JCL Bioassay Corporation

Nishiwaki, Japan

JCL Bioassay Corporation

Nishiwaki, Japan
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Kusuhara H.,University of Tokyo | Furuie H.,Osaka Pharmacology Clinical Research Hospital | Inano A.,Association for Promoting Drug Development | Sunagawa A.,JCL Bioassay Corporation | And 11 more authors.
British Journal of Pharmacology | Year: 2012

BACKGROUND AND PURPOSE: An ATP-binding cassette (ABC) transporter, breast cancer resistance protein (BCRP)/ABCG2, limits oral bioavailability of sulphasalazine. Here we examined the effect of curcumin, the principal curcuminoid of turmeric, on oral bioavailability of microdoses and therapeutic doses of sulphasalazine in humans. EXPERIMENTAL APPROACH:Effects of curcumin were measured on the ATP-dependent sulphasalazine uptake by hBCRP-expressing membrane vesicles and on oral bioavailability of sulphasalazine in wild-type and Bcrp(-/-) mice. Eight healthy Japanese subjects received an oral dose of sulphasalazine suspension (100 μg) or tablets (2 g) alone or after curcumin tablets (2 g). Uptake of sulphasalazine was studied in HEK293 cells transfected with the influx transporter (OATP)2B1. KEY RESULTS:Curcumin was a potent hBCRP inhibitor in vitro (K i 0.70 ± 0.41 μM). Curcumin increased the area under the curve (AUC) 0-8 of plasma sulphasalazine eightfold in wild-type mice at 300 and 400 mg·kg -1, but not in Bcrp(-/-) mice. Curcumin increased AUC 0-24 of plasma sulphasalazine 2.0-fold at microdoses and 3.2-fold at therapeutic doses in humans. Non-linearity of the dose - exposure relationship was observed between microdoses and therapeutic doses of sulphasalazine. Sulphasalazine was a substrate for OATP2B1 (K m 1.7 ± 0.3 μM). Its linear index (dose/K m) at the therapeutic dose was high and may saturate OATP2B1. CONCLUSIONS AND IMPLICATIONS: Curcumin can be used to investigate effects of BCRP on oral bioavailability of drugs in humans. Besides the limited dissolution, OATP2B1 saturation is a possible mechanism underlying non-linearity in the dose-exposure relationship of sulphasalazine. © 2012 The Authors British Journal of Pharmacology © 2012 The British Pharmacological Society.

Ishikado A.,Shiga University of Medical Science | Ishikado A.,Sunstar Inc. | Morino K.,Shiga University of Medical Science | Nishio Y.,Kagoshima University | And 16 more authors.
PLoS ONE | Year: 2013

Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 PUFAs) have direct antioxidant and anti-inflammatory effects in vascular tissue, explaining their cardioprotective effects. However, the molecular mechanisms are not yet fully understood. We tested whether n-3 PUFAs showed antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes. C57BL/6 or Nrf2-/- mice were fed a fish-oil diet for 3 weeks. Fish-oil diet significantly increased the expression of heme oxygenase-1 (HO-1), and endothelium-dependent vasodilation in the aorta of C57BL/6 mice, but not in the Nrf2-/- mice. Furthermore, we observed that 4-hydroxy hexenal (4-HHE), an end-product of n-3 PUFA peroxidation, was significantly increased in the aorta of C57BL/6 mice, accompanied by intra-aortic predominant increase in docosahexaenoic acid (DHA) rather than that in eicosapentaenoic acid (EPA). Human umbilical vein endothelial cells were incubated with DHA or EPA. We found that DHA, but not EPA, markedly increased intracellular 4-HHE, and nuclear expression and DNA binding of Nrf2. Both DHA and 4-HHE also increased the expressions of Nrf2 target genes including HO-1, and the siRNA of Nrf2 abolished these effects. Furthermore, DHA prevented oxidant-induced cellular damage or reactive oxygen species production, and these effects were disappeared by an HO-1 inhibitor or the siRNA of Nrf2. Thus, we found protective effects of DHA through Nrf2 activation in vascular tissue, accompanied by intra-vascular increases in 4-HHE, which may explain the mechanism of the cardioprotective effects of DHA. © 2013 Ishikado et al.

