Therapeutic effect of CS-706, a specific cyclooxygenase-2 inhibitor, on gallbladder carcinoma in BK5.ErbB-2 mice.

Biliary tract cancer is still challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect the early tumor formation. The most well known risk factor for gallbladder cancer is the presence of chronic inflammation, usually related to gallstones. It has been suggested that cyclooxygenase-2 (COX-2) plays a variety of roles in the gastrointestinal tract, including pathogenic processes such as neoplasia. Recently, we have generated transgenic mice that overexpress rat ErbB-2 under the control of bovine keratin 5 promoter (BK5.ErbB-2 mice). Homozygous BK5.ErbB-2 mice develop adenocarcinoma of gallbladder with an approximately 90% incidence. In addition to the activation of ErbB-2 and epidermal growth factor receptor, mRNA and protein levels of COX-2 were up-regulated in the gallbladder carcinomas that developed in these transgenic mice. The aim of this study was to examine the effects of a COX-2 inhibitor, CS-706, on the development of gallbladder carcinomas using the BK5.ErbB-2 mouse model. Ultrasound image analysis as well as histologic evaluation revealed a significant therapeutic effect of CS-706 on the gallbladder tumors, either as reversion to a milder phenotype or inhibition of tumor progression. The antitumor effect was associated with inhibition of prostaglandin E(2) synthesis. CS-706 treatment also down-regulated the activation of ErbB-2 and epidermal growth factor receptor, resulting in decreased levels of phosphorylated Akt and COX-2 in gallbladder cancers of BK5.ErbB-2 mice. Based on our results, targeting COX-2 could provide a potentially new and effective therapy alone or in combination with other therapeutic agents for patients with biliary tract cancer.

Inhibition of VEGF mRNA by 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides and their influence on off-target gene expressions.

We investigated 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found in the GenBank database by BLAST and CLUSTAL W searches. Three of these genes were actually inhibited by the ENA AON. In shorter ENA AONs with fewer matched sequences of these genes, inhibitiory activities were decreased and off-target effects were improved. These results indicate that ENA AONs act in a sequence-specific manner and could be used as effective antisense drugs.

Syntheses of glucose derivatives of E5564-related compounds and their LPS-antagonistic activities.

Glucose analogues 6, 12, 17b, 19a, and 19b of E5564 were synthesized, and their LPS-antagonistic activities were measured. The antagonistic activities (IC(50)) on LPS-induced TNFalpha production of these five compounds toward human whole blood were 72.8, 3.0, 0.9, 7.5, and 1.4nM, respectively. Inhibitory doses (ID(50)) of compounds 12, 17b, 19a, and 19b on TNFalpha production induced by co-injection of galactosamine and LPS in C3H/HeN mice in vivo were measured. The values of these compounds were 0.9, ND (not determined), 1.6, and 0.9mg/kg, respectively.

Syntheses of glucose analogues of E5564 as a highly potent anti-sepsis drug candidate.

Glucose analogues 5 and 9 of E5564 were synthesized, and their LPS-antagonistic activities were measured. The inhibitory activities (IC50) on LPS-induced TNFalpha production of these two compounds towards human whole blood cells were 0.06 and 0.83 nM, respectively. Inhibitory doses (ID50) of compounds 5 and 9 on TNFalpha production induced by coinjection of galactosamine and LPS in C3H/HeN mice in vivo were measured and were 0.55 and <0.20 mg/kg, respectively. And also C3H/HeN mice preinjected with compounds 5 and 9 were protected from lethality induced by coinjection of galactosamine and LPS; out of eight mice preinjected with 1 mg/kg of the compounds, one-six and three of eight mice were protected, respectively.

2'-O,4'-C-ethylene-bridged nucleic acid (ENA) for effective antisense formation.

ENA antisense oligonucleotides for vascular endothelial growth factor (VEGF) mRNA were synthesized and evaluated in A549 lung cancer cells. It was found that the VEGF ENA-antisense inhibited not only the expression of VEGF, but also the expression of three genes, which were found in Genbank by BLAST and Clustal W search and considered likely to bind to the VEGF ENA-antisense. These results indicate that ENA-antisense oligonucleotides act in a sequence-specific manner, and could be used as effective antisense drugs.

Synthesis of lipid A type carboxymethyl derivatives with ether chains instead of ester chains and their LPS-antagonistic activities.

Synthesis of lipid A type carboxymethyl derivatives having ether chains at both the C-3 and C-3' positions and their LPS-antagonistic activities toward human U937 cells are described.

Synthesis of ceramidated GLA-60 derivatives.

Ceramidated GLA-60 derivatives 11 and 11' were synthesized from 1 via glycosidation of ceramide derivative 12 as a glycosyl acceptor with GLA-60 derivative 5 as a glycosyl donor, and successive conversion. Compound 11' showed only weak LPS-antagonistic activity without showing any LPS-agonistic activity.

Practical stereoselective synthesis of alpha-linked C-glucosamine propionic acid esters: conversion to GLA-60 derivatives.

Radical C-glycosylation of glucosamine derivatives by acrylic acid esters gave the corresponding 3-(alpha-C-glucosyl)-propionate derivatives in moderate yields. One of them was used as a versatile synthon for GLA-60 derivatives. However, the biological activity of these compounds as LPS-antagonists was disappointing.

Down-regulation of VEGF mRNA expression by 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) antisense oligonucleotides and investigation of non-target gene expression.

We studied the properties of 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) oligonucleotides as antisense molecules. Compared to a phosphorothioate (PS) DNA and RNA heteroduplex, a duplex of an ENA/PS/ENA gapmer with RNA was a more effective substrate for RNase H-mediated cleavage. We designed ENA antisense oligonucleotides (AON) targeting human vascular endothelial growth factor (VEGF) mRNA. ENA AON with a cationic polymer considerably down-regulated VEGF mRNA expression, while no down-regulation by PS AON was observed. We also investigated the influence on the gene expression of genes other than VEGF by a gene database homology search and RT-PCR analysis. This method turned out to be an efficient approach to search for non-target genes sequence-specifically down-regulated by AON. In rat plasma, an ENA/PS/ENA gapmer was very stable after 24 hr, while a bridged nucleic acid (BNA) or locked nucleic acid (LNA) oligonucleotide and a PS oligonucleotide were half degraded in 4 hr. The high stability of ENA oligonucleotides in plasma would make ENA oligonucleotides ideal for in vivo studies.