Long-term safety and efficacy of fasiglifam (TAK-875), a G-protein-coupled receptor 40 agonist, as monotherapy and combination therapy in Japanese patients with type 2 diabetes: a 52-week open-label phase III study.

This multicentre, open-label, phase III study investigated the safety and efficacy of the G-protein-coupled receptor 40 agonist fasiglifam. Japanese patients with type 2 diabetes and inadequate glycaemic control despite diet and/or exercise (n = 282), or despite diet and/or exercise plus one oral antidiabetic agent [sulphonylurea (n = 262), rapid-acting insulin secretagogue (n = 124), α-glucosidase inhibitor (n = 141), biguanide (n = 136), thiazolidinedione (n = 139) or dipeptidyl peptidase-4 inhibitor (n = 138)] were randomized to treatment with fasiglifam 25 or 50 mg once daily for 52 weeks. The primary endpoints were safety variables. The overall incidence of treatment-emergent adverse events (TEAEs) was 75.4-85.1% in the 25 mg group and 78.9-89.9% in the 50 mg group; most TEAEs were mild. Hypoglycaemia was negligible with fasiglifam monotherapy and most common with sulphonylurea combination therapy (12.4 and 9.1% for 25 and 50 mg groups, respectively). Abnormal liver-related laboratory values were uncommon. Glycated haemoglobin levels decreased from week 2 in all groups and were maintained to week 52. Although fasiglifam as monotherapy or in combination regimens was well tolerated during long-term treatment, global concerns about liver safety led to termination of its development after study completion.

Efficacy and safety of fasiglifam (TAK-875), a G protein-coupled receptor 40 agonist, in Japanese patients with type 2 diabetes inadequately controlled by diet and exercise: a randomized, double-blind, placebo-controlled, phase III trial.

AIM: To assess the efficacy and safety of fasiglifam 25 and 50 mg in Japanese patients with type 2 diabetes inadequately controlled by diet and exercise. METHODS: This phase III, double-blind, placebo-controlled, multicentre study included 192 patients randomized to once-daily treatment with fasiglifam 25 mg (n = 63) or 50 mg (n = 62) or placebo (n = 67) for 24 weeks. The primary efficacy endpoint was the change from baseline in glycated haemoglobin (HbA1c) at week 24. RESULTS: At week 24, both fasiglifam groups had significantly reduced HbA1c levels compared with the placebo group (p < 0.0001). The least squares mean change from baseline in HbA1c was 0.16% with placebo, -0.57% with fasiglifam 25 mg and -0.83% with fasiglifam 50 mg. The percentage of patients who achieved an HbA1c target of <6.9% at week 24 was also significantly higher (p < 0.05) for fasiglifam 25 mg (30.2%) and 50 mg (54.8%) compared with placebo (13.8%). Fasiglifam significantly reduced fasting plasma glucose levels at all assessment points, starting from week 2. The incidence and types of treatment-emergent adverse events in each fasiglifam group were similar to those in the placebo group, and hypoglycaemia was reported in 1 patient receiving fasiglifam 50 mg. There were no clinically meaningful changes in body weight in any treatment group. CONCLUSIONS: Fasiglifam significantly improved glycaemic control and was well tolerated, with a low risk of hypoglycaemia in Japanese patients with type 2 diabetes inadequately controlled by diet and exercise; however, in a recent review of data from overall fasiglifam global clinical trials, concerns about liver safety arose and the clinical development of fasiglifam was terminated after this trial was completed.

Human epidermal growth factor receptor-2 and estrogen receptor expression, a demonstration project using the residual tissue repository of the Surveillance, Epidemiology, and End Results (SEER) program.

