Multinational, double-blind, randomised, placebo-controlled, prospective study of esomeprazole in the prevention of recurrent peptic ulcer in low-dose acetylsalicylic acid users: the LAVENDER study.

OBJECTIVES: To evaluate if esomeprazole prevents recurrent peptic ulcer in adult patients with a history of peptic ulcer receiving low-dose acetylsalicylic acid (ASA, aspirin) for cardiovascular protection in East Asia. METHODS: In this prospective, randomised, double-blind, placebo-controlled trial conducted in Japan, Korea and Taiwan, eligible patients receiving low-dose ASA for cardiovascular protection (81-324 mg/day) were randomised to esomeprazole 20 mg/day or placebo for ≤72 weeks. All patients received concomitant mucosal protection (gefarnate 100 mg/day). The primary endpoint was time to ulcer recurrence (Kaplan-Meier analysis). Efficacy findings are presented up to week 48, as per a planned interim analysis within the study protocol. RESULTS: A total of 364 patients (79.9% men; mean age, 67.1 years) comprised the full analysis set (esomeprazole, n=182; placebo, n=182). There was a statistically significant difference in the time to ulcer recurrence between esomeprazole and placebo (HR 0.09; 96.65% CI 0.02 to 0.41; p<0.001). The estimated ulcer-free rate at week 12 was 99.3% (esomeprazole) and 89.0% (placebo). The high estimated ulcer-free rate for esomeprazole was maintained through to week 48 (98.3% vs. 81.2% of placebo-treated patients). No factors, other than female gender, reduced time to ulcer recurrence in addition to the effect of esomeprazole (p<0.001). Treatment with esomeprazole was generally well tolerated. CONCLUSIONS: Daily esomeprazole 20 mg is efficacious and well tolerated in reducing the recurrence of peptic ulcer in East-Asian patients with a history of ulcers who are taking low-dose ASA for cardiovascular protection. CLINICALTRIALGOV IDENTIFIER: NCT01069939.

Effects of omeprazole on symptoms and quality of life in Japanese patients with reflux esophagitis: final results of OMAREE, a large-scale clinical experience investigation.

BACKGROUND: For patients with reflux esophagitis (RE), endoscopic findings alone (without the frequency and severity of symptoms) may not fully reflect the associated impact on health-related quality of life (QOL). There is not enough data about symptoms and QOL of Japanese patients with RE. The present study therefore investigated the epidemiological characteristics of such patients, and evaluated the efficacy and safety of omeprazole (and other gastrointestinal drugs, except proton pump inhibitors [PPIs]) in terms of improving patients' symptoms and QOL. METHODS: In a large-scale, specific clinical experience investigation of Japanese patients with RE, epidemiological characteristics, QOL and symptoms of the disease in relation to treatment with omeprazole and other gastrointestinal drugs, except PPIs, and safety data of omeprazole were collected. The Quality Of Life in Reflux and Dyspepsia questionnaire (QOLRAD) was used for QOL assessment. RESULTS: 9967 patients were included in the analysis (omeprazole: 7888). At baseline, 75.2% of patients had three or more upper gastrointestinal symptoms, and 31.5% of patients had six or more upper gastrointestinal symptoms. The overall mean QOLRAD score at baseline was 5.14 (the best score is 7). In the omeprazole group, the rate of satisfactory improvement in subjective symptoms was 61.7% and 81.8% at Weeks 4 and 8, respectively, and these were both significantly higher than those of patients treated with other drugs. In both the omeprazole group and the other drugs group, the QOLRAD score at Week 4 improved significantly from baseline, and the degree of improvement was significantly greater in the omeprazole group than in the other drugs group. The favourable tolerability profile of omeprazole was confirmed. CONCLUSION: In a large-scale survey, omeprazole improved symptoms and QOL more effectively in Japanese patients with RE than other investigated drugs, and had a good tolerability profile. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00859287.

