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.

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.

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.

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.

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.

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.

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.

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.

Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage.

Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage.

Nicorandil stimulates a Na⁺/Ca²âº exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 µM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 µM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 µM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

Preventive Effect of Hydrogen Water on the Development of Detrusor Overactivity in a Rat Model of Bladder Outlet Obstruction.

PURPOSE: Bladder ischemia and oxidative stress contribute to the pathogenesis of bladder dysfunction caused by bladder outlet obstruction. H2 reportedly acts as an effective antioxidant. We investigated whether oral ingestion of H2 water would have a beneficial effect on bladder function in a rat model of bladder outlet obstruction. MATERIALS AND METHODS: H2 water was made by dissolving H2 gas in ordinary drinking water using a hydrogen water producing apparatus. The bladder outlet obstruction model was surgically induced in male rats. Rats with obstruction were fed H2 water or ordinary drinking water. On week 4 postoperatively cystometry was performed. Oxidative stress markers and the bladder nerve growth factor level were determined. Bladder tissues were processed for pharmacological studies and histological analysis. RESULTS: The micturition interval and micturition volume significantly decreased in obstructed rats given ordinary drinking water. These decreases were significantly suppressed by oral ingestion of H2 water. Increased post-void residual volume in obstructed rats was significantly reduced by H2 water. Obstruction led to a significant increase in bladder weight, oxidative stress markers and nerve growth factor. H2 water significantly suppressed these increases without affecting bladder weight. There was no significant difference in histological findings between rats with bladder obstruction given H2 water and ordinary drinking water. Decreased responses of detrusor muscle strips from obstructed bladders to KCl, carbachol and electrical field stimulation were reversed by H2 water ingestion. CONCLUSIONS: Results suggest that H2 water could ameliorate bladder dysfunction secondary to bladder outlet obstruction by attenuating oxidative stress.

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.

Effects of renal Na+/Ca2+ exchanger 1 inhibitor (SEA0400) treatment on electrolytes, renal function and hemodynamics in rats.

BACKGROUND: Na(+)/Ca(2+) exchanger 1 (NCX1) controls intracellular Ca(2+) concentration in various cell types. In the kidney, NCX1 is expressed mainly in the distal tubular basolateral membrane as well as in vascular smooth muscle. Tubular NCX1 is involved in Ca(2+) reabsorption, and NCX1 in renal arterioles may control intraglomerular pressure. However, the functions of renal NCX1 have not been studied in vivo. Therefore, this study examined the effects of renal NCX1 blockade on water and solute metabolism, renal function and blood pressure in rats. METHODS: Wistar-Kyoto rats were uninephrectomized, and an osmotic mini pump was implanted to infuse the remnant kidney cortex with a specific NCX1 inhibitor, SEA0400 (SEA), or vehicle for 7 days. RESULTS: Serum Ca(2+) concentration and urinary Ca(2+) excretion were similar between the vehicle- and SEA-treated groups. However, serum phosphate was significantly decreased by 8 % in the SEA group, with similar urinary phosphate excretion between the two groups. Systolic blood pressure was higher in the SEA group (117 ± 3 vs. 126 ± 1 mmHg, n = 9-11), with a 1.6-fold increase in plasma aldosterone concentration. However, SEA significantly reduced urinary protein excretion and the glomerular sectional area by 16 and 8 %, respectively. Similar experiment in spontaneously hypertensive rats produced different results. CONCLUSION: Renal SEA treatment reduced serum phosphate concentration, urinary protein and glomerular size with higher systemic blood pressure compared to control Wistar-Kyoto rats. Further study on renal NCX1 may be beneficial in delineating the pathophysiology of glomerular pressure control and calcium/phosphate regulations.

Upregulation of N-methyl-D-aspartate receptor subunits and c-Fos expressing genes in PC12D cells by nobiletin.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Nobiletin, a flavone from Citrus depressa, induces gene expression and increases the protein level and activity of neprilysin in SK-N-SH cells.