BACKGROUND: In 2001, the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program established Residual Tissue Repositories (RTR) in the Hawaii, Iowa, and Los Angeles Tumor Registries to collect discarded tissue blocks from pathologic laboratories within their catchment areas. To validate the utility of the RTR for supplementing SEER's central database, we assessed human epidermal growth factor receptor-2 (HER2) and estrogen receptor expression (ER) in a demonstration project. MATERIALS: Using a prepared set of tissue microarrays (TMAs) residing in the Hawaii Tumor Registry (HTR), we performed standard immunohistochemistry. Breast cancers in the TMA were diagnosed in 1995, followed through 2006, and linked to SEER's main database. RESULTS: The TMA included 354 cases, representing 51% of 687 breast cancers in the HTR (1995). The HTR and TMA cases were similar with respect to patient demographics and tumor characteristics. Seventy-six percent (76%, 268 of 354) of TMA cases were HER2+ and/or ER+, i.e., 28 HER2+ER-, 12 HER2+ER+, and 228 HER2-ER+. There were 67 HER2-ER- cases and 19 were unclassified. Age distributions at diagnosis were bimodal with dominant early-onset modes for HER2+ER- tumors and dominant late-onset modes for HER2-ER+ breast cancers. Epidemiologic patterns for concordant HER2+ER+ (double-positive) and HER2-ER- (double-negative) were intermediate to discordant HER2+ER- and HER2-ER+. CONCLUSION: Results showed contrasting incidence patterns for HER2+ (HER2+ER-) and ER+ (HER2-ER+) breast cancers, diagnosed in 1995. Though sample sizes were small, this demonstration project validates the potential utility of the RTR for supplementing the SEER program.

Oxidation of linoleic acid encapsulated with gum arabic or maltodextrin by spray-drying.

Linoleic acid was emulsified with gum arabic or maltodextrin at various weight ratios of the acid to the polysaccharide in the presence or absence of a small-molecule emulsifier. The emulsions were spray-dried to produce microcapsules. Emulsions prepared with gum arabic were smaller in droplet size and more stable than those prepared with maltodextrin, and linoleic acid in a gum arabic-based microcapsule was also most resistant to oxidation than that in a maltodextrin-based microcapsule. Although the oil droplet size in the emulsion with maltodextrin decreased and the emulsion stability was improved by addition of a small-molecule emulsifier to linoleic acid, the oxidative stability of the encapsulated linoleic acid was not significantly improved. Encapsulated linoleic acid of small droplet size oxidized more slowly than that of large droplet size.

Effects of medium-chain fatty acids on intracellular calcium levels and the cytoskeleton in human intestinal (Caco-2) cell monolayers.

The effects of medium-chain fatty acids (MCFA) on intracellular calcium (Ca2+) levels and actin filaments in the Caco-2 monolayer were investigated. A site-dependent increase in intracellular Ca2+ levels caused by decanoic acid (C10) at 13 mM was observed by confocal laser scanning microscopy. The area in which the intracellular Ca2+ levels was increased was measured by image analysis, and increased to 11% of the total area of the monolayer within 1 minute. This was maintained for 5 minutes, and decreased thereafter. The other MCFAs did not significantly increase the intracellular Ca2+ levels. Obvious morphological changes of actin filaments were induced by only C10 among C8-C14. The area in which actin filaments were depleted was also quantified, and the increase in area became significant after 40 minutes. The area of the actin-depleted spot corresponded to the area occupied by 5 to 10 cells as well as that in which the intracellular Ca2+ level was increased. The effectiveness of only C10 suggested that the mechanism of the absorption enhancement by C10 would be different from that by the other MCFAs, or that C10 has some additional physiological functions although the mechanism of the enhancement is the same as for the other MCFAs.

Involvement of TGF-beta in inhibitory effects of negatively charged liposomes on nitric oxide production by macrophages stimulated with lps.