Efficacy and Safety of Oral Budesonide in Patients with Active Crohn's Disease in Japan: A Multicenter, Double-Blind, Randomized, Parallel-Group Phase 3 Study.

BACKGROUND: US and European guidelines recommend budesonide for the treatment of mild-to-moderate active ileocolic Crohn's disease (CD). However, budesonide has not been approved, and mesalazine is widely used as first-line treatment in Japan. The objective of this study was to evaluate the efficacy and safety of budesonide in patients with mild-to-moderate active CD in Japan. METHODS: In this phase 3 noninferiority study (NCT01514240), 112 patients with a baseline Crohn's Disease Activity Index (CDAI) score of 180-400 were randomized to budesonide or mesalazine for 8 weeks. Assessments included remission rate (CDAI score ≤150) at weeks 2, 4, and 8, change in CDAI score, health-related quality of life (measured using the Inflammatory Bowel Disease Questionnaire [IBDQ]), and tolerability. RESULTS: The remission rate at week 8 was numerically higher in the budesonide group (30.4%) than in the mesalazine group (25.0%), and the noninferiority of budesonide to mesalazine was shown. The mean total CDAI score decreased to a greater extent with budesonide than with mesalazine. Mean IBDQ scores improved from baseline to weeks 2, 4, 8, and 10 in both groups; improvements were numerically higher with budesonide than with mesalazine. No safety concerns were found. CONCLUSION: Budesonide is comparably effective to mesalazine in the treatment of Japanese patients with mild-to-moderate active CD.

Efficacy and tolerability of oral budesonide in Japanese patients with active Crohn's disease: a multicentre, double-blind, randomized, parallel-group Phase II study.

BACKGROUND AND AIMS: Current treatments for Japanese patients with active Crohn's disease have not proved optimal, and new treatment options are required. The present study therefore evaluated the efficacy and tolerability of oral budesonide in Japanese patients with mild-to-moderate active Crohn's disease. METHODS: In this multicentre, double-blind, randomized, parallel-group, Phase II study, patients (18-65 years) with baseline Crohn's Disease Activity Index (CDAI) score≥200 were randomized to once-daily (od) oral budesonide 9 mg or 15 mg, or matching placebo, for 8 weeks. Concomitant therapy with sulfasalazine or 5-aminosalicylic acid, and nutritional therapy, was allowed. The rate of remission (defined as CDAI score≤150) after 8 weeks' treatment (primary variable), health-related quality of life (assessed using the Inflammatory Bowel Disease Questionnaire [IBDQ]), and tolerability were assessed. RESULTS: 77 patients were randomized and 63 completed the study. The proportion of budesonide-treated patients with remission after 8 weeks' treatment was higher compared with placebo (23.1%, 28.0%, and 11.5% for budesonide 9 mg, 15 mg, and placebo, respectively; no significant difference). The mean change from baseline to week 8 in CDAI total score (-48.0, -58.2, and -27.2, respectively) and IBDQ total score (10.8, 23.2, and 6.5, respectively) was greater for budesonide-treated patients than placebo recipients. While budesonide 9 mg and 15 mg demonstrated similar efficacy, budesonide 9 mg caused fewer drug- and glucocorticosteroid-related adverse events and less adrenal suppression. CONCLUSIONS: Oral budesonide 9 mg od (for up to 8 weeks) may offer a new treatment option for Japanese patients with mild-to-moderate active Crohn's disease.

Transdermal Delivery of Small Interfering RNA with Elastic Cationic Liposomes in Mice.

