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Cancer cells require oxygen and nutrients for growth, making angiogenesis one of the essential components of tumor growth. Gangliosides, constituting membrane lipid rafts, regulate intracellular signal transduction and are involved in the malignancy of cancer cells. While endothelial cells, as well as cancer cells, express vast amounts of gangliosides, the precise function of endothelial gangliosides in angiogenesis remains unclear. In this study, we focused on gangliosides of vascular endothelial cells and analyzed their functions on tumor angiogenesis. In human breast cancer, GM3 synthase was highly expressed in vascular endothelial cells as well as immune cells. Angiogenesis increased in GM3S-KO mice. In BAEC, RNA interference of GM3S showed increased cellular invasion and oxidative stress tolerance through activation of ERK. In the breast cancer model, GM3-KO mice showed an increase in tumor growth and angiogenesis. These results suggest that the endothelial ganglioside GM3 regulates tumor angiogenesis by suppressing cellular invasion and oxidative stress tolerance in endothelial cells.

Background: Tumor hypoxia drastically changes cancer phenotypes, including angiogenesis, invasion, and cell death. Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on plasma membranes and are involved in many biological processes, such as the endoplasmic reticulum stress response and apoptosis. In this study, we investigated the regulation and function of glycosphingolipids, which associate with lipid raft on mammalian plasma membranes under hypoxic condition. Methods: B16F10 melanoma cells were subjected to chemical hypoxia and low pO2 condition, and the effect of hypoxia on expression of GM3 synthase were analyzed. Cellular resistance to oxidative stress was analyzed in GM3S-KO B16F10 cells. Results: Hypoxia treatment decreased the expression of ganglioside GM3 synthase (GM3S; ST3GAL5), which synthesizes the common substrate of ganglioside biosynthesis. RNA interference of hypoxia inducible factor 1 subunit alpha (HIF-1α) inhibited hypoxia-induced GM3S suppression. Additionally, GM3S deficiency increased cellular resistance to oxidative stress and radiation therapy via upregulation of ERK. Conclusions: Altered synthesis of glycosphingolipids downstream of HIF-1α signaling increased the resistance of melanoma cells to oxidative stress. Furthermore, GM3 has important role on cellular adaptive response to hypoxia. General significance: This study indicates that tumor hypoxia regulates therapy-resistance via modulation of ganglioside synthesis.

Introduction The pharmacokinetics, pharmacodynamics, and safety and tolerability profile of etelcalcetide (ONO-5163/AMG 416), a novel, i.v., long-acting calcium-sensing receptor agonist, were studied in Japanese hemodialysis patients with secondary hyperparathyroidism. Methods This multicenter, randomized, double-blind, placebo-controlled, parallel-group study consisted of a single dose part and a multiple dose (3 times weekly for 4 weeks) part. Major inclusion criteria were hemodialysis for at least 90 days, serum intact parathyroid hormone (iPTH) ≥ 300 pg/ml, and serum albumin-corrected Ca (cCa) ≥ 9.0 mg/dl. There were 3 single-dose cohorts (n = 6 each) randomized 2:1 to 5, 10, or 20 mg etelcalcetide or placebo, and 2 multiple-dose cohorts (n = 11 each) randomized 8:3 to 2.5 or 5 mg etelcalcetide or placebo. Results Etelcalcetide plasma concentration decreased rapidly after i.v. administration, generally remained stable from 24 hours postdose to the next dialysis, and then decreased by dialysis. Etelcalcetide exposure increased dose proportionally. Etelcalcetide plasma predialysis concentration reached almost steady state at week 4. A single dose of etelcalcetide dose-dependently reduced serum iPTH in 30 minutes, and the reduction reached a plateau at 1 hour that lasted until 8 hours. The percent change from baseline serum iPTH thereafter showed a trend to gradually decrease; it was still −30% or greater on day 3. Similar results were obtained at the last injection (days 27–29) of the multiple dose. The effect of the multiple dose was sustained during the interdialytic period. Etelcalcetide decreased serum cCa in a more gradual but dose-dependent and sustained manner. Discussion Etelcalcetide dose-dependently reduced serum iPTH and serum cCa. Moreover, the effect was sustained in the interdialytic period.