Kondo K.,Shiga University of Medical Science | Morino K.,Shiga University of Medical Science | Nishio Y.,Kagoshima University | Kondo M.,Shiga University of Medical Science | And 10 more authors.
Metabolism: Clinical and Experimental | Year: 2014

Objective The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM). Materials/Methods Twenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs 3.0 g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR). Results A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase. Conclusions A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon. © 2014 Elsevier Inc.

PubMed | Shiga University of Medical Science, Kagoshima University and JCL Bioassay Corporation
Type: Journal Article | Journal: Biochemical and biophysical research communications | Year: 2014

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo. In this study, we examined the effect of fish-oil dietary supplementation on the distribution of fatty acids and their peroxidative metabolites and on the expression of HO-1 in multiple tissues (liver, kidney, heart, lung, spleen, intestine, skeletal muscle, white adipose, brown adipose, brain, aorta, and plasma) of C57BL/6 mice. Mice were divided into 4 groups, and fed a control, safflower-oil, and fish-oil diet for 3 weeks. One group was fed a fish-oil diet for just 1 week. The concentration of fatty acids, 4-hydroxy hexenal (4-HHE), and 4-hydroxy nonenal (4-HNE), and the expression of HO-1 mRNA were measured in the same tissues. We found that the concentration of 4-HHE (a product of n-3 PUFAs peroxidation) and expression of HO-1 mRNA were significantly increased after fish-oil treatment in most tissues. In addition, these increases were paralleled by an increase in the level of docosahexaenoic acid (DHA) but not eicosapentaenoic acid (EPA) in each tissue. These results are consistent with our previous results showing that DHA induces HO-1 expression through 4-HHE in vascular endothelial cells. In conclusion, we hypothesize that the HO-1-mediated protective effect of the fish oil diet may be through production of 4-HHE from DHA but not EPA in various tissues.

Fang Y.,China Pharmaceutical University | Fang Y.,Kobe University | Hu L.,China Pharmaceutical University | Hu L.,Kobe University | And 9 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a Schizosaccharomyces pombe gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-D-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Goto R.,Wakayama Medical University | Goto R.,JCL Bioassay Corporation | Nakamura Y.,Wakayama Medical University | Takami T.,JCL Bioassay Corporation | And 2 more authors.
PLoS ONE | Year: 2015

The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms.We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis. © 2015 Fujii et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Yamazaki A.,Kitasato University | Yamazaki A.,Tokyo Metroplitan University | Kumagai Y.,Kitasato University | Yamane N.,JCL Bioassay Corporation | And 5 more authors.
Journal of Clinical Pharmacy and Therapeutics | Year: 2010

Objective: Fexofenadine is a P-glycoprotein substrate of low bioavailability. It is primarily excreted into faeces as a parent drug via biliary excretion. The predictability from microdose data for the drug absorbed via transporters such as P-glycoprotein is not known. Therefore, this study assessed the predictability of therapeutic-dose pharmacokinetics of fexofenadine from microdosing data using non-radioisotope-labelled drug and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Method: In a single dose, randomized, two-way crossover study, eight subjects received a microdose (100 μg) or a therapeutic dose (60 mg) of fexofenadine. Blood samples were collected until 12 h after dosing, and assayed using LC/MS/MS. Results: Plasma concentration-time curves of fexofenadine between microdose and therapeutic dose were similar. The mean ± SD of C max normalized to 60 mg dose after microdose and therapeutic dose were 379 ± 147 and 275 ± 145 ng/mL respectively. The mean AUC last normalized to 60 mg dose after microdose and therapeutic dose were 1914 ± 738 and 1431 ± 432 ng/h/mL respectively. The mean dose-adjusted Cmax and AUClast after microdose were higher compared with those after therapeutic dose. Individual plots of Cmax and AUClast normalized to 60 mg dose, were similar for microdose and therapeutic dose. None of the pharmacokinetic parameters were statistically different using anova. Overall, the microdose pharmacokinetics profile was similar to, and hence predictive of, that of the therapeutic dose. Conclusion: For the P-glycoprotein substrate fexofenadine, the predictability of therapeutic-dose pharmacokinetics from microdose data was good. A microdose study using a non-radioisotope-labelled drug and LC/MS/MS is convenient, and has the potential to aid the early selection of drug candidates. © 2010 Blackwell Publishing Ltd.