We developed elastic cationic liposomal vectors for transdermal siRNA delivery. These liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid and sodium cholate (NaChol) or Tween 80 as an edge activator. When NaChol or Tween 80 was included at 5, 10, and 15% (w/w) into DOTAP liposomal formulations (C5-, C10-, and C15-liposomes and T5-, T10-, and T15-liposomes), C15- and T10-liposomes showed 2.4- and 2.7-fold-higher elasticities than DOTAP liposome, respectively. Although the sizes of all elastic liposomes prepared in this study were about 80-90 nm, the sizes of C5-, C10- and C15-liposome/siRNA complexes (lipoplexes) were about 1,700-1,800 nm, and those of T5-, T10-, and T15-lipoplexes were about 550-780 nm. Their elastic lipoplexes showed strong gene suppression by siRNA without cytotoxicity when transfected into human cervical carcinoma SiHa cells. Following skin application of the fluorescence-labeled lipoplexes in mice, among the elastic lipoplexes, C15- and T5-lipoplexes showed effective penetration of siRNA into skin, compared with DOTAP lipoplex and free siRNA solution. These data suggest that elastic cationic liposomes containing an appropriate amount of NaChol or Tween 80 as an edge activator could deliver siRNA transdermally.

Cell line-dependent internalization pathways determine DNA transfection efficiency of decaarginine-PEG-lipid.

Previously, we have reported that decaarginine-conjugated PEG-lipids (R10B) efficiently delivered plasmid DNA (pDNA) into human cervical carcinoma HeLa cells via macropinocytosis; however, the mechanism of cellular uptake by R10B was not evaluated in other cell lines. In this study, we investigated the internalization mechanism by R10B/pDNA complex (R10B-lipoplex) in human prostate tumor PC-3 and human nasopharyngeal tumor KB cells, and compared with that in HeLa cells. Although it was necessary for R10B-lipoplex to associate with heparan sulfate (HS) on the cell surface in all cell lines, the R10B-lipoplex was internalized primarily through clathrin-mediated endocytosis in PC-3 and KB cells, and macropinosytosis in HeLa cells. In HeLa cells, treatment with the R10B-lipoplex induced the formation of lamellipodia for macropinocytosis, but did not in KB and PC-3 cells. Furthermore, the highest transfection efficiency by R10B-lipoplex was observed in HeLa cells. These findings indicated that the R10B-lipoplex induced the formation of lamellipodia in HeLa cells after binding to HS on the cells and was then internalized by macropinocytosis, which could induce high gene expression because of escaping degradation in lysosomes. Cell physiology might be a critical factor in cellular internalization and efficient transfection by cell penetration peptide.

Preclinical study of a "tailor-made" combination of NK4-expressing gene therapy and gefitinib (ZD1839, Iressa) for disseminated peritoneal scirrhous gastric cancer.

We evaluated the effect of a "tailor-made" chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailor-made approach, we first selected gefitinib (epidermal growth factor receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric fibroblasts, NF22, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between fibroblasts and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n = 8), 11.9; treated (n = 8), 17.3; p = 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p = 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p = 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, 22; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination.

15-Deoxy-delta12,14-prostaglandin J2, a ligand for peroxisome proliferators-activated receptor-gamma, induces apoptosis in human hepatoma cells.

BACKGROUND/AIMS: 15-deoxy-delta12,14-prostaglandin J2 (15-d-PGJ2) induces apoptosis in several carcinoma cell lines and is a potent activator of peroxisome proliferators-activated receptor-gamma (PPAR-gamma). In the present study, we examined the effect of 15-d-PGJ2 on human hepatoma cells. METHODS: HuH-7 and HepG2 cell lines were used in all the experiments. The mRNA expression of PPAR-gamma was studied by reverse transcriptase-polymerase chain reaction. The cell viability was determined by a modified MTT assay. Two methods were used for the determination of apoptosis in hepatoma cells: the TUNEL assay, and detection of fragmented mono- and oligo-nucleosomes by ELISA. RESULTS: The expression of PPAR-gamma mRNA and protein was detected in HuH-7 and HepG2. Treatment with 15-d-PGJ2 decreased cell viability in a time- and dose-dependent manner. 15-d-PGJ2 induced apoptosis and this effect was time-dependent. Exposure of cells to 15-d-PGJ2 induced caspase-3 and -9 activation. Furthermore, co-treatment with the pan-caspase inhibitor Z-VAD-FMK or the caspase-3 inhibitor Z-DEVD-FMK blocked apoptosis of human hepatoma cells that had been treated with 15-d-PGJ2. CONCLUSIONS: Our study demonstrates that PPAR-gamma is expressed in human hepatoma cell lines and that treatment with 15-d-PGJ2 inhibits the growth of these cells by inducing apoptosis through caspase activation.