ONO-2952, a novel antagonist of translocator protein 18 kDa (TSPO), binds with high affinity to TSPO in rat brain and human tumor cell line membrane preparations. This study used the TSPO-specific PET radioligand [

We examined the efficacy and safety of inactivated influenza vaccine when the amount of HA influenza vaccination in children was increased to the dose recommended by the WHO. The purpose of this study was to obtain basic evidence to review the vaccination dose in Japanese children. HA influenza vaccine produced by the Research Foundation for Microbial Diseases of Osaka University (Biken) licenced in Japan was administered through vaccination at the international dose, and split HA influenza vaccine produced by Sanofi Pasteur corp. (Sanofi) was used as control. Children from 6 months to less than 13 years of age were registered, and vaccinated with doses of 0.25 mL or 0.5 mL. Clinical symptoms during the influenza season were monitored to investigate vaccine efficacy, and information on adverse reactions was collected to evaluate safety profile. Paired serum HI and NT antibody titers were measured at pre first dose and post second dose of vaccination. Both HI and NT antibody titers for H1N1 subtype were satisfactory elevated after administration of both vaccines. Elevation of the NT antibody titer for the H3N2 subtype was observed for both vaccines, but the H3N2 HI antibody titer for the Biken vaccine was not so high. For the subtype B virus, the NT titer had a better response than the HI titer for both vaccines. As only the H1N1 virus was prevalent in the area during the study period, we performed factor analysis concerning influenza contraction only for the H1N1 antibody titer. An HI titer of 1 : 40 or more at post-vaccination was a significant factor to lower the risk of influenza contraction. The relative risk for fever among children with an HI titer of 1 : 20 or less was significantly higher than those with an HI titer of 1 : 40 or more. Children with a higher HI titer had better prevention against fever, so that both vaccines were considered to be effective. As for the appearance of adverse reactions, both vaccines were considered to be safe. From the above-mentioned results, vaccination with the Japanese Biken vaccine at an international dose was thought to be an effective and safe procedure.

We analyzed the current status of clinical pharmacokinetic (PK) studies in new drug applications (NDA) by evaluating 17 cardiovascular (CV) drugs that were approved between 2000 and 2007 in Japan since CV drugs were one of the largest categories to be submitted. All summaries of data had been submitted by applicants and were freely accessible on the website of the Pharmaceuticals and Medical Devices Agency (PMDA). The clinical PK studies were divided into 12 categories: single-dose, multiple-dose, food-effect, elderly, gender difference, mass balance, absolute bioavailability, target patients, liver impairment, renal impairment, drug-drug interaction, and population PK studies. The median number of clinical PK studies per new molecular entity (NME) excluding single-dose, multiple-dose, and food-effect studies was 13.5, and foreign data accounted for 86% of the study results. A relatively high proportion of PK studies in Japanese subjects were observed in elderly volunteers and target patients; however, a dependency on foreign data was noted in the other PK study categories. The effect of the guidance“Clinical PK Studies of Pharmaceuticals”by the Ministry of Health, Labour and Welfare on implementation of clinical PK studies could not be fully assessed, since there were only three NMEs approved prior to issuance of the guidance. The median number of clinical PK studies per NME was approximately three times higher for drugs for lifestyle-related diseases than that for other drugs. In recent NDA reviews by the PMDA, the trend to emphasize a relationship between PK and safety with respect to drug-drug interactions and/or special populations was noted.

Pranlukast is a cysteinyl leukotriene receptor antagonist that has been used to treat bronchial asthma and allergic rhinitis. In vitro data suggest that pranlukast is a substrate of CYP3A4. Thus, the effect of clarithromycin, a potent CYP3A4 inhibitor, on the pharmacokinetics of pranlukast was examined in an open-label, randomized, two-way crossover study in 16 healthy male volunteers. In treatment A, volunteers received a single, 225 mg dose of pranlukast. In treatment B, 200 mg of clarithromycin was administered twice daily for 7 days and a single, 225 mg dose of pranlukast was coadministered on day 7. Blood samples were collected up to 24 hours after treatment, and pranlukast concentrations in the plasma were measured. The geometric mean ratios [GMR] (90% confidence intervals [CIs]) for pranlukast AUC(0-infinity) and C(max) (with/without clarithromycin) were 1.06 (0.91, 1.24) and 1.17 (0.95, 1.45), respectively. In conclusion, clarithromycin and pranlukast could be coadministered without dose adjustment because clarithromycin minimally affected the pharmacokinetics of pranlukast.

