinal fluid(CSF)and the effect of intravenous administration of 20%mannitolon total Pt distribution in the plasma and CSF. Total Pt levels in the plasma and CSF were determined immediately after intravenous infusion of CDDP(80mg/m2)for 1 hour, with or without pre-intravenous infusion of 20% mannitol(200mL), in 11 patients with brain metastasis from lung cancer. CSF samples were obtained via Ommaya reservoirs placed in the anterior horn of the lateral ventricle(CSF-V)and the postoperative cavity(CSF-C). Spinal CSF (CSF-L)was also obtained in the last 4 patients of the series via spinal drainage. CDDP was administered intravenously without mannitol 10 days after brain tumor excision, and 1 week after the initial administration of CDDP, CDDP was intravenously administered again after intravenous mannitol administration. CDDP was always administered intravenously at 1:00 PM to rule out the influence of circadian variation. Plasma and CSF were sequentially sampled after intravenous CDDP administration, and their Pt levels were analyzed for Pt content by using atomic absorption spectroscopy. The area under the concentration time curve(AUC)was calculated for plasma and CSF using the moment method. The AUC for total Pt in plasma showed a significantly higher level with the administration of mannitol than without the administration of mannitol(p0.01, paired t-test). Total Pt levels(AUCs and peak concentrations)in CSF-C were much higher than were those in CSF-V and CSF-L both with and without the administration of mannitol. The ratio(%)of CDDP penetration into CSF(CSF AUC/plasma AUC)was much higher for CSF-C than for CSF-V(p0.0001)both with and without mannitol administration. However, the CSF penetration ratio with mannitol administration did not differ significantly from that without mannitol administration. Thus, the administration of mannitol did not significantly increase the penetration ratio of total Pt. Creatinine clearance was moderately reduced in all patients during the first administration of CDDP. However, 7 of the 11 patients showed an increase in creatinine clearance after the second administration of CDDP with mannitol.

We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P₁ receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P₁ and S1P₃ agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P₁/S1P₃ selectivity. These changes of the S1P₁ and S1P₃ agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P₁ X-ray crystal structure and S1P₃ homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P₁/S1P₃ selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

This article concerns experiments with a free-floating underwater robot with a two-dimensional, horizontal planar, two-link manipulator. Some dynamic models of underwater manipulators have been proposed, but only a few experiments have been carried out. Here, we derive a dynamic model for a free-floating underwater robot with a two-link manipulator, including the hydrodynamic forces, and validate the effectiveness of the model by simulation and experiment. We also show an experimental result using a resolved acceleration control method. These experimental results show the effectiveness the model and the control method.

We have previously shown that the Fe(II) tetrakis-N,N,N′,N′-(2-pyridylmethyl)ethylenediamine complex (Fe(II)TPEN) has high superoxide dismutase (SOD) activity, using the xanthine–xanthine oxidase–cytochrome c method (cyt. c method) [J. Biol. Chem. 264 (1989) 9243–9249]. X-ray analysis showed that Fe(II)TPEN has two different coordination structures, one in which Fe(II) is coordinated with six nitrogens of TPEN and one in which Fe(II) is coordinated with five nitrogens of TPEN and one oxygen of sulfate anion as the counter anion [Chem. Pharm. Bull. 48 (2000) 223–230]. To investigate the relationship between these two structures and SOD activity, we synthesized novel Fe(II) complexes of TPEN analogues and measured their SOD activity by the cyt. c method. The Fe(II) tetrakis-N,N,N′,N′-(2-pyridylmethyl)trimethylenediamine complex (Fe(II)TPTN) and the Fe(II) tris(2-pyridylmethyl)triazacyclononane complex (Fe(II)TPTCN) had no SOD activity (IC50=>100 μM), probably because these two complexes have undistorted steric structure with no easily substituted ligand. On the other hand, other Fe(II) complexes with unsaturated coordination or an easily substituted ligand had high SOD activity (IC50=0.4–20 μM). The results indicate that the substitution reaction of a ligand with superoxide or the coordination of superoxide is essential for Fe(II)TPEN analogue complexes to have SOD activity. Moreover, we examined the effect of steric hindrance of the ligands on the SOD activity and the stability of the iron complexes to oxygen.

