Effectiveness, safety, and methotrexate dose-tapering pattern over two years of treatment with adalimumab and ≥12 mg/week methotrexate for early rheumatoid arthritis: Results of the HAWK postmarketing surveillance study in Japan.

Objectives: To investigate the long-term effectiveness, safety, and methotrexate (MTX) dose-tapering patterns in patients with rheumatoid arthritis (RA) receiving adalimumab plus high-dose MTX.Methods: In this prospective, postmarketing study (2012-2017), conducted at 128 sites in Japan, biologic-naïve patients with RA (duration ≤2 years) previously treated with MTX for ≥3 months, initiated treatment with adalimumab and MTX (≥12 mg/week). Effectiveness by Disease Activity Score in 28 joints using C-reactive protein (DAS28-CRP), safety, and MTX dose-tapering were assessed from baseline to 104 weeks.Results: In the effectiveness analysis set (n = 292), DAS28-CRP remission (<2.6) was achieved in 92.3% (n = 120/130) of patients at week 104. The proportions of patients receiving MTX dose <10 mg/week increased to 32.3% (n = 50/155) and ≥12 mg/week reduced to 52.9% (n = 82/155) by week 104. Per univariate regression analysis, MTX dose tapering was associated with longer adalimumab drug survival. Of 70 patients with joint X-rays available, 59 (84.3%) achieved Δ modified total Sharp score ≤1.0 at 104 weeks. In the safety analysis set (n = 300), 143 adverse drug reactions were reported in 92 patients (30.7%, non-serious; 24.7%, serious 8.7%).Conclusion: The long-term effectiveness and safety of adalimumab with high-dose MTX was confirmed in biologic-naïve patients with early RA in a real-world setting in Japan.Clinical Trial Registration: This study is registered at ClinicalTrials.gov (identifier: NCT01736189; retrospectively registered 29 November 2012, due to administrative reasons).

Glucocorticoids attenuate the sensitivity of glucocorticoid-resistant lymphoid cells to doxorubicin via reduction in OCTN2.

Glucocorticoid (GC) resistance is associated with poor response to the following chemotherapy in lymphoid malignancies, such as lymphoma and leukemia. However, it remains unclear whether GCs interfere with the cytotoxic effects of anti-cancer drugs on GC-resistant cells. In this study, we examined whether GCs affected the sensitivities to vincristine (VCR)/doxorubicin (DOX) and the expression of drug transporters in GC-resistant cells. The dexamethasone (DEX)/prednisolone (PSL)-resistant lymphoid and non-lymphoid cell lines Raji and HL60 were cultured with DEX for 7 days and then treated with VCR or DOX for 3 days. Seven days of DEX treatment increased the IC50s of both VCR and DOX in Raji cells but not in HL60 cells. The mRNA and protein expression levels of organic cation/carnitine transporter (OCTN) 2, one of the drug uptake transporters expressed in both cell lines, were decreased only in Raji cells. When Raji cells were cultured with PSL, the IC50 of DOX but not VCR increased as the expression of OCTN2 decreased. No significant increases in efflux transporter expression were induced by DEX or PSL. When siRNA against OCTN2 was introduced into Raji cells, the IC50 of DOX but not VCR increased significantly. These data suggested that both DEX and PSL decreased the sensitivity of the DEX/PSL-resistant Raji cells to DOX, a change that was at least partially due to reductions in OCTN2. Thus, the continuous usage of GCs may interfere with the effects of chemotherapy on GC-resistant lymphoid cells.

Patient preference for biologic treatments of psoriasis in Japan.