Neprilysin (NEP) is one of the candidate amyloid ß protein (Aß) degrading enzymes affecting brain Aß clearance. This enzyme declines in the brain with age, which leads to the increased Aß deposition in Alzheimer's disease (AD). Pharmacological activation of NEP during the aging process, therefore, represents a potential strategy to prevent the development of AD. To examine the influence of nobiletin on neprilysin activity, we measured cellular NEP activity in SK-N-SH cells. Moreover, NEP expression was examined by using reverse transcription - polymerase chain reaction and Western blotting. Measurement of cellular NEP activity showed that nobiletin stimulated this in a dose- and time-dependent manner in SK-N-SH cells. Moreover, nobiletin increased the expression of NEP mRNA, and then the levels of NEP protein, also in a dose- and time-dependent manner. Our findings showed that nobiletin promoted NEP gene and protein expression, resulting in enhancement of cellular NEP activity in SK-N-SH cells. This compound could be a novel Aß-degrading compound for use in the development of disease-modifying drugs to prevent and (or) cure AD.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines.

Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines - 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells - were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 µM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.

Mechanism of statin-induced rhabdomyolysis.

Statins, a group of drugs used for the treatment of hypercholesterolemia, have adverse effects on skeletal muscle. The symptoms of these effects range from slight myalgia to severe rhabdomyolysis. The number of patients currently taking statins is estimated to be several millions worldwide. However, the mechanism of statins' myotoxic effects is unclear. Statins inhibit biosynthesis of mevalonate, a rate-limiting step of cholesterol synthesis, by inhibiting HMG-CoA reductase. Mevalonate is also an essential precursor for producing isoprenoids such as farnesylpyrophosphate and geranylgeranylpyrophosphate. These isoprenoids are especially important for anchoring small GTPases to the membrane before they function; e.g., Ras GTPases modulate proliferation and apoptosis, Rho GTPases control cytoskeleton formation, and Rab GTPases are essential for intracellular vesicle trafficking. Inactivation of these small GTPases alters cellular functions. Recently, we successfully reproduced statin-induced myotoxicity in culture dishes using in vitro skeletal muscle systems (e.g., skeletal myotubes and myofibers). This review summarizes our findings that statins induce depletion of isoprenoids and inactivation of small GTPases, especially Rab, which are critical for statin-induced myotoxicity. Although further study is required, our findings may contribute to the prevention and treatment of statins' adverse effects on skeletal muscle and development of safer anti-hypercholesterolemia drugs.

Loss of ectonucleotidases from the coronary vascular bed after ischemia-reperfusion in isolated rat heart.

BACKGROUND: Ectonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart. METHODS: Langendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography. RESULTS: ATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5'-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious. CONCLUSION: Ectonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.

6-demethoxynobiletin, a nobiletin-analog citrus flavonoid, enhances extracellular signal-regulated kinase phosphorylation in PC12D cells.

We previously demonstrated that nobiletin, a polymethoxylated flavone isolated from citrus peels, has the potential to improve cognitive dysfunction in patients with Alzheimer's disease (AD). Recent studies suggest that the generation of intraneuronal amyloid-beta (Aß) oligomers is an early event in the pathogenesis of AD. Aß oligomers cause deficits in the regulation of the extracellular signal-regulated kinase (ERK) signaling which is critical for consolidation of the memory. Our previous studies revealed that nobiletin activated ERK signaling and subsequent cyclic AMP response element-dependent transcription. In this study, the effects of five nobiletin analogs, 6-demethoxynobiletin, tangeretin, 5-demethylnobiletin, sinensetin, and 6-demethoxytangeretin, isolated from citrus peels were assessed on ERK phosphorylation in PC12D cells, and the structure-activity relationships were examined. PC12D cells were treated with nobiletin or its analogs, and the cell extracts were analyzed by Western blotting using an antibody specific to phosphorylated ERK. 6-Demethoxynobiletin markedly enhanced ERK phosphorylation in a concentration-dependent manner. These results may be useful in developing drugs and functional foods using citrus peels for the treatment of dementia including AD.