Experiments were carried out in a nude mouse model of human glioblastoma to determine whether gamma-knife radiosurgery combined with herpes simplex virus thymidine kinase (tk) suicide gene therapy and tumor necrosis factor alpha (TNFalpha) gene transfer provided an improved multimodality treatment of this disease. Animals were inoculated intracerebrally with 2 x 10(5) U-87MG human glioblastoma cells to establish brain tumors. At 3 days postinoculation, the tumor region was injected with 2 x 10(6) infectious particles of highly defective herpes simplex viral vectors expressing the viral tk gene with the kinetics of a viral immediate early gene either alone (T.1) or together with TNF alpha (TH:TNF). Subgroups of animals were given daily intraperitoneal injections of ganciclovir (GCV) for 10 days and/or subjected to gamma-knife radiosurgery on the fifth day post tumor-cell implantation. Comparisons of animal survival showed that the TH:TNF vector in combination with radiosurgery and GCV administration provided the most effective therapy; eight of nine animals survived for 75 days compared to four of eight using the next best protocol. These findings suggest that gene therapy in combination with more conventional therapeutic methods may provide an improved strategy for extending the life expectancy of patients afflicted with this ultimately fatal disease.

It is well known that phenytoin can cause impairment of cellular immunity. The authors investigated the potential role of other anticonvulsant drugs in the development of antitumor immunity in murine malignant glioma models. The survival rate was determined in murine glioma models using syngeneic 203 glioma cells following treatment with four anticonvuisants, which are most commonly administered to glioma patients, i.e., phenytoin, phenobarbital, valproate and zonisamide. In a second set of experiments, we further examined the effect of these drugs on interferon-γ (IFN-γ) secretion by lymphocytes prepared from cervical lymph nodes (CLN) in the same models. The IFN-γ production of CLN lymphocytes as measured by ELISA method was markedly impaired in the early stage of tumor-bearing mice treated with phenytoin or zonisamide, and the median survival time (MST) of controls and of mice treated with either phenytoin or zonisamide was 13, 10 and 11 days, respectively, which was not a statistically significant difference. Phenobarbital and valproate did not affect either IFN-γ production or their survival rate. In addition, immunohistochemistry showed a reduction in tumor-infiltrating lymphocytes containing CD4 and CD8 antigens in the mice treated with phenytoin and zonisamide. Two anticonvulsants, phenytoin and zonisamide, showed a significant inhibitory effect on IFN-γ production by CLN lymphocytes in murine glioma models, although there was no statistically significant difference in MST between controls and the anticonvulsant-treated mice. These drugs might have some detrimental influence on the prognosis of brain tumor patients when combined with the latent immune dysfunction accompanying the tumor-bearing state.

We report herein, efficient and practical synthetic methods for the preparation of enantiomerically pure spiro[((2 S )-hydroxy)indane-1,4′-piperidine] ( S )- 1 , a key intermediate for the synthesis of a tachykinin receptor antagonist, using catalytic asymmetric reduction or catalytic asymmetric epoxidation as a key step.

The intracranial migration of transduced glioma cells was investigated in order to improve the treatment of malignant glioma by gene therapy using retroviral vectors. In this study, about half the volume of the tumor mass could be transduced in 14 days after only a single implantation of 3 x 10(5) retrovirus-producing cells into a tumor mass with a diameter of 5 mm. Moreover, we were able to follow the migration of glioma cells transduced by the lacZ-harboring retroviruses originating from the high-titer retrovirus-producing cells. Besides the importance of using a high-titer retroviral vector system, our results also indicate that the implantation site of the virus-producing cells and the interval between the implantation of the virus-producing cells and the subsequent administration of ganciclovir are important factors for the efficient killing of glioma cells.