We examined the role of TGF-beta in the inhibitory effects of negatively charged liposomes composed of phosphatidylserine (PS-liposomes) on nitric oxide (NO) production by macrophages stimulated with LPS. The expression of TGF-beta mRNA increased when mouse peritoneal macrophages were treated with PS-liposomes. The inhibitory effect of PS-liposomes on NO production was restored by treatment with anti-TGF-beta antibody. Furthermore, NO production, iNOS mRNA expression, and iNOS protein induction by LPS were inhibited by treatment of macrophages with TGF-beta as well as PS-liposomes. These results indicated that PS-liposomes down-regulate NO production by macrophages through the induction of TGF-beta and suggested that TGF-beta may suppress NO production upstream of the transcription of iNOS mRNA.

Involvement of p38 MAP Kinase in the inhibitory effects of phosphatidylserine liposomes on nitric oxide production from macrophages stimulated with LPS.

The mechanism by which liposomes composed of phosphatidylserine (PS-liposomes) inhibit nitric oxide (NO) production was investigated in vitro using mouse peritoneal macrophages stimulated with LPS. The expression of inducible NO synthase (i-NOS) mRNA was completely inhibited by PS-liposomes. PS-liposomes inhibited tyrosine phosphorylation of p38 MAP kinase, which is required for the activation of p38 MAP kinase. NO production was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, there was no effect on the activation of transcription factor NF-kappaB, a primary transcription factor involved in induction of i-NOS. These results suggested that PS-liposomes inhibit NO production up stream of the transcription of i-NOS mRNA, and that the inhibition of p38 MAP kinase is crucial for this effect.

Microbial production of 2-chloro-alpha-methylbenzyl alcohol and its adsorptive recovery.

An adsorptive process was combined with yeast-mediated production of chiral 2-chloro-alpha-methylbenzyl alcohol (o-Cl-1-PA) for effective product recovery and reuse of the reaction medium. Low temperature was suitable for long-term reactor operation, and continuous production using a shallow-bed reactor was achieved for at least 22 days while maintaining a high conversion. The appropriate size of the adsorption column for product recovery from the reactor effluent was estimated through measurement of breakthrough curves of o-Cl-1-PA in a packed bed of the resin at various adsorbate concentrations and feed flow rates. Using the adsorption column, 98% of the product and the residual substrate were recovered from the reactor effluent, and the effluent from the adsorption column was successfully reused as the reaction medium after microfiltration to save the medium consumption.

Effect of the molar ratio of an energy source to the substrate on yeast-mediated production of 2-chloro-alpha-methylbenzyl alcohol.

Pachysolen tannophilus cells immobilized in Ca-alginate gels were shown to catalyze the asymmetric reduction of acetophenone (AP) and chloroacetophenones (Cl-APs) to their corresponding alcohols. The position of the Cl-group on the aromatic ring of AP greatly affected the reaction rate, and o-Cl-AP was the most readily reduced. For the reduction of o-Cl-AP to 2-chloro-alpha-methylbenzyl alcohol, the effect of the molar ratio of the energy source, glucose, to the substrate was examined in both batch and continuous operations, and a molar ratio much lower than that conventionally used was found to be sufficient for the reduction.

Macrosialin may not be involved with the regulation of nitric oxide production from mouse peritoneal macrophages stimulated with LPS.

We reported previously that nitric oxide (NO) production from mouse peritoneal macrophages stimulated with LPS was inhibited by negatively charged liposomes composed of phosphatidylserine (PS-liposomes) or oxidized LDL (Ox-LDL) which have an affinity for scavenger receptors (SRs). In this study, to clarify whether macrosialin, an SR, participates in the inhibitory effects of PS-liposomes or Ox-LDL on NO production, the effects of anti-macrosialin monoclonal antibody (FA/11) on inhibition of NO production by PS-liposomes or Ox-LDL were investigated. FA/11 itself showed no effect on NO production induced by LPS. Binding of FA/11 to macrophages was inhibited by the addition of PS-liposomes or Ox-LDL, indicating that PS-liposomes or Ox-LDL have an affinity for macrosialin. However, the inhibitory effects of these ligands on NO production were not reduced in the presence of FA/11. These findings suggest that macrosialin does not contribute to the inhibitory effects of PS-liposomes or Ox-LDL on NO production from mouse peritoneal macrophages stimulated with LPS.