Enhanced photodynamic antitumor effect on gastric cancer by a novel photosensitive stealth liposome.

Lipophilic photosensitizers hold potential for cancer photodynamic therapy. We sought to develop a novel photosensitive stealth liposome (PSSL) which incorporating a lipophilic photosensitizer into its lipid bilayer and to examine its photoactivity. We prepared PSSL composed of lipophilic chlorin e6 (Ce6) ester, dilauroylphosphatidylcholine, dioleoylphosphatidylethanolamine and distearoyl-phosphoethanolamine-N-[poly (ethylene glycol) 2000] and evaluated its photodynamic effect against gastric cancer cell lines and tumor-bearing nude mice models. In gastric cancer cell lines, LC(80) of PSSL was a maximum of 53 times as low as that of Ce6 sodium salt (Ce6-Na). PSSL completely destroyed all tumors in animal models and tumor recurrence levels were minimal (1.5 +/- 0.9%). PSSL achieved greater photodynamic effects in gastric cancer cell lines and in murine models than Ce6-Na. PSSL holds promise for photodynamic therapy for gastric cancer.

Therapeutic effect of photodynamic therapy using PAD-S31 and diode laser on human liver cancer cells.

BACKGROUND/AIMS: Photodynamic therapy (PDT) is an effective local cancer treatment which a photosensitizer is administered and the tumor is irradiated with light. We examined the effect of PDT using PAD-S31 as the photosensitizer, and the 670 nm diode laser on human hepatocellular carcinomas (HCCs). METHODS: Huh-7, HepG2 and Hep3B cell lines were used in the all experiments. Cell viability was determined by a modified MTT assay. Two methods were used for the determination of apoptosis: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay and detection of fragmented mono- and oligo-nucleosomes by enzyme-linked immunosorbent assay. The caspase activity was measured by fluorometric assay. Cytochrome c in cytosolic fraction was determined using a human cytochrome c immunoassay. Xenografts of human oral HCC cells were generated in KSN S1c nude mice. RESULTS: In vitro PDT showed excellent cytotoxicity that was a function of laser energy, drug concentration and time to the hepatoma cell lines. The combined use of PAD-S31 and laser irradiation showed excellent anti-tumor activity without severe side-effect against human hepatoma xenografts in nude mice. PDT-mediated cell death occurred predominantly by apoptosis in vitro and in vivo. Furthermore, this treatment initiates early cytochrome c release, followed by late caspase-3 and -9 activation. CONCLUSION: Our study demonstrates that PDT using PAD-S31 and the diode laser induces apoptosis that is mediated by cytochrome c release and caspase activation in human liver cancer cell lines. It is expected that this therapy will be clinically useful for the treatment of patients with HCC.

Possible involvement of p38 in mechanisms underlying acceleration of proliferation by 15-deoxy-Delta(12,14)-prostaglandin J2 and the precursors in leukemia cell line THP-1.