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

We investigated the delivery of foreign genes into mouse glioma cells in vivo using hemagglutinating virus of Japan (HVJ)-liposomes, which are coated by Sendai virus envelope protein. HVJ-liposomes, containing lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene-bearing plasmid DNA were applied in the meningeal gliomatosis (MG) mouse model system. Highly efficient delivery was observed in disseminated glioma cells, and 80% of MG mice expressing the HSVtk gene were cured by treatment with ganciclovir. These results suggest that this novel gene delivery system may be applicable for the in vivo gene therapy of human malignant glioma.

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

The "bystander effect" describes the killing of nearby unmodified cells and herpes simplex thymidine kinase (HTK)-transduced cells by ganciclovir (GCV) treatment. This effect is thought to be produced by contact between these cells. In this study, we showed that injected glioma cells migrated rapidly to a place distant from the injection point whereas injected virus-producing fibroblast cells did not migrate in a murine brain model. Moreover, the initially injected glioma cells and glioma cells injected at a later time mix very well, even at a place distant from the injection point. This suggested that glioma cells migrating after injection could be targeted by HTK-modified glioma cells introduced in a second injection and be killed together by GCV treatment. Therefore, we injected HTK-modified glioma cells 3 days after injection of wild glioma cells to investigate whether wild-type glioma cells that migrated to a place distant from the injection point could also be killed by GCV treatment. Tumor growth was suppressed after the GCV treatment. Suppression of tumor growth of wild glioma cells is not solely mediated by the immune response, which may be triggered by the killing of HTK-modified glioma cells with GCV, because inoculation of HTK-modified glioma to the contralateral side followed by GCV treatment did not cure the initial wild glioma. Moreover, the migration of the second inoculum of glioma cells is necessary for effective killing, because early administration of GCV resulted in insufficient killing.

Intravenous gene transfer using recombinant retroviruses tends to suffer from a low infectious viral titer when conducted in vivo. This is, in part, caused by complement‐mediated proteolytic inactivation of the retrovirus in human serum. However, if the retroviruses were directly injected into the brain, they might not be inactivated. Supernatant from amphotropic retrovirus‐producing cells harboring the BAG vectors was incubated with sera or cerebrospinal fluid (CSF) of patients with gliomas or unrelated disorders. The retroviruses were severely inactivated in sera. However, no such inactivation was noted in CSF or fluid from the tumor bed of glioma patients. These data suggest that gene transfer using recombinant retroviruses could be done into the cavity after removal of the tumor in glioma patients.

We report herein, efficient and practical synthetic methods for the preparation of enantiomerically pure spiro[((2 S )-hydroxy)indane-1,4′-piperidine] ( S )- 1 , a key intermediate for the synthesis of a tachykinin receptor antagonist, using catalytic asymmetric reduction or catalytic asymmetric epoxidation as a key step.

Intrathecal administration of MTX and/or Ara-C through the Ommaya system placed in a ventricle has been accepted as a standard therapy for leptomeningeal carcinomatosis. Recently, sustained-release cytosine arabinoside, a depot cytosine arabinoside liposomal injection: DepoCyt, has been reported to be useful for the treatment of leptomeningeal carcinomatosis in Canada, Europe, and the USA. Phase I study of NS-101, DepoCyt in Japan, was performed in patients with leptomeningeal carcinomatosis from solid tumors using the continuous reassessment method (CRM). NS-101 was administered twice through the Ommaya system placed in the lateral ventricle with an interval of 2 weeks. A dose of 25 mg was initially tried in a patient and then a dose of 50 mg was administered in 8 patients. The recommended dose of NS-101 was estimated from pharmacodynamics and tolerance from CRM, and the clinical effectiveness of NS-101 was also assessed. We also compared the present data with the reported data from Europe and the USA. As a result, free Ara-C was maintained over the estimated effective concentration for 2 weeks, and no pharmacodynamic differences were confirmed among Japan, Europe and the USA. Maximum tolerated dose was determined to be 50 mg. Complete cytological response of cerebrospinal fluid (CSF) was obtained in a patient, and CSF cytological conversion of either ventricle or lumbar CSF from positive to negative was observed in four patients. Neurological improvements in 3 pts and no sign of worsening in one patient were observed, and grade IV toxicity based on National Cancer Institute-Common Toxicity Criteria was not encountered. In conclusion, NS-101 was effective for leptomeningeal carcinomatosis from solid tumors and the estimated maximum tolerated dose was 50 mg as approved in Europe and the USA.