Autoxidation of Linoleic Acid Encapsulated with Polysaccharides of Differing Weight Ratio.

Linoleic acid was encapsulated with pullulan, maltodextrin and gum arabic at various weight ratios of the fatty acid to wall material by the hot-air-drying method. The autoxidative process of the encapsulated linoleic acids was observed at 37°C and at a relative humidity of 75%. The weight ratio strongly affected the autoxidative process, autoxidation being more suppressed with smaller ratios. The autoxidation process of encapsulated linoleic acid leveled off at fraction Y∞ of the unoxidized substrate within 15 days. The Y∞ value strongly depended on both the ratio and the wall material, and steeply decreased near the ratio of 0.75 for every wall material. The dependence of the Y∞ value on the weight ratio was analyzed by the two- and three-dimensional models for percolation theory. The two-dimensional model expressed well the experimentally observed dependence.

Recovery of Caco-2 Cell Monolayers to Normal from the Transport-enhanced State Induced by Capric Acid Sodium Salt and its Monoacylglycerol.

Caco-2 cell monolayers were used as a model of the intestinal epithelium to investigate the recovery profile from the transport-enhanced state induced by the transport enhancers, capric acid sodium salt (C10FANa) and capric acid monoacylglycerol (C10MG). The transepithelial electrical resistance, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay, and lactate dehydrogenase (LDH) release rate were investigated. The cell monolayer recovered depending on the concentration of the enhancer and on the exposure time. The MTT assay revealed that the cells recovered their mitochondrial dehydrogenase activity without proliferation. The cell monolayer exposed to C10FANa released LDH to both the apical and basolateral sides, but to C10MG, only to the apical side. The results were compared with those for SDS and taurocholic acid sodium salt, and the effect of C10FANa was found to be different. These results suggest that the damage by MCFA compounds is recoverable and that the recovery can be assessed by an MTT assay, but that the LDH-release behavior is different among the enhancers.

Contribution of CR3 to nitric oxide production from macrophages stimulated with high-dose of LPS.

The contribution of the complement receptor type 3 (CR3) to nitric oxide (NO) production from macrophages stimulated by LPS was investigated. When thioglycollate-elicited mouse peritoneal macrophages were stimulated with a high dose of LPS (10 micrograms/ml) in both the presence and absence of fetal calf serum, a source of LPS binding protein (LBP) necessary for the binding of LPS to CD14, NO production was observed. These findings suggest that CD14-dependent and CD14-independent signaling pathways for NO production are present in macrophages. Because binding and phagocytosis of bacteria by macrophages through the CR3 has been previously reported, we investigated whether the CR3 acts in CD14-independent signaling pathway for NO production. By flow cytometric analysis, the binding of FITC-labeled anti-CR3 monoclonal antibody (anti-CR3 mAb) to macrophages was inhibited by LPS. Anti-CR3 mAb induced iNOS protein and produced NO in a dose dependent manner. Further, NO production induced by anti-CR3 mAb was also inhibited by zymocel, beta-glucan with a high affinity to CR3. These results suggest that the CR3 molecule acts in a CD14-independent signaling pathway, and contributes to NO production by macrophages stimulated with high doses of LPS.

Physico-chemical Properties of Fatty Acids for Assessing the Threshold Concentration to Enhance the Absorption of a Hydrophilic Substance.

The effects of natural and non-natural fatty acids on enhancing the absorption of a hydrophilic marker through a human epithelial cell (Caco-2) monolayer were measured to elucidate the properties of the fatty acids. Fatty acids from C9 to C14 enhanced the absorption depending on the concentration and the carbon chain length. Those fatty acids with longer chains gave a higher permeability coefficient at low concentrations and a lower toxicity than those with shorter chains. The surface energy lowering coefficient (SELC), an intrinsic physico-chemical property, and the critical micellar concentration (CMC) were good criteria for identifying the threshold concentrations of a fatty acid to significantly enhance absorption.