To characterize Japanese encephalitis virus (JEV) strains recently prevalent in Japan, JEV surveillance was performed in pigs from 2002 to 2004. Eleven new JEV isolates were obtained and compared with previous isolates from Japan and other Asian countries. All of the isolates were classified into genotype 1 by nucleotide sequence analysis of the E gene. Two new isolates with different levels of neurovirulence and neuroinvasiveness, but with only one nucleotide difference in the E gene, Sw/Mie/34/2004 and Sw/Mie/40/2004, were isolated at the same farm on the same day. Sw/Mie/40/2004 displayed higher neurovirulence and neuroinvasiveness in mice than the other four new isolates. Another new isolate, Sw/Hiroshima/25/2002, was neutralized by antiserum to Beijing-1 at a level similar to the homologous Beijing-1 strain, whilst seven other new isolates were neutralized at 10-fold-lower titres. However, there were no amino acid differences in the E protein among these eight isolates. The present study indicated that the 11 new JEV isolates were genetically similar, but biologically and serologically heterogeneous.

The liquid chromatography-mass spectrometry (LC-MS) methods were developed and validated for the determination of testosterone (T) in the brain and serum of rats and of 5alpha-androstane-3alpha,17beta-diol (ADIOL), a metabolite of T, in the brain of rats. After derivatization of T with 2-hydrazino-1-methylpyridine and of ADIOL with p-nitrobenzoyl chloride, the detection sensitivities of T and ADIOL using LC-MS were increased 70- and 400-times superior to those of intact T and intact ADIOL, respectively. Those LC-MS methods are specific and reliable for the analysis of trace amounts of T and ADIOL in small amounts of samples. The animal studies using the developed methods showed that the brain and serum levels of T and the brain levels of ADIOL were not changed by stress or ethanol administration but the concentration ratio of the brain T to serum T in the stressed rats was higher than that in untreated rats. The low levels of endogenous AIDOL in brain of stressed and unrestrained rats found in this study demonstrated that the contribution to anesthetic and anxiolytic effects of ADIOL via gamma-aminobutyric acid type A receptors may be negligible.

Derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) has been examined. Under APCI conditions, gas-phase electrons are provided by the corona discharge and captured by electron-affinitive compounds. In negative APCI-MS, therefore, ultrahigh sensitivity can be obtained by tagging neutral steroids, whose ionization efficiencies are low in the conventional APCI-MS, with electron -capturing moieties, such as a nitro group. We synthesized various boronic acid and hydrazine derivatives having electron-capturing moieties as derivatization reagents for 1,2-diol compounds and oxosteroids, respectively. Among reagents examined, those having the 2-nitro-4-trifluoromethylphenyl moiety were most effective in increasing sensitivity. That is, the detection responses of the derivatives with these reagents were increased by several to more than 200-fold over intact steroids, where limits of detection were some picograms. The developed derivatization procedures were applied to analyses of small amounts of steroids in human plasma and gave satisfactory results.

ONO-2952, a novel antagonist of translocator protein 18 kDa (TSPO), binds with high affinity to TSPO in rat brain and human tumor cell line membrane preparations. This study used the TSPO-specific PET radioligand [11 C]PBR28 to confirm binding of ONO-2952 to brain TSPO in human subjects, and evaluate brain TSPO occupancy and its relationship with ONO-2952 plasma concentration. Sixteen healthy subjects received a single oral dose of 200, 60, 20, or 6 mg ONO-2952 (n = 4 per dose). Two PET scans with [11 C]PBR28 were conducted ≤7 days apart: at baseline and 24 h after ONO-2952 administration. [11 C]PBR28 regional distribution volume (VT ) was derived with kinetic modeling using the arterial input function and a two tissue compartment model. Nonspecific binding (VND ) was obtained on an individual basis for each subject using linear regression as the x-intercept of the Lassen plot. The binding potential relative to VND (BPND ) was derived as the difference between VT in the ROI (VT ROI) and VND , normalized to VND ; BPND = (VT ROI - VND )/VND . TSPO occupancy was calculated as the change in BPND (ΔBPND ) from individual's baseline scan to the on-medication scan to the baseline BPND value. TSPO occupancy by ONO-2952 was dose dependent between 20-200 mg, approaching saturation at 200 mg both in the whole brain and in 15 anatomic regions of interest (ROI). Estimated Ki values ranged from 24.1 to 72.2 nM. This open-label, single-center, single-dose study demonstrated engagement of ONO-2952 to brain TSPO. The relationship between pharmacokinetics and TSPO occupancy observed in this study support the hypothesis that ONO-2952 could potentially modulate neurosteroid production by binding to brain TSPO.

Reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR were used to detect 14 (6.6%) influenza C virus (InfC) among 213 clinical samples collected from children with respiratory symptoms in Mie Prefecture, Japan, between January 2012 and December 2012. Virus isolation using Madin-Darby canine kidney cells and/or embryonated chicken eggs was also successful for 3 of the 14 PCR-positive samples. Eleven patients (78.6%) were aged

Landiolol hydrochloride is a newly developed cardioselective, ultra short-acting beta(1)-adrenergic receptor blocking agent used for perioperative arrhythmia control. The objective of this study was to characterize the population pharmacokinetics of landiolol hydrochloride in healthy male subjects. A total of 420 blood concentration data points collected from 47 healthy male subjects were used for the population pharmacokinetic analysis. NONMEM was used for population pharmacokinetic analysis. In addition, the final pharmacokinetic model was evaluated using a bootstrap method and a leave-one-out cross validation method. The concentration time course of landiolol hydrochloride was best described by a two-compartment model with lag time. The final parameters were total body clearance (CL: 36.6 mL/min/kg), distribution volume of the central compartment (V1: 101 mL/kg), inter-compartmental clearance (16.1 mL/min/kg), distribution volume of the peripheral compartment (55.6 mL/kg), and lag time (0.82 min). The inter-individual variability in the CL and V1 were 21.8% and 46.3%, respectively. The residual variability was 22.1%. Model evaluation by the two different methods indicated that the final model was robust and parameter estimates were reasonable. The population pharmacokinetic model for landiolol hydrochloride in healthy subjects was developed and was shown to be appropriate by both bootstrap and leave-one-out cross validation methods.

The liquid chromatography-mass spectrometry (LC-MS) methods were developed and validated for the determination of testosterone (T) in the brain and serum of rats and of 5alpha-androstane-3alpha,17beta-diol (ADIOL), a metabolite of T, in the brain of rats. After derivatization of T with 2-hydrazino-1-methylpyridine and of ADIOL with p-nitrobenzoyl chloride, the detection sensitivities of T and ADIOL using LC-MS were increased 70- and 400-times superior to those of intact T and intact ADIOL, respectively. Those LC-MS methods are specific and reliable for the analysis of trace amounts of T and ADIOL in small amounts of samples. The animal studies using the developed methods showed that the brain and serum levels of T and the brain levels of ADIOL were not changed by stress or ethanol administration but the concentration ratio of the brain T to serum T in the stressed rats was higher than that in untreated rats. The low levels of endogenous AIDOL in brain of stressed and unrestrained rats found in this study demonstrated that the contribution to anesthetic and anxiolytic effects of ADIOL via gamma-aminobutyric acid type A receptors may be negligible.

A practical liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of prostatic 5alpha-dihydrotestosterone (DHT) and testosterone (T) has been developed. The prostatic androgens were extracted with MeOH-H2O (3:7, v/v), purified with an Oasis HLB cartridge, derivatized with the permanently charged reagent 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-MS-MS analysis using electrospray ionization (ESI) operated in the positive ion mode. The derivatization with HMP was very effective at increasing the detectability using the positive-ESI-MS. The method allowed the reproducible and accurate quantification of ng g(-1) tissue levels of prostatic androgens in 10 mg of tissue. That is, the intra- and inter-assay coefficients of variation were below 8.1 and 9.3%, respectively, and the analytical recoveries of the androgens were quantitative. The limits of quantitation for DHT and T were both 1.0 ng g(-1) tissue. The developed method was used to determine DHT and T in the prostates of patients with benign prostatic hyperplasia and prostate cancer, and satisfactory results were obtained.