Experimental results of impurity behavior in the closed loop experimental facility (CLEF) are presented in this paper. The closed loop has been constracted to demonstrate an MHD power generation under a long duration time. In order to make the power generation successful, a molecular impurity level in a working gas of argon should be kept as low as possible. The present experimental results have shown that the procedure of replacing contaminated argon with pure argon is very useful to reduce impurity concentrations. At the same time, the results have shown a good performance of O2 removal system and of molecular sieve H2O removal system. Furthermore, it was found that the concentration of H2O decreased with an increase of ceramic temperature in the high temperature argon heater. It is suggested that the concentrations of H2O, N2 and O2 will be kept within a maximum permissible level for MHD power generation if the pressure inside the closed loop is kept higher than the atmospheric pressure.

OBJECTIVE: Previously, we reported a good clinical treatment effect of intrathecal chemotherapy by repeated bolus administration of 5-fluoro-2′-deoxyuridine (FdUrd) for neoplastic meningitis (NM). Moreover, we detected no side effects or neurotoxicity despite the long-term repetition of intrathecal administration. On the basis of these findings, continuous intrathecal chemotherapy (CIC) with FdUrd for patients with NM was attempted using a simple pump system. We evaluated the usefulness of CIC with FdUrd for the treatment of NM. METHODS: A total of 25 patients were enrolled in this study. FdUrd (1.0 mg/d) was administered using a balloon pump system. CIC was continued as long as possible. Eight patients received whole-brain irradiation (3 Gy × 10) simultaneously with CIC. The effects of the treatment were analyzed in terms of improvement in neurological signs and symptoms and the findings of ventricular and lumbar cerebrospinal fluid analysis 2 and 4 weeks after CIC was initiated and on magnetic resonance imaging scans 2 months after CIC began. RESULTS: No apparent toxicity has been observed to date. Evidence of a cerebrospinal fluid response was observed in 13 patients. Headache and nausea were improved in all patients, and cranial nerve impairment was improved in 12 patients. A magnetic resonance imaging response was observed in only 5 patients. Overall response was observed in 15 patients when cases of stable disease were excluded from the responding cases. Survival time from the commencement of CIC (mean ± standard error of the mean) was 255 ± 30 days in 25 patients. CONCLUSION: This therapy may be useful, especially as a maintenance therapy for NM.

Gliomatosis cerebri (GC), which mainly consists of oligodendroglial tumour cells, is rare and six cases have been documented to date. We present a new case of oligodendroglial gliomatosis cerebri with a favourable response to combination treatment using surgery, radiotherapy and chemotherapy.

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.

Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate-early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor-bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect.

Implantation of retrovirus-producing cells within a tumor has been demonstrated to eliminate malignant brain tumors effectively in animal models. In our previous study, the implantation of high-titer retrovirus-producing fibroblasts into tumors resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. It is also possible, however, that the implantation of murine fibroblast-derived virus-producing cells may induce an immune response in patients. In this study, we prepared retroviruses carrying the herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4--2.5 x 10(11) colony-forming units (c.f.u.)/ml, and stereotactically inoculated only 3 microl of the HTK-bearing retroviruses into the brain tumors of mice. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty percent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas.

