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Filgotinib, a preferential Janus Kinase‐1 inhibitor, is approved in Europe and Japan for treatment of rheumatoid arthritis and is being developed for treatment of other chronic inflammatory diseases. Three drug‐drug interactions studies were conducted in healthy subjects to evaluate the effect of P‐glycoprotein (P‐gp) modulation (study 1: P‐gp inhibition by itraconazole and study 2: P‐gp induction by rifampin) on filgotinib pharmacokinetics and the potential of filgotinib to impact exposure of metformin, an organic cation transporter (OCT) 2 and multidrug and toxin extrusion (MATE) 1/2K substrate (study 3). Co‐administration of filgotinib with itraconazole increased filgotinib exposure (maximum concentration [Cmax] by 64% and area under the curve to infinity [AUCinf] by 45%) but had no effect on the exposure of GS‐829845, filgotinib’s primary metabolite. Rifampin moderately reduced exposures of filgotinib and GS‐829845 (Cmax by 26% and AUCinf by 27% for filgotinib; Cmax by 19% and AUCinf by 38% for GS‐829845). The data confirmed that filgotinib is a P‐gp substrate. However, the magnitude of change in filgotinib/GS‐829845 exposure by P‐gp modulators is not deemed to be clinically relevant based on filgotinib exposure‐response analyses in subjects with rheumatoid arthritis. Filgotinib did not alter metformin exposures, indicating that filgotinib and GS‐829845 do not inhibit OCT2 and MATE1/2K at the clinical doses. Filgotinib was generally well‐tolerated when administered alone or with the co‐administered drugs in the studies. Results from these studies were the basis to enable the use of P‐gp modulators and substrates of OCT2, MATE1, and MATE2K with filgotinib without the need for dose modifications in the current approved rheumatoid arthritis population.

BackgroundCluster of differentiation (CD)73-adenosine and transforming growth factor (TGF)-β pathways are involved in abrogated antitumor immune responses and can lead to protumor conditions. This Phase 1 study (NCT03954704) evaluated the safety, pharmacokinetics, pharmacodynamics, and efficacy of dalutrafusp alfa (also known as GS-1423 and AGEN1423), a bifunctional, humanized, aglycosylated immunoglobulin G1 kappa antibody that selectively inhibits CD73-adenosine production and neutralizes active TGF-β signaling in patients with advanced solid tumors.MethodsDose escalation started with an accelerated titration followed by a 3+3 design. Patients received dalutrafusp alfa (0.3, 1, 3, 10, 20, 30, or 45 mg/kg) intravenously every 2 weeks (Q2W) up to 1 year or until progressive disease (PD) or unacceptable toxicity.ResultsIn total, 21/22 patients received at least one dose of dalutrafusp alfa. The median number of dalutrafusp alfa doses administered was 3 (range 1–14). All patients had at least one adverse event (AE), most commonly fatigue (47.6%), nausea (33.3%), diarrhea (28.6%), and vomiting (28.6%). Nine (42.9%) patients had a Grade 3 or 4 AE; two had Grade 5 AEs of pulmonary embolism and PD, both unrelated to dalutrafusp alfa. Target-mediated drug disposition appears to be saturated at dalutrafusp alfa doses above 20 mg/kg. Complete CD73 target occupancy on B cells and CD8+ T cells was observed, and TGF-β 1/2/3 levels were undetectable at dalutrafusp alfa doses of 20 mg/kg and higher. Free soluble (s)CD73 levels and sCD73 activity increased with dalutrafusp alfa treatment. Seventeen patients reached the first response assessment, with complete response, partial response, stable disease, and PD in 0, 1 (4.8%), 7 (33.3%), and 9 (42.9%) patients, respectively.ConclusionsDalutrafusp alfa doses up to 45 mg/kg Q2W were well tolerated in patients with advanced solid tumors. Additional evaluation of dalutrafusp alfa could further elucidate the clinical utility of targeting CD73-adenosine and TGF-β pathways in oncology.