Psoriasis is a chronic autoimmune disease affecting skin which may also manifest in nails and joints. Several biologic treatments have been approved in Japan for psoriasis. Each biologic has a different profile for efficacy and safety, including different dosing regimens and co-payment considerations which may complicate treatment decisions made by patients and physicians during short consultations. Elucidating patient preference is expected to contribute to shared decision-making between patients and physicians to optimize treatment satisfaction and outcomes. However, the number of studies investigating this in Japan is very limited. The study used a discrete choice experiment methodology to elicit patient preferences for hypothetical options in an experimental framework. Participants were asked to choose their preferred treatment option from two hypothetical choices, defined by different levels of six attributes (i.e. early onset of efficacy, long-term efficacy, sustained efficacy after drug withdrawal, dosing convenience, co-payment and risk of serious infection). The survey included 16 treatment choice scenarios and was completed by 395 participants. Across all participants, the attribute regarded as most important was sustained efficacy after drug withdrawal, followed by dosing convenience, co-payment, long-term efficacy, early onset of efficacy and risk of serious infection. The study found that patients prefer treatments which have durable efficacy and lower treatment burden characterized as fewer injection frequency and lower co-payment. These results may be helpful to understand patient preference for biologic treatments used for psoriasis in Japan and contribute to shared decision-making between patients and physicians to improve patient satisfaction and treatment outcomes.

Real-World Postmarketing Study of the Impact of Adalimumab Treatment on Work Productivity and Activity Impairment in Patients with Psoriatic Arthritis.

INTRODUCTION: This study investigated the effectiveness of adalimumab treatment in improving Work Productivity and Activity Impairment (WPAI) in patients with psoriatic arthritis (PsA) in real-world settings in Japan. METHODS: This 24-week, single-arm, postmarketing surveillance study (2014-2017), conducted at 75 centers in Japan, enrolled adalimumab-naïve patients (paid workers, including part-time) meeting ClASsification criteria for Psoriatic ARthritis (CASPAR). The primary endpoint was improvement in overall work impairment (OWI) scores from baseline to week 24. Secondary endpoints included changes in WPAI-PsA (OWI, absenteeism, presenteeism, and activity impairment), Psoriasis Area and Severity Index (PASI), psoriatic arthritis screening and evaluation (PASE) scores, Disease Activity Scores in 28 joints using C-reactive protein (DAS28[CRP]), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) scores, Health Assessment Questionnaire-Disability Index (HAQ-DI) scores, and PASI75/90 and American College of Rheumatology (ACR) 20/50/70 rates. RESULTS: In the effectiveness population (n = 106; 72.6% men; mean ± standard deviation [SD] age, 49.3 ± 10.7 years), OWI scores significantly improved (mean ± SD change, - 25.2 ± 35.3; p < 0.0001) from baseline to week 24. Other WPAI domain scores also improved significantly. Changes in OWI were significantly correlated (p < 0.0001) with PASE (r = 0.6284), DAS28(CRP) (r = 0.6059), BASDAI (r = 0.7281), and HAQ-DI (r = 0.6161) scores and were significantly influenced by previous nonsteroidal anti-inflammatory drug use (p = 0.0142), and baseline PASE (p = 0.0098), DAS28(CRP) (p = 0.0026), HAQ-DI (p = 0.0004), and BASDAI (p < 0.0001) scores. At the last evaluation, rate (95% confidence interval) of PASI 75 and 90 (n = 100) was 58.0% (47.7-67.8) and 39.0% (29.4-49.3), respectively, and that of ACR 20, 50, and 70 (n = 58) was 86.2% (74.6-93.9), 70.7% (57.3-81.9), and 53.4% (39.9-66.7), respectively. No new safety signals were observed in the safety population (n = 148). CONCLUSION: Adalimumab treatment improved WPAI in patients with PsA. Improvements in OWI and joint symptoms were significantly associated. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02414633. FUNDING: AbbVie GK and Eisai Co., Ltd.

Effectiveness and safety of initiating adalimumab plus ≥12 mg/week methotrexate with adjustable dosing in biologic-naïve patients with early rheumatoid arthritis: HAWK study postmarketing surveillance in Japan.