15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ2), which is a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), induced apoptosis of several human tumors including gastric, lung, colon, prostate, and breast. However, the role of PPARgamma signals in other types of cancer cells (e.g., leukemia) except solid cancer cells is still unclear. The aim of this study is to evaluate the ability of 15dPGJ2 to modify the proliferation of the human leukemia cell line THP-1. 15dPGJ2 at 5 microM stimulated the proliferation in THP-1 at 24 to 72 h after incubation. In contrast, 15dPGJ2 at concentrations above 10 microM inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2, and Delta12-PGJ2 (DeltaPGJ2), precursors of 15dPGJ2, had similar proliferative effects at lower concentrations, whereas they induced apoptosis at high concentrations. 15dPGJ2 and three precursors failed to induce the differentiation in THP-1 as assessed by using the differentiation marker CD11b. FACScan analysis revealed that PGD2 at 5 microM, PGJ2 at 1 microM, DeltaPGJ2 at 1 microM and 15dPGJ2 at 5 microM all accelerated cell cycle progression in THP-1. Immunoblotting analysis revealed that PGD2 at 5 microM and 15dPGJ2 at 5 microM inhibited the expression of phospho-p38, phospho-MKK3/MKK6, and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p21, and p27 in THP-1. In contrast, PGJ2 at 1 microM and DeltaPGJ2 at 1 microM did not affect their expressions. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 mitogen-activated protein kinase pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of the THP-1 proliferation.

Induction of proliferation by 15-deoxy-delta12,14-prostaglandin J2 and the precursors in monocytic leukemia U937.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in several human tumors including gastric, lung, colon, prostate and breast. However, the role of PPARgamma signals in leukemia is still unclear. The aim of this study is to evaluate the ability of 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2), that is a ligand for PPARgamma, on proliferation of human leukemia cell line U937. 15dPGJ2 at 5 micromol/l stimulated the proliferation. In contrast, 15dPGJ2 at concentrations of >10 micromol/l inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2 and Delta12-PGJ2 (DeltaPGJ2), those are precursors of 15dPGJ2, had similarly proliferative effects, whereas they showed antiproliferative effects at high concentrations. FACScan analysis revealed that PGD2 at 5 micromol/l, PGJ2 at 1 micromol/l, DeltaPGJ2 at 1 micromol/l and 15dPGJ2 at 5 micromol/l, all accelerated cell cycle progression. Immunoblotting analysis revealed that PGD2 at 5 micromol/l and 15dPGJ2 at 5 micromol/l inhibited the expression of phospho-p38, phospho-MKK3/MKK6 and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p27. In contrast, PGJ2 at 1 micromol/l and DeltaPGJ2 at 1 micromol/l did not affect the expression of them. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 MAPK pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of proliferation.

Photodynamic therapy for human oral squamous cell carcinoma and xenografts using a new photosensitizer, PAD-S31.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of photosensitizer and visible light. We examined the effect of PDT using a new photosensitizer, PAD-S31, and the 670-nm diode laser in human oral squamous cell carcinomas (SCC). STUDY DESIGN/MATERIALS AND METHODS: SAS and HSC-4 cell lines were used in all the experiments. Cell viability was determined by a modified MTT assay. Two methods were used for the determination of apoptosis in human oral SCC cells: TUNEL assay and detection of fragmented mono- and oligo-nucleosomes by ELISA. Xenografts of human oral SCC cells were generated in KSN S1c nude mice. RESULTS: In vitro PDT using PAD-S31 and the 670-nm diode laser showed cytotoxicity that was a function of laser energy, drug concentration, and time to the SAS and HSC-4 cell lines. On the other hand, PAD-S31 without irradiation had no effect on cell viability. The combinated use of PAD-S31 and the laser irradiation showed excellent anti-tumor activity against tumor xenografts without severe side effects. PDT-mediated cell death occurred predominantly by apoptosis in vitro and in vivo. CONCLUSIONS: The present study demonstrates that PAD-S31 may serve as a potent photosensitizer for PDT. Furthermore, it is expected that this therapy will be clinically useful for the treatment of patients with oral carcinoma.

Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury.

During chronic liver injury, transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-beta-induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-beta signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-beta increased plasminogen activator inhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-beta were observed in HSCs. Thereafter, Smad 7 induced by TGF-beta negatively regulated the Smad 2 action. These results indicated that endogenous TGFbeta-mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-beta signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-beta signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-beta signal requiring Smad 2 phosphorylation by activated TGF-beta receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-beta under a low level of Smad 7 could be involved in the progression of liver fibrosis.