Sofosbuvir-velpatasvir-voxilaprevir is a pangenotypic regimen for chronic HCV infection. In the USA and Europe, sofosbuvir-velpatasvir-voxilaprevir once daily for 12 weeks is indicated for adults who previously received an HCV NS5A inhibitor. In Europe, sofosbuvir-velpatasvir-voxilaprevir is also indicated in the absence of prior HCV direct-acting antiviral (DAA) therapy as an 8-week or 12-week regimen. In an open-label study, we evaluated the safety, efficacy, and pharmacokinetics of sofosbuvir-velpatasvir-voxilaprevir in adolescents 12 to 17 years with chronic HCV of any genotype.In this Phase 2, multicenter study, sofosbuvir-velpatasvir-voxilaprevir 400/100/100 mg daily was administered to adolescents for 8 weeks if DAA-naïve or for 12 weeks for cirrhosis or prior DAA failure. The key efficacy endpoint was sustained virologic response 12 weeks after therapy (SVR12). Intensive pharmacokinetic sampling was done in 14 patients at week 2 or 4, and samples for population pharmacokinetics were collected in all patients.All patients (n = 21) were naïve to HCV DAAs, and none had cirrhosis. HCV genotype 3a infection was most common, occurring in 43% of patients. Overall, 100% of patients (21 of 21) reached SVR12. The most common adverse events were abdominal pain and headache (24% each) and nausea (19%); no adverse events led to discontinuation. The only serious adverse event, hypotension, was considered related to study drug and resolved the same day without interruption of treatment. Sofosbuvir-velpatasvir-voxilaprevir exposures were similar to those observed in adults.The pangenotypic regimen of sofosbuvir-velpatasvir-voxilaprevir is highly efficacious and well-tolerated in treating chronic HCV infection in adolescents.

Tirabrutinib is an irreversible, small-molecule Bruton's tyrosine kinase (BTK) inhibitor, which was approved in Japan (VELEXBRU) to treat B-cell malignancies and is in clinical development for inflammatory diseases. As an application of model-informed drug development, a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model for irreversible BTK inhibition of tirabrutinib was developed to support dose selection in clinical development, based on clinical PK and BTK occupancy data from two phase I studies with a wide range of PK exposures in healthy volunteers and in subjects with rheumatoid arthritis. The developed model adequately described and predicted the PK and PD data. Overall, the model-based simulation supported a total daily dose of at least 40 mg, either q.d. or b.i.d., with adequate BTK occupancy (> 90%) for further development in inflammatory diseases. Following the PK/PD modeling and simulation, the relationship between model-predicted BTK occupancy and preliminary clinical efficacy data was also explored and a positive trend was identified between the increasing time above adequate BTK occupancy and better efficacy in treatment for RA by linear regression.

For children under 12 years of age who have chronic hepatitis C virus (HCV) infection, there are currently no approved treatments with direct‐acting antiviral agents. We therefore evaluated the safety and efficacy of ledipasvir‐sofosbuvir in HCV‐infected children aged 3 to