Tumor cell transduction with the herpes simplex virus (HSV) thymidine kinase (tk) gene and treatment with ganciclovir (GCV) is a widely studied cancer gene therapy. Connexin (Cx)-dependent gap junctions between cells facilitate the intercellular spread of TK-activated GCV, thereby creating a bystander effect that improves tumor cell killing. However, tumor cells often have reduced connexin expression, thus thwarting bystander killing and the effectiveness of TK/GCV gene therapy. To improve the effectiveness of this therapy, we compared an HSV vector (TOCX) expressing Cx43 in addition to TK with an isogenic tk vector (TOZ.1) for their abilities to induce bystander killing of Cx-positive U-87 MG human glioblastoma cells and Cx-negative L929 fibrosarcoma cells in vitro and in vivo. The results showed that low-multiplicity infection of U-87 MG cells with TOCX only minimally increased GCV-mediated cell death compared with infection by TOZ.1, consistent with the endogenous level of Cx in these cells. In contrast, bystander killing of L929 cells was markedly enhanced by vector-mediated expression of Cx. In vivo experiments in which U-87 MG cells were preinfected at low multiplicity and injected into the flanks of nude mice showed complete cures of all animals in the TOCX group following GCV treatment, whereas untreated animals uniformly formed fatal tumors. TOCX injection into U-87 MG intradermal and intracranial tumors resulted in prolonged survival of the host animals in a GCV-dependent manner. Together, these results suggest that the combination of TK and Cx may be beneficial for the treatment of human glioblastoma.

Retroviral vector is the most commonly used vector for gene therapy. However, its use is limited because of the low titer. To overcome this problem, we improved the packaging cell line and concentrated the viral supernatant by the low-speed centrifugation and obtained retroviral titers up to 1012cfu/ml. This high titer preparation enabled us to transduce foreign genes efficiently into the neural cells, which are generally resistant to the transduction. It was also possible to massively transduce RSV- glioma cells transplanted into the mouse brain in vivo. This study showed that high-titer retrovirus was able to overcome the limitation of infectivity to the nervous system both in vitro and in vivo.

Serum-induced inactivation of retroviruses is the most critical limitation for in vivo gene transfer therapy. To solve this problem, we searched for reagents that protect retroviruses from inactivation. The effects of the protease inhibitors FOY-007 and FOY-305 and of an inhibitor of the complement pathway FUT-175, all of which have been used clinically, were investigated. All of these agents protected against the inactivation of retroviruses by human serum, with 1 microM FUT-175 providing the most effective protection. Thus, the co-administration of FUT-175 with retroviruses may make retrovirus-mediated in vivo gene transfer feasible for the treatment of patients. FUT-175 dose-dependently inhibited the classical pathway of complement in a hemolysis protection assay of sensitized sheep erythrocytes with guinea pig serum or by cell-lysis assay of mouse fibroblasts with human serum. However, increasing the FUT-175 concentration by 10-fold (10 microM) did not produce further protection against retroviral inactivation in most human sera. There was also no correlation between the serum-induced inactivation of retroviruses and either the amount of anti-alpha-galactosyl (anti-alpha-Gal) antibody or the complement activity in human serum. These results suggest that retroviruses are not inactivated by utilizing the same pathway leading to cell lysis by the classical complement system.