The Relationship between Transport-enhancement Effects and Cell Viability by Capric Acid Sodium Salt, Monocaprin, and Dicaproin.

Using Caco-2 cell monolayers and MTT assay, the relationship between cell viability (a) and transport-enhancement effect of 1,2-dicaproin (C6DG), monocaprin (C10MG), and capric acid sodium salt (C10FANa) was examined. Transport enhancement effect was assessed by apparent permeability (P app) of penicillin V. There was a linear relationship between (P app-aS a) and (1-a) values, where S a was the apparent permeability for the viable cells. The apparent permeability for the damaged cells (S d) was evaluated from the slope of the line. Each of the enhancer compounds gave a different S d value 2.00×10(-4), 0.82×10(-4), and 0.10×10(-4) cm/s for C6DG, C10MG, and C10FANa, respectively, but the value was independent of its concentration for C10MG and C10FANa. C6DG would be the safest enhancer among the three compounds because of its high S d value at the low level of cell damage. S d could be used as a criterion for estimating the safety of enhancers.

NADPH-dependent reduction of ethyl acetoacetate coupled with ethanol oxidation in Kloeckera magna.

We evaluated the catalytic ability of 29 yeast strains to reduce ethyl acetoacetate (EA) in the presence of ethanol or glucose. In 18 yeast strains, the reduction in the presence of ethanol proceeded as well as in the presence of glucose. Among them, Kloeckera magna (AKU 4704) effectively catalyzed the NADPH-dependent reduction of EA in the presence of ethanol. In this reduction, 1 mol of EA was reduced by consuming 1 mol of ethanol. We found that the NADPH regeneration system responsible for EA reduction in K. magna was coupled with oxidation of acetaldehyde to acetic acid catalyzed by an NADP(+)-dependent aldehyde dehydrogenase.

Scavenger receptors may regulate nitric oxide production from macrophages stimulated by LPS.

The effects of scavenger receptor (SR) ligands on nitric oxide (NO) production were investigated using mouse peritoneal macrophages stimulated by LPS. Pretreatment of macrophages with oxidized LDL, heparin, maleylated BSA, or liposomes composed of phosphatidylserine (PS-liposomes) inhibited NO production, but native LDL, acetyl LDL dextran sulfate, did not. Immunoblotting analysis suggests that the inhibitory effects could be a result of the inhibition of inducible NO synthase (iNOS) induction, but not enzyme activity. Further, tyrosine phosphorylation of a 41 kDa protein was also inhibited by OxLDL, heparin, maleylated BSA, and PS-liposomes. Chloroquine did not affect the extent of inhibition of NO production induced by these ligands, suggesting that the binding of these ligands to SR generates a signal(s) which is involved in the inhibition of NO production from macrophages stimulated by LPS. SR, which has an affinity to these ligands, may strictly regulate NO production from macrophages, and this inhibitory effect may be due to the inhibition of LPS-induced tyrosine phosphorylation of 41 kDa protein.

Effects of medium-chain fatty acids and their acylglycerols on the transport of penicillin V across Caco-2 cell monolayers.

The transport-enhancing effects of medium-chain fatty acids (caproic, caprylic, and capric acids) and their acylglycerols (mono-, di-, and triacylglycerols) were investigated by using Caco-2 cell monolayers as a model of the human intestinal epithelium. Penicillin V was used as a model for a hydrophilic bioactive compound. Among the fatty acids and acylglycerols tested, 1,2-dicaproin, monocaprin, monocaprylin, and capric acid sodium salt effectively enhanced the transport rate, whereas other substances enhanced the rate only slightly or not at all. With each of these four substances, the rate of enhancement was proportional to the concentration at low concentrations, but leveled off at high concentrations. The transport-enhancing effects were well correlated with the reduction in surface tension and with a physico-chemical parameter, denoted by the surface energy-lowering coefficient, characterizing the surface activity of a substance.