Currently, the only approved hepatitis C virus (HCV) treatment for children aged

The global prevalence of ulcerative colitis is increasing, and induction and maintenance of remission is a crucial therapeutic goal. We assessed the efficacy and safety of filgotinib, a once-daily, oral Janus kinase 1 preferential inhibitor, for treatment of ulcerative colitis.This phase 2b/3, double-blind, randomised, placebo-controlled trial including two induction studies and one maintenance study was done in 341 study centres in 40 countries. Eligible patients were aged 18-75 years with moderately to severely active ulcerative colitis for at least 6 months before enrolment (induction study A: inadequate clinical response, loss of response to or intolerance to corticosteroids or immunosuppressants, naive to tumour necrosis factor [TNF] antagonists and vedolizumab [biologic-naive]; induction study B: inadequate clinical response, loss of response to or intolerance to any TNF antagonist or vedolizumab, no TNF antagonist or vedolizumab use within 8 weeks before screening [biologic-experienced]). Patients were randomly assigned 2:2:1 to receive oral filgotinib 200 mg, filgotinib 100 mg, or placebo once per day for 11 weeks. Patients who had either clinical remission or a Mayo Clinic Score response at week 10 in either induction study entered the maintenance study. Patients who received induction filgotinib were rerandomised 2:1 to continue their induction filgotinib regimen or to placebo. Patients who received induction placebo continued receiving placebo. The primary endpoint was clinical remission by Mayo endoscopic, rectal bleeding, and stool frequency subscores at weeks 10 and 58. For the induction studies, efficacy was assessed in all randomised patients who received at least one dose of study drug or placebo within that study. For the maintenance study, efficacy was assessed in all patients randomised to any filgotinib treatment group in the induction studies who received at least one dose of study drug or placebo in the maintenance study. Patients who received placebo throughout the induction and maintenance study were not included in the full analysis set for the maintenance study. Safety was assessed in all patients who received at least one dose of the study drug or placebo within each study. This trial is registered with ClinicalTrials.gov, NCT02914522.Between Nov 14, 2016, and March 31, 2020, we screened 2040 patients for eligibility. 659 patients enrolled in induction study A were randomly assigned to receive filgotinib 100 mg (n=277), filgotinib 200 mg (n=245), or placebo (n=137). 689 patients enrolled into induction study B were randomly assigned to receive filgotinib 100 mg (n=285), filgotinib 200 mg (n=262), or placebo (n=142). 34 patients in induction study A and 54 patients in induction study B discontinued the study drug before week 10. After efficacy assessment at week 10, 664 patients entered the maintenance study (391 from induction study A, 273 from induction study B). 93 patients continued to receive placebo. 270 patients who had received filgotinib 100 mg in the induction study were randomly assigned to receive filgotinib 100 mg (n=179) or placebo (n=91). 301 patients who had received filgotinib 200 mg in the induction study were randomly assigned to receive filgotinib 200 mg (n=202) or placebo (n=99). 263 patients discontinued treatment in the maintenance study. At week 10, a greater proportion of patients given filgotinib 200 mg had clinical remission than those given placebo (induction study A 26·1% vs 15·3%, difference 10·8%; 95% CI 2·1-19·5, p=0·0157; induction study B 11·5% vs 4·2%, 7·2%; 1·6-12·8, p=0·0103). At week 58, 37·2% of patients given filgotinib 200 mg had clinical remission versus 11·2% in the respective placebo group (difference 26·0%, 95% CI 16·0-35·9; p0·0001). Clinical remission was not significantly different between filgotinib 100 mg and placebo at week 10, but was significant by week 58 (23·8% vs 13·5%, 10·4%; 0·0-20·7, p=0·0420). The incidence of serious adverse events and adverse events of interest was similar between treatment groups. In the induction studies, serious adverse events occurred in 28 (5·0%) of 562 patients given filgotinib 100 mg, 22 (4·3%) of 507 patients given filgotinib 200 mg, and 13 (4·7%) of 279 patients given placebo. In the maintenance study, serious adverse events were reported in eight (4·5%) of 179 patients given filgotinib 100 mg, seven (7·7%) of 91 patients in the respective placebo group, nine (4·5%) of 202 patients in the filgotinib 200 mg group, and no patients in the respective placebo group. No deaths were reported during either induction study. Two patients died during the maintenance study; neither was related to treatment.Filgotinib 200 mg was well tolerated, and efficacious in inducing and maintaining clinical remission compared with placebo in patients with moderately to severely active ulcerative colitis.Gilead Sciences.