Goto R.,Wakayama Medical University | Goto R.,JCL Bioassay Corporation | Nakamura Y.,Wakayama Medical University | Shio Yama S.,JCL Bioassay Corporation | And 2 more authors.
Journal of the Wakayama Medical Society | Year: 2014

For diagnostic and prognostic purposes, and to understand the mechanism of metastasis, identifying and quantifying metastasis-specific proteins are important. The purpose of this study was to identify and quantify proteins involved in metastasis of breast cancer. To identify metastasis-specific proteins, breast cancer tissues of the non-metastasis group (n=6) were compared with those of the metastasis group of patients with massive lymph node involvement (n=13). By comprehensive analysis using two-dimensional liquid chromatography- electrospray ionization -tandem mass spectrometry (2D-LC-ESI-MS/MS), 34 proteins were identified as protein candidates highly expressed in breast cancer with massive lymph node involvement. The most positive protein candidate was Type XIV Collagen; that is, Type XIV Collagen seemed to be expressed only in specimens from the massive lymph node metastasis group. Immunohistochemistry study also showed intense protein expression of Type XIV Collagen in the specimens from the massive lymph node metastasis group. The increased expression of these proteins including Type XIV Collagen may be related to metastasis. Further studies are desired to evaluate their usefulness as biomarkers.

Maeda K.,University of Tokyo | Ikeda Y.,Kitasato University | Fujita T.,Kitasato University | Yoshida K.,University of Tokyo | And 5 more authors.
Clinical Pharmacology and Therapeutics | Year: 2011

Clearance of atorvastatin occurs through hepatic uptake by organic anion transporting polypeptides (OATPs) and subsequent metabolism by cytochrome P450 (CYP) 3A4. To demonstrate the relative importance of OATPs and CYP3A4 in the hepatic elimination of atorvastatin in vivo, a clinical cassette microdose study was performed. A cocktail consisting of a microdose of atorvastatin along with probe substrates for OATPs (pravastatin) and CYP3A4 (midazolam) was orally administered to eight healthy volunteers. The pharmacokinetics of this cocktail was observed at baseline, after an oral dose of 600 mg rifampicin (an inhibitor of OATPs), and after an intravenous dose of 200 mg itraconazole (a CYP3A4 inhibitor). Rifampicin increased the pravastatin dose-normalized area under the plasma concentration-time curve (AUC) (4.6-fold), and itraconazole significantly increased the midazolam dose-normalized AUC (1.7-fold). The atorvastatin dose-normalized AUC increased 12-fold when coadministered with rifampicin but did not change when coadministered with itraconazole. These results indicate that hepatic uptake via OATPs makes the dominant contribution to the hepatic elimination of atorvastatin at a subtherapeutic microdose. © 2011 American Society for clinical Pharmacology and therapeutics.

JCL Bioassay Corporation | Date: 2010-11-29

A detection agent for high malignancy breast cancer includes an antibody against collagen XIV, or a variant or derivative or fragment of the antibody. A therapeutic agent for high malignancy breast cancer includes a conjugate of an anticancer drug and an antibody against that protein, or a variant or derivative or fragment thereof. Accordingly, it is possible to easily and accurately detect and diagnose high malignancy breast cancer.

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