Objectives: This real-world study assessed the effectiveness and safety outcomes of initiating adalimumab and methotrexate (≥12 mg/week) with adjustable dosing in Japanese patients with early rheumatoid arthritis (RA). Methods: This single-arm, prospective postmarketing observational study (conducted from September 2012 to March 2017 at 119 sites) enrolled biologic-naïve patients with early RA (≤2 years duration) and a Disease Activity Score in 28 joints using C-reactive protein (DAS28-CRP) >3.2 who were treated with methotrexate for ≥3 months and had initiated treatment with adalimumab and methotrexate (≥12 mg/week). This report presents 52-week data. The primary outcome was the proportion of patients who achieved DAS28-CRP scores <2.6 at week 52. Results: Overall, 293 of 346 enrolled patients were included in the effectiveness population: women, 73%; mean (standard deviation) age, 54.3 (13.9) years; DAS28-CRP score, 4.51 (0.90); and modified total Sharp score (mTSS), 7.69 (9.98). At week 52, 77% of patients achieved clinical remission (DAS28-CRP <2.6), 92.3% achieved low disease activity (DAS28-CRP ≤3.2), and 86% of evaluable patients experienced structural remission (ΔmTSS ≤0.5). Conclusion: Adalimumab plus methotrexate (≥12 mg/week) with adjustable dosing was well tolerated, and could be a beneficial treatment option for Japanese patients with early RA.

Real-world effectiveness and safety of adalimumab for treatment of ankylosing spondylitis in Japan.

Objective: To evaluate the real-world effectiveness and safety of adalimumab for the treatment of ankylosing spondylitis (AS) in Japan.Methods: All AS patients initiated on adalimumab from 27 October 2010 to 28 May 2015, were enrolled. Patient characteristics at baseline, changes in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) scores, and adverse drug reactions (ADRs) for 24 weeks from the first adalimumab treatment were evaluated.Results: Of 403 enrolled patients, 396 and 374 comprised the safety and effectiveness analysis sets, respectively. In the safety analysis set, 266/396 (67.2%) were males, with a mean ± standard deviation (SD) age of 46.3 ± 15.6 years and mean ± SD disease duration of 9.8 ± 9.8 years. Of 236 patients examined for human leukocyte antigen (HLA)-B27, 131 (55.5%) were HLA-B27-positive. In the effectiveness analysis set, the mean ± SD BASDAI score was 4.9 ± 2.3 at baseline (n = 292). Overall, 216 patients had BASDAI data pre- and post-baseline. At 24 weeks, 143 patients had BASDAI scores, and the mean ± SD decrease was -2.0 ± 2.6 (p < .0001). Fifteen serious ADRs occurred in 15 (3.79%) patients; 30 (7.58%) ADRs of infections were reported, of which, five (1.26%) were serious.Conclusion: Safety and effectiveness of adalimumab in this postmarketing observational study were similar to that in previous clinical trials.

Pre-Operative Planning Using Real-Time Virtual Sonography, an MRI/Ultrasound Image Fusion Technique, for Breast-Conserving Surgery in Patients with Non-Mass Enhancement on Breast MRI: A Preliminary Study.

The purpose of this retrospective study was to evaluate the effect of pre-operative planning using real-time virtual sonography (RVS), a magnetic resonance imaging (MRI)/ultrasound (US) image fusion technique on breast-conserving surgery (BCS) in patients with non-mass enhancement (NME) on breast MRI. Between 2011 and 2015, we enrolled 12 consecutive patients who had lesions with NME that exceeded the US hypo-echoic area, in which it was particularly difficult to evaluate the tumor margin. During pre-operative planning before breast-conserving surgery, RVS was used to delineate the enhancing area on the breast surface after additional supine breast MRI was performed. We analyzed both the surgical margin positivity rate and the re-operation rate. All NME lesions corresponded to the index cancer. In all patients, the diameter of the NME lesion was greater than that of the hypo-echoic lesion. The median diameters of the NME and hypo-echoic lesions were 24 mm (range: 12-39 mm) and 8.0 mm (range: 4.9-18 mm), respectively (p = 0.0002). After RVS-derived skin marking was performed on the surface of the affected breast, lumpectomy and quadrantectomy were conducted in 7 and 5 patients, respectively. The surgical margins were negative in 10 (83%) patients. Two patients with positive margins were found to have ductal carcinoma in situ in 1 duct each, 2.4 and 3.2 mm from the resection margin, respectively. None of the patients required additional resection. Although further prospective studies are required, the findings of our preliminary study suggest that it is very well possible that the use of RVS-derived skin marking during pre-operative planning for BCS in patients with NME would have resulted in surgical outcomes similar to or better than those obtained without the use of such marking.