Physiologically-based pharmacokinetic (PBPK) modeling in predicting metabolic drug–drug interactions (mDDIs) is routinely used in drug development. Currently, the US FDA endorses the use of PBPK to potentially support dosing recommendations for investigational drugs as enzyme substrates of mDDIs, and to inform a lack of mDDIs for investigational drugs as enzyme modulators. We systematically evaluated the performance of PBPK modeling in predicting mDDIs published in the literature. Models developed to assess both investigational drugs as enzyme substrates (Groups 1 and 2, as being inhibited and induced, respectively) or enzyme modulators (Groups 3 and 4, as inhibitors and inducers, respectively) were evaluated. Predicted ratios of the area under the curve (AUCRs) and/or maximum plasma concentration (CmaxRs) with and without comedication were compared with the observed ratios. For Groups 1, 2, 3, and 4, 62, 50, 44, and 43% of model-predicted AUCRs, respectively, were within a predefined threshold of 1.25-fold of observed values (0.8–1.25x). When the threshold was widened to twofold, the values increased to 100, 80, 81, and 86% (0.5–2.0x). For Groups 3 and 4, prediction for mDDI liability (the existence or lack of mDDIs) using PBPK appears to be satisfactory. Our analysis supports the FDA’s current recommendations on the use of PBPK to predict mDDIs.

Chronic kidney disease (CKD) is characterized by the accumulation of uremic solutes; however, little is known about how these solutes affect drug absorption and disposition. The goal of this study is to evaluate the effect of uremic solutes on the organic cation transporter, OCT2, which plays a key role in the renal secretion of many basic drugs. As a second goal, we reviewed the literature to determine whether there was evidence for the effect of CKD on the renal secretion of basic drugs. We first screened 72 uremic solutes as inhibitors of [14C]-labeled metformin uptake by OCT2. Seven were identified as inhibitors and 3 of them were determined to be clinically relevant. Of the 7 solutes, dimethylamine, malondialdehyde, trimethylamine, homocysteine, indoxyl-β-d-glucuronide, and glutathione disulfide were novel OCT2 inhibitors. For 6 drugs that are known OCT2 substrates, both secretory clearance and glomerular filtration rate declined in parallel with progression of CKD from stage 2 to 4, suggesting that selective effects of uremic solutes on net tubular secretion of organic cations do not occur. Further clinical studies are warranted with a broader range of OCT2 substrates to determine whether CKD may differentially affect tubular secretion of drugs especially in patients with advanced CKD.

Background The melanin synthesis enzyme dopachrome tautomerase (Dct) regulates intracellular Ca 2+ in melanocytes. Homozygous Dct knockout (Dct −/− ) adult mice are vulnerable to atrial arrhythmias (AA). Objective The purpose of this study was to determine whether apamin-sensitive small conductance Ca 2+ -activated K + (SK) currents are upregulated in Dct −/− mice and contribute to AA. Methods Optical mapping was used to study the membrane potential of the right atrium in Langendorff perfused Dct −/− (n = 9) and Dct +/− (n = 9) mice. Results Apamin prolonged action potential duration (APD) by 18.8 ms (95% confidence interval [CI] 13.4–24.1 ms) in Dct −/− mice and by 11.5 ms (95% CI 5.4–17.6 ms) in Dct +/− mice at a pacing cycle length of 150 ms ( P = .047). The pacing cycle length threshold to induce APD alternans was 48 ms (95% CI 34–62 ms) for Dct −/− mice and 21 ms (95% CI 12–29 ms) for Dct +/− mice ( P = .002) at baseline, and it was 35 ms (95% CI 21–49 ms) for Dct −/− mice and 22 ms (95% CI 11–32 ms) for Dct +/− mice ( P = .025) after apamin administration. Apamin prolonged post-burst pacing APD by 8.9 ms (95% CI 3.9–14.0 ms) in Dct −/− mice and by 1.5 ms (95% CI 0.7–2.3 ms) in Dct +/− mice ( P = .005). Immunoblot and quantitative polymerase chain reaction analyses showed that protein and transcripts levels of SK1 and SK3 were increased in the right atrium of Dct −/− mice. AA inducibility (89% vs 11%; P = .003) and duration (281 seconds vs 66 seconds; P = .008) were greater in Dct −/− mice than in Dct +/− mice at baseline, but not different (22% vs 11%; P = 1.00) after apamin administration. Five of 8 (63%) induced atrial fibrillation episodes in Dct −/− mice had focal drivers. Conclusion Apamin-sensitive SK current upregulation in Dct −/− mice plays an important role in the mechanism of AA.