We have demonstrated that retrovirus-mediated herpes simplex virus type I (HTK) genes transferred to mouse glioma cells caused those glioma cells to be selectively sensitive to ganciclovir (GCV) in which myelin basic protein (MBP) promoter controlled these killer genes (MBP 1.3/pNT230) in vitro. For this study, to know what percentage of retrovirus-transduced glioma cells is sufficient to kill most other wild glioma cells in vivo, we made an in vivo glioma model that contained several percentages (0%, 10%, 25%, 50%, and 100%) of retrovirus-transduced glioma cells. Mouse glioma models with 1 × 105 wild-type Rous sarcoma virus (RSV-M) glioma cells developed a large tumor mass in their brain even with the GCV treatment. However, the tumor mass was much smaller in a model containing 25% HTK-transduced cells, and the glioma model containing 50% HTK-transduced cells caused small tumor mass formation only in half of this group. Control mice that received only HTK-transduced glioma cells without GCV treatment suffered large tumor masses in the same way as did mice that received totally wild-type RSV-M glioma cells with GCV treatment. Therefore, glioma-affected mice may be rescued if one-fourth of the glioma cells are transduced with the MBP 1.3/pNT230 retroviruses. We will investigate mechanisms of this phenomenon (bystander effect).

To construct recombinant retroviruses with only a single active promoter, we introduced point mutations into the TATA box region of the 3'-LTR, and successfully obtained high-titer virus with sufficient self-inactivating activity. However, the viral titer could not be determined by the number of G418 resistant colon es since the neomycin resistance gene was under 5'-LTR control, because of inactivation of the selection marker in target glioma cells. To overcome this problem, we constructed PCR primers with homology to a gene under the control of the internal promoter of recombinant retrovirus, and to retrovirus-specific sequences. There was good correlation between the amount of PCR-amplified product and the number of colony forming units when glioma cells were transduced with the retroviruses containing both the neomycin resistance gene and the HTK gene. Amplified PCR products quantitated by densitometry after glioma cells were transduced with SIV retrovirus vectors, and there was good correlation between density and sensitivity to GCV following transduction. Therefore, detection of HTK PCR products from glioma cells transduced with HTK-bearing retroviruses is useful for determining the appropriate packaging cell for efficient production of viral particles. This detection system is especially useful for isolating high titer clones producing SIV-type retroviruses.

Glioblastomas are characterized by aggressive invasion into normal brain tissues, thus limiting complete removal by surgical resection resulting in recurrence. Our identified molecule involved in glioma invasion is important for developing targeted effective therapies in the malignant glioma. Cancer stem cells are capable for self-renewing and are thought to be responsible for tumor metastasis and dissemination. However, the invasive activities of cancer stem cells have not been fully understood. To investigate a role of glioma stem cells in the tumor invasion, tumor sphere culture was established from mouse glioma RSV-M cells. The high tumorigeneity and aggressive migration of RSV-M tumor sphere were observed in intracranial transplantation. Quantitative RT-PCR analysis showed the enhanced expression of metastasis-related genes in RSV-M tumor sphere. Of these, MUC18/MCAM also highly expressed in tumor sphere derived from human glioma cell lines. Importantly, CD133-positive fraction of glioma sphere culture isolated from glioblastoma patients contained a cell population expressing MUC18 but CD133-negative fraction not. To examine a function of MUC18 in glioma cells, the expression or knockdown construct for the gene were introduced into human glioma cells. Over-expression of MUC18 in the glioma cells increased their invasive capacity and clonogenicity in comparison to the parent or knockdown cells. In addition, expression of some metastasis-related genes was up-regulated in MUC18-over-expressing cells. These results suggest that enhanced MUC18 expression of glioma stem cells may contribute to the aggressive invasion of this tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3402. doi:10.1158/1538-7445.AM2011-3402

Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We first searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes by in silico and RT-PCR screening. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter activity was measured in glioma, telomerase-immortalized fibroblast and NHA cells. The SSX4 promoter but not MAGEA3 showed the tumor-specific activity. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomerase-immortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mouse. The administration of GCV suppressed the tumor growth completely. These results suggest that trans-acting factors may play a role for tumor-specific activity of SSX4 promoter while the expression of most CTA genes is regulated by epigenetic factors and the retrovirus vector expressing HSVtk under control of SSX4 promoter is a promising therapeutic reagent for malignant brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 587.