Nobiletin Reduces Intracellular and Extracellular ß-Amyloid in iPS Cell-Derived Alzheimer's Disease Model Neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with progressive memory impairment. Recently, neprilysin, a ß-amyloid (Aß)-degrading enzyme has become featured as a drug target for AD. Previously, we identified nobiletin from citrus peels as a natural compound possessing anti-dementia activity. In addition, we demonstrated that nobiletin improved memory in memory-impaired animals and, further, that Aß levels were markedly decreased in the brains of these animals. We demonstrated in vitro that nobiletin up-regulates neprilysin expression and activity in human neuroblastoma cells. However, the action of nobiletin with regard to Aß degradation under in vitro AD pathological conditions remains unclear. In this study, we examined whether nobiletin could enhance the degradation of intra- and extracellular Aß using human induced pluripotent stem cell-derived AD model neurons, which generate an excess of Aß1-42 due to the familial AD presenilin-1 mutation. The neurons were treated in the presence or absence of nobiletin. The results of real-time quantitative RT-PCR indicated that neprilysin mRNA levels were significantly up-regulated by nobiletin. Furthermore, immunostaining with an anti-Aß antibody revealed that nobiletin substantially reduced the intraneuronal content of Aß. Interestingly, the results of Aß1-42 immunoassays confirmed that nobiletin also significantly decreased the levels of Aß1-42 released into the cellular medium. These results suggest that nobiletin enhanced the reduction of intra- and that extracellular Aß levels under AD pathologic conditions, and this is associated with the up-regulation of neprilysin expression. Collectively, nobiletin appears to be a promising novel prophylactic seed drug or functional food for AD.

Role of (-)-epigallocatechin gallate in the pharmacokinetic interaction between nadolol and green tea in healthy volunteers.

PURPOSE: The aim of the present study is to investigate a possible role of a single dose of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, for the pharmacokinetic interaction between green tea and nadolol in humans. METHODS: In a randomized three-phase crossover study, 13 healthy volunteers received single doses of 30 mg nadolol orally with water (control), or an aqueous solution of EGCG-concentrated green tea extract (GTE) at low or high dose. Plasma concentrations and urinary excretion of nadolol were determined up to 48 h. In addition, blood pressure and pulse rate were monitored. In vitro transport kinetic experiments were performed using human embryonic kidney 293 cells stably expressing organic anion transporting polypeptide (OATP)1A2 to evaluate the inhibitory effect of EGCG on OATP1A2-mediated substrate transport. RESULTS: Single coadministration of low and high dose GTE significantly reduced the plasma concentrations of nadolol. The geometric mean ratios with 90% CI for area under the plasma concentration-time curves from 0 to infinity of nadolol were 0.72 (0.56-0.87) for the low and 0.60 (0.51-0.69) for the high dose. There were no significant differences in Tmax, elimination half-life, and renal clearance between GTE and water phases. No significant changes were observed for blood pressure and pulse rate between phases. EGCG competitively inhibited OATP1A2-mediated uptake of sulphobromophthalein and nadolol with Ki values of 21.6 and 19.4 µM, respectively. CONCLUSIONS: EGCG is suggested to be a key contributor to the interaction of green tea with nadolol. Moreover, even a single coadministration of green tea may significantly affect nadolol pharmacokinetics.