Background and Objectives Recent studies showed that, in addition to parasympathetic nerves, cervical vagal nerves contained significant sympathetic nerves. We hypothesized that cervical vagal nerve stimulation (VNS) may capture the sympathetic nerves within the vagal nerve and activate the stellate ganglion. Materials and Methods We recorded left stellate ganglion nerve activity (SGNA), left thoracic vagal nerve activity (VNA), and subcutaneous electrocardiogram in seven dogs during left cervical VNS with 30 seconds on-time and 30 seconds off time. We then compared the SGNA between VNS on and off times. Results Cervical VNS at moderate (0.75 mA) output induced large SGNA, elevated heart rate (HR), and reduced HR variability, suggesting sympathetic activation. Further increase of the VNS output to >1.5 mA increased SGNA but did not significantly increase the HR, suggesting simultaneous sympathetic and parasympathetic activation. The differences of integrated SGNA and integrated VNA between VNS on and off times (ΔSGNA) increased progressively from 5.2 mV-s {95% confidence interval (CI): 1.25-9.06, p=0.018, n=7} at 1.0 mA to 13.7 mV-s (CI: 5.97-21.43, p=0.005, n=7) at 1.5 mA. The difference in HR (ΔHR, bpm) between on and off times was 5.8 bpm (CI: 0.28-11.29, p=0.042, n=7) at 1.0 mA and 5.3 bpm (CI 1.92 to 12.61, p=0.122, n=7) at 1.5 mA. Conclusion Intermittent cervical VNS may selectively capture the sympathetic components of the vagal nerve and excite the stellate ganglion at moderate output. Increasing the output may result in simultaneously sympathetic and parasympathetic capture.

Background Purkinje cells ( PC s) are important in cardiac arrhythmogenesis. Whether small‐conductance calcium‐activated potassium ( SK ) channels are present in PC s remains unclear. We tested the hypotheses that subtype 2 SK ( SK 2) channel proteins and apamin‐sensitive SK currents are abundantly present in PC s. Methods and Results We studied 25 normal rabbit ventricles, including 13 patch‐clamp studies, 4 for Western blotting, and 8 for immunohistochemical staining. Transmembrane action potentials were recorded in current‐clamp mode using the perforated‐patch technique. For PC s, the apamin (100 nmol/L) significantly prolonged action potential duration measured to 80% repolarization by an average of 10.4 ms (95% CI , 0.11–20.72) (n=9, P =0.047). Voltage‐clamp study showed that apamin‐sensitive SK current density was significantly larger in PC s compared with ventricular myocytes at potentials ≥0 mV. Western blotting of SK 2 expression showed that the SK 2 protein expression in the midmyocardium was 58% ( P =0.028) and the epicardium was 50% ( P =0.018) of that in the pseudotendons. Immunostaining of SK 2 protein showed that PCs stained stronger than ventricular myocytes. Confocal microscope study showed SK 2 protein was distributed to the periphery of the PC s. Conclusions SK 2 proteins are more abundantly present in the PC s than in the ventricular myocytes of normal rabbit ventricles. Apamin‐sensitive SK current is important in ventricular repolarization of normal PC s.

Background A secondary rise of intracellular Ca2+(Cai) and an upregulation of apamin-sensitive K+current (IKAS) are characteristic findings of failing ventricular myocytes. We hypothesize that apamin, a specific IKASblocker, may induce torsades de pointes (TdP) ventricular arrhythmia from failing ventricles exhibiting secondary rises of Cai. Objective To test the hypothesis that small conductance Ca2+activated IKASmaintains repolarization reserve and prevents ventricular arrhythmia in a rabbit model of heart failure (HF). Methods We performed Langendorff perfusion and optical mapping studies in 7 hearts with pacing-induced HF and in 5 normal control rabbit hearts. Atrioventricular block was created by cryoablation to allow pacing at slow rates. Results The left ventricular ejection fraction reduced from 69.1% [95% confidence interval 62.3%-76.0%] before pacing to 30.4% [26.8%-34.0%] (N = 7; P