A derivatization reagent, 2-hydrazino-1-methylpyridine, was developed for the liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of oxosteroids. The reagent quantitatively reacted with oxosteroids at 60 degrees C within 1h and the resulting derivatives of the mono-oxosteroids provided a 70-1600-fold higher sensitivity compared to intact steroids. However, HMP was unsuitable for di-oxosteroids, such as androstenedione and progesterone. The developed derivatization procedure was applied to the LC-ESI-MS analysis of 5alpha-dihydrotestosterone in human prostate, and allowed the reproducible quantification of nanogram/gram level of the androgen with a 10-mg sample.

The utility of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione (NPTAD) as a derivatization reagent in the analysis of 25-hydroxyvitamin D3 [25(OH)D3] using liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry (LC/ECAPCI-MS) was examined. The derivatives of 25(OH)D3 with NPTAD underwent electron capture in the APCI source in the negative-ion mode and provided 30-fold higher sensitivity compared to an intact compound. This derivatization-LC/ECAPCI-MS method was applied to a plasma 25-hydroxyvitamin D3 assay, and gave satisfactory results in sensitivity, specificity, measurable range and throughput of the analysis.

The Norwalk virus(NV) is widely known as a cause of nonbacterial food poisoning, infant diarrhea, and acute gastroenteritis in the winter months between November and March. While it is strongly suspected that NV that is excreted by humans flows into coastal seawaters via rivers and wastewater treatment facilities to contaminate oysters that are grown in farms in the area, light has yet to be shed on the behavior of this virus in the natural environment. We therefore conducted a polymerase chain reaction (PCR) survey of NV levels in the aquatic environment of the oyster bed area of the Shima region in Mie Prefecture, whereupon the NV was detected in marine sediment, oysters, and mule clams even during the summer months, when food poisoning is infrequent. In order to assess their similarity to human-derived strains, the detected viruses and their human-derived counterparts were subjected to genetic analysis, whereupon some of the detected viruses were found to be remarkably similar to those that were previously detected in humans infected with NV. In the interests of examining methods for decontaminating NV-contaminated oysters, we also conducted an assessment on a system of virus decontamination that focuses on seawater temperature and oyster metabolism, using Poliovirus Sabin strain. The decontamination system mentioned above was a closed loop, water circulating system, built on the same principles as those actually in use at oyster farms. Our experiment indicated that at seawater temperatures of both 10 degrees C and 20 degrees C, virus placed into the water tank was rapidly incorporated into the midgut glands of the oysters. Thereafter, when seawater irradiated with UV was circulated, the virus count in the oysters fell from 1/1,000 to 1/10,000 within 6 hours. These results indicated the utility of this system for virus decontamination, suggesting the possibility of significantly alleviating the risk of NV infection in humans by using this system to maintain the seawater temperature within the decontamination tank above a certain temperature, and to perform decontamination with an adequate water flow.

Vibrio parahaemolyticus isolated from the feces of patients with food poisoning contains in most cases either thermostable direct hemolysin (TDH) and/or TDHrelated hemolysin (TRH), both important pathogenic factors carried by this microorganism [1–3]. However, most organisms from brackish water, estuary mud or fish that cause food poisoning have neither the TDH production gene (tdh) nor the TRH production gene (trh) [1]. This seems to be because TDH-producing V. parahaemolyticus occurs in only small amounts in nature or foods compared to other Vibrio spp. In addition, it is difficult to grow in artificial media, since TDH-producing V. parahaemolyticus is in the viable but nonculturable (VNC) state due to nutrient limitation, low temperature, etc. [4, 5]. Therefore, TDH-producing V parahaemolyticus is rarely isolated from foods assumed to cause food poisoning that have a high probability of containing TDH-producing V. parahaemolyticus Accordingly, it is very difficult to quantitate TDH-producing V. parahaemolyticus by culture. In order to estimate the content of TDH-producing V. parahaemolyticus in suspect foods, we have developed a LightCycler assay to quantitate tdh.