Lack of pharmacokinetic interaction between fluvastatin and green tea in healthy volunteers.

PURPOSE: The objective of this study is to assess the effects of green tea and its major catechin component, (-)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. METHODS: The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. RESULTS: EGCG inhibited diclofenac 4'-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 µM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration-time curve (AUC0-∞) of fluvastatin were 0.993 (0.963-1.024, vs. brewed green tea) and 0.977 (0.935-1.020, vs. GTE). CONCLUSIONS: Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.

Urinary Sodium-to-Potassium Ratio Tracks the Changes in Salt Intake during an Experimental Feeding Study Using Standardized Low-Salt and High-Salt Meals among Healthy Japanese Volunteers.

The Na/K ratio is considered to be a useful index, the monitoring of which allows an effective Na reduction and K increase, because practical methods (self-monitoring devices and reliable individual estimates from spot urine) are available for assessing these levels in individuals. An intervention trial for lowering the Na/K ratio has demonstrated that a reduction of the Na/K ratio mainly involved Na reduction, with only a small change in K. The present study aimed to clarify the relationship between dietary Na intake and the urinary Na/K molar ratio, using standardized low- and high-salt diets, with an equal dietary K intake, to determine the corresponding Na/K ratio. Fourteen healthy young adult volunteers ingested low-salt (3 g salt per day) and high-salt (20 g salt per day) meals for seven days each. Using a portable urinary Na/K meter, participants measured their spot urine at each voiding, and 24-h urine was collected on the last day of each diet period. On the last day of the unrestricted, low-salt, and high-salt diet periods, the group averages of the 24-h urine Na/K ratio were 4.2, 1.0, and 6.9, while the group averages of the daily mean spot urine Na/K ratio were 4.2, 1.1, and 6.6, respectively. The urinary Na/K ratio tracked changes in dietary salt intake, and reached a plateau approximately three days after each change in diet. Frequent monitoring of the spot urine Na/K ratio may help individuals adhere to an appropriate dietary Na intake.

Second-look US Using Real-time Virtual Sonography, a Coordinated Breast US and MRI System with Electromagnetic Tracking Technology: A Pilot Study.

Our aim was to retrospectively evaluate the utility of second-look ultrasound (US) using real-time virtual sonography (RVS) for detection of conventional B-mode (cB-mode) occult magnetic resonance imaging (MRI)-detected breast lesions. Between July 2011 and May 2015, 53 consecutive patients who underwent second-look US to identify lesions detected by prone MRI were enrolled in this study. Second-look US using RVS was performed for cB-mode occult MRI-detected breast lesions after an additional supine MRI. In the 53 patients, 59 lesions were initially detected by prone MRI, followed by second-look US. Of the 59 lesions, 20 (34%) were identified by second-look US using cB-mode. Of the 39 (66%) cB-mode occult lesions, 38 (97%) were detected in supine MRI and 33 (85%) were detected by second-look US using RVS. MRI morphology types of the 33 lesions were as follows: mass, 16; non-mass enhancement, 5; and focus, 12. US-guided biopsy under RVS or excisional biopsy demonstrated that of the 33 lesions, 8 (24%) were malignant and the remaining 25 (76%) were benign. A total of 53 (90%) MRI-detected lesions were sonographically identified using both cB-mode and RVS (p < 0.001). All five remaining US-occult lesions could be followed up under RVS after the enhancing area was marked on the breast surface using RVS. Although further prospective studies are required, the findings of our pilot study suggest that second-look US using RVS with additional supine MRI may improve the sonographic and histopathologic detection rate of cB-mode occult MRI-detected breast lesions.

Low disease activity for up to 3 years after adalimumab discontinuation in patients with early rheumatoid arthritis: 2-year results of the HOPEFUL-3 Study.