Small conductance calcium activated potassium (SK) currents are abundantly present in the neurons1, 2 and in atrial cardiomyocytes.3–6 However, little or no apamin-sensitive K currents are present in normal ventricles.3, 7, 8 These channels are activated by increases in intracellular Ca2+ (Cai) and are blocked by apamin.2 In the nervous system, activation of apamin-sensitive K+ current (IKAS) is responsible for slow afterhyperpolarizations, which help terminate neuronal action potential bursts.9 Similar to rapid neuronal discharges, ventricular fibrillation (VF) also causes Cai accumulation that may persist minutes after successful defibrillation.10 Cai accumulation and acute postshock action potential duration (APD) shortening facilitated the development of late phase 3 early afterdepolarization (EAD)11 (also known as Cai transient triggered firing)12, 13 and electrical storm in that model. The acute postshock APD shortening in failing ventricles was shown to be due to IKAS activation.8 A more recent study by Chang et al14 showed that both the IKAS and the SK protein are increased in the native hearts of transplant recipients, and that apamin significantly prolongs the APD in failing human ventricular myocytes but not in normal control ventricular myocytes. These findings suggest that IKAS is important in ventricular repolarization and arrhythmogenesis in failing ventricles by shortening APD during Cai accumulation. However, the ability to accelerate the repolarization may also be antiarrhythmic. The redundancy in the complexities of myocardial repolarization (repolarization reserve)15 is important in maintaining normal and orderly ventricular repolarization, while reduced repolarization reserve underlies the mechanisms of afterdepolarization and ventricular arrhythmias in congenital or acquired long-QT syndromes.16 Myocardial infarction (MI) is followed by significant arrhythmogenic ion-channel remodeling including downregulation of multiple K currents in the peri-infarct zone as well as in the subendocardial Purkinje fibers.17 These changes were thought to underlie the mechanisms of afterdepolarizations and ventricular arrhythmias in MI ventricles. While K current remodeling after MI has been extensively studied, none of these studies included an evaluation of IKAS after MI. If there is an increased IKAS, it would counterbalance the downregulation of other K currents, hence maintaining repolarization reserve. Inhibition of IKAS by apamin would prolong the APD and reduce the repolarization reserve. The purpose of the present study was to perform optical mapping studies and patch clamp studies to test the hypothesis that IKAS is increased in rabbit ventricles with chronic MI and contributes significantly to ventricular repolarization in MI ventricles.

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10-8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established.

One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.

"No one believes modeling results except the one who performed the calculation; ...everyone believes the experimental results except the one who did the measurements." P. J. Roache - Computational Physicist.

To investigate whether rimonabant, a cannabinoid receptor antagonist, had inhibitory effects on inflammatory reactions in human umbilical vein endothelial cells (HUVEC). TNF-α-induced IL-6 production was measured by ELISA and effects on related signaling pathways were investigated by immunoblot analysis. Cellular cAMP level was measured using kinase-coupled luciferase reaction. Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IκB kinase (IKK) α/β and IκB-α degradation. ACEA, a cannabinoid receptor subtype 1 (CB1) agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of cAMP-dependent protein kinase (PKA), abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant had anti-inflammatory effects on endothelial cells and inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect was related to CB1 antagonism and PKA activation.

Background and purpose: Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts. Experimental approach: Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca2+ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts. Key results: In rat ventricular cells, piceatannol (3–30 µmol·L−1) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (Vmax) without altering Ca2+ transients. Piceatannol decreased peak INa and slowed INa inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L−1) decreased peak INa without slowing INa inactivation. The inhibition of peak INa or Vmax was associated with a negative shift of the voltage-dependent steady-state INa inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L−1, piceatannol could inhibit ICa,L, Ito, IKr, Ca2+ transients and Na+-Ca2+ exchange except IK1. Piceatannol (1–10 µmol·L−1) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury. Conclusions and implications: The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in INa inhibition and uniquely slowing INa inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L−1.

Mutations of the KCNH2 with decreased channel activity lead to congenital long QT syndrome (LQTS). We studied the electrophysiological, glycosylation, trafficking and assembly properties of three novel KCNH2 mutations identified in Taiwanese patients with LQTS (p.N633D, p.R744fs, and p.P923fs). When expressed in HEK293T cells, p.N633D and p.R744fs HERG channels displayed no HERG current while p.P923fs HERG channel showed HERG current with significantly lower (34%) current density and faster inactivation kinetics. In Western blot analysis, pR744fs was the only one with glycosylation defect, which was in consistence with the confocal microscopic findings showing that p.R744fs-GFP was the only one with trafficking defect. However, p.R744fs-GFP differed from pR744fs in being fully glycosylated while p.R744fs fusion with GFP at the N-terminus revealed glycosylation defect. To access the assembly capacity of each mutant, co-immunoprecipitation was conducted. Wild type (WT), p.N633D, and p.P923fs HERG protein showed assembly ability while p.R744fs failed to assemble with neither WT nor itself. In conclusion, we identified three novel LQTS-related KCNH2 mutations and each had a distinct mechanism of channel defect. For p.R744fs mutant, adding GFP to the C-terminus rescued the glycosylation defect but the channel was still assembly defective indicating a dissociation between glycosylation and assembly defects.

Paralysis periodica paramyotonica is an overlapping disease that shares the features of paramyotonia characteristic of paramyotonia congenita (PC) and periodic paralysis characteristic of hyperkalemic periodic paralysis. We report the case of a 23-year-old man with paralysis periodica paramyotonica. His father and a younger brother also exhibited a similar phenotype. A SCN4A Arg1448Cys mutation was detected in this family. The affected family members exhibited marked shifts in compound muscle action potential amplitudes on exercise test, and muscle weakness could be induced by potassium loading and cold exposure. This case demonstrates that SCN4A Arg1448Cys can produce paralysis periodica paramyotonica. Other genetic or environmental factors may modulate the manifestation of SCN4A Arg1448Cys mutation.

Patients with CKD have an increased risk of cardiovascular mortality from arrhythmias and sudden cardiac death. We used a rat model of CKD (Cy/+) to study potential mechanisms of increased ventricular arrhythmias. Rats with CKD showed normal ejection fraction but hypertrophic myocardium. Premature ventricular complexes occurred more frequently in CKD rats than normal rats (42% versus 11%, P=0.18). By optical mapping techniques, action potential duration (APD) at 80% of repolarization was longer in CKD rats (78±4ms) than normal rats (63±3 ms, P

Brugada syndrome (BrS) is an inheritable sudden cardiac death disease mainly caused by SCN5A mutations. Traditional approaches can be costly and time-consuming if all candidate variants need to be validated through in vitro studies. Therefore, we developed a new approach by combining multiple in silico analyses to predict functional and structural changes of candidate SCN5A variants in BrS before conducting in vitro studies. Five SCN5A non-synonymous variants (1651G>A, 1776C>G, 1673A>G, 3269C>T and 3578G>A) were identified in 14 BrS patients using direct DNA sequencing. Several bioinformatics algorithms were applied and predicted that 1651G>A (A551T) and 1776C>G (N592K) were high-risk SCN5A variants (odds ratio 59.59 and 23.93). The results were validated by Mass spectrometry and in vitro electrophysiological assays. We concluded that integrating sequence-based information and secondary protein structures elements may help select highly potential variants in BrS before conducting time-consuming electrophysiological studies and two novel SCN5A mutations were validated.

An 18-year-old woman presented with exercise induced sudden collapse. Series of cardiac work up revealed no structural cardiac abnormalities. Bidirectional ventricular tachycardia occurred during a treadmill exercise test. Under the impression of catecholaminergic polymorphic ventricular tachycardia, we screened the cardiac ryanodine receptor gene for mutation. We identified a novel heterozygous mutation at the 169th amino acid (Arg169Gln). This amino acid is highly conserved among many species and this mutation was not present in 50 normal control subjects. This patient was treated with a β-block with good response.

Background— Apamin-sensitive K currents ( I KAS ) are upregulated in heart failure. We hypothesize that apamin can flatten action potential duration restitution (APDR