BACKGROUND: This study was conducted to evaluate the feasibility of long-term adalimumab (ADA) discontinuation after achievement of low disease activity (LDA) in Japanese patients with early rheumatoid arthritis (RA) and to identify predictors of LDA maintenance. METHODS: In the HOPEFUL-1 study, patients received initial therapy with either ADA plus methotrexate (MTX; intensive therapy) or MTX alone (standard therapy) for 26 weeks, followed by ADA + MTX for 26 weeks. In the HOPEFUL-2 study, patients received ADA + MTX (ADA continuation) or MTX alone (ADA discontinuation) for 52 weeks. HOPEFUL-3 was an observational study that enrolled patients who had completed HOPEFUL-2; these patients were followed for an additional 104 weeks. RESULTS: Of the 172 patients enrolled in the HOPEFUL-3 study, 135 (ADA continuation, n = 61; ADA discontinuation, n = 74) with 28-joint Disease Activity Score using C-reactive protein (DAS28-CRP) values at both week 52 (start of HOPEFUL-2) and week 208 (end of HOPEFUL-3) were included in the effectiveness analysis. At week 208, 58 (95.1%) of 61 patients and 59 (79.7%) of 74 patients who continued or discontinued ADA, respectively, had LDA (DAS28-CRP <3.2). Initial intensive therapy was associated with a better outcome than standard therapy in terms of change in modified total Sharp score from week 0 to week 208, which was ≤0.5 (64% vs. 30%). The incidence of adverse events was significantly lower in the ADA discontinuation group than in the ADA continuation group (9.7% vs. 32.9%; p < 0.001). CONCLUSIONS: Approximately 80% of patients who discontinued ADA for 3 years after achieving LDA with ADA + MTX were still in LDA, with a lower incidence of adverse events than patients who continued ADA. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01346501. Registered 29 April 2011.

Mechanisms of Impaired Neutrophil Migration by MicroRNAs in Myelodysplastic Syndromes.

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8-induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Suppressive Effect of Carvedilol on Na+/Ca2+ Exchange Current in Isolated Guinea-Pig Cardiac Ventricular Myocytes.

BACKGROUND AND AIMS: Carvedilol ((+/-)-1-(carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol), a ß-adrenoceptor-blocker, has multi-channel blocking and vasodilator properties. This agent dose-dependently improves left ventricular function and reduces mortality in patients with arrhythmia and chronic heart failure. However, the effect of carvedilol on the cardiac Na+/Ca2+ exchanger (NCX1) has not been investigated. METHODS AND RESULTS: We examined the effects of carvedilol and metoprolol, 2 ß-blockers, on Na+/Ca2+ exchange current (INCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed INCX in a concentration-dependent manner but metoprolol did not. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 µmol/l inhibited INCX in CCL39 cells expressing wild type NCX1 similar to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 µmol/l abolished ouabain-induced delayed afterdepolarizations. CONCLUSION: Carvedilol inhibited cardiac NCX in a concentration-dependent manner in isolated cardiac ventricles, but metoprolol did not. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

[Validation Study of Analytical Method for Determination of Amnesic Shellfish Poison in Bivalves].

Amnesic shellfish poison (ASP) is regarded as one of the shellfish poison groups in the EU, though it is not subject to regulation in Japan. We have developed an analytical method of ASP based on the report by Hatfield et al. and other methods. Validation studies were carried out with certified compositional reference materials (CRM). Performance parameters were estimated based on 17 analytical results. The estimate of trueness was 97.5%, and the estimate of intralaboratory reproducibility (RSD) was 1.5%. The HorRat(r) value was 0.16. These performance parameters meet the criteria in the Codex Procedural Manual. Furthermore, internal quality control was performed by using the CRM. The action limits were set based on the performance parameters of the method. Most of the results of the internal quality control were within the action limit range. The results confirmed that the quality of the analyses was well maintained. The purpose of the analytical method is to confirm that the level of ASP in scallop is less than 4.6 mg/kg. The applicability of the analytical method to scallops was confirmed by using spiked samples.

Inhibitory effect of YM-244769, a novel Na+/Ca2+ exchanger inhibitor on Na+/Ca2+ exchange current in guinea pig cardiac ventricular myocytes.

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na+/Ca2+ exchange (NCX) inhibitor by using various cells transfected with NCX using the 45Ca2+ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (INCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional INCX in a concentration-dependent manner. The IC50 values of YM-244769 for the bidirectional outward and inward INCX were both about 0.1 µM. YM-244769 suppressed the unidirectional outward INCX (Ca2+ entry mode) with an IC50 value of 0.05 µM. The effect on the unidirectional inward INCX (Ca2+ exit mode) was less potent, with 10 µM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na+ concentration, YM-244769 suppressed INCX more potently than it did at 0 mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode, and inhibition by YM-244769 is [Na+]i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.

Reduction of c-Fos via Overexpression of miR-34a Results in Enhancement of TNF- Production by LPS in Neutrophils from Myelodysplastic Syndrome Patients.

Although increased TNF-α has been considered to cause ineffective hematopoiesis in myelodysplastic syndromes (MDS), the mechanisms of TNF-α elevation are not known. We recently found that c-Fos mRNA stabilization under translation-inhibiting stimuli was impaired in MDS-derived neutrophilic granulocytes. In the current study, we identified overexpression of c-Fos-targeting miR-34a and miR-155 as the cause of impairment. Expression levels of miR-34a but not miR-155 inversely correlated with ratios of c-Fos-positive cells in MDS-derived CD16+ neutrophils (r = -0.618, P<0.05), which were analyzed by flow cytometry. Among the seventeen patients, c-Fos was detectable in less than 60% of CD16+ cells in eight patients (Group A), while five (Group B) expressed c-Fos in more than 80% of CD16+ cells, which was consistent with the controls (88.6 ± 7.8%). Group A-derived granulocytes secreted more TNF-α in response to 1 µM LPS for 3 hours (735.4 ± 237.5 pg/mL) than Group B (143.5 ± 65.7 pg/mL, P<0.05) and healthy controls (150.8 ± 91.5 pg/mL, P<0.05). Knockdown of c-Fos in neutrophil-like differentiated HL60 increased the binding of NF-κB p65 to the promoter region of TNF-α DNA. Thus, c-Fos reduction via overexpression of miR-34a contributes to TNF-α overproduction under inflammatory stimuli in MDS.

DEC2 is a negative regulator for the proliferation and differentiation of chondrocyte lineage-committed mesenchymal stem cells.

Differentiated embryo chondrocyte 2 (DEC2) is a basic helix-loop-helix-Orange transcription factor that regulates cell differentiation in various mammalian tissues. DEC2 has been shown to suppress the differentiation of mesenchymal stem cells (MSCs) into myocytes and adipocytes. In the present study, we examined the role of DEC2 in the chondrogenic differentiation of human MSCs. The overexpression of DEC2 exerted minimal effects on the proliferation of MSCs in monolayer cultures with the growth medium under undifferentiating conditions, whereas it suppressed increases in DNA content, glycosaminoglycan content, and the expression of several chondrocyte-related genes, including aggrecan and type X collagen alpha 1, in MSC pellets in centrifuge tubes under chondrogenic conditions. In the pellets exposed to chondrogenesis induction medium, DEC2 overexpression downregulated the mRNA expression of fibroblast growth factor 18, which is involved in the proliferation and differentiation of chondrocytes, and upregulated the expression of p16INK4, which is a cell cycle inhibitor. These findings suggest that DEC2 is a negative regulator of the proliferation and differentiation of chondrocyte lineage-committed mesenchymal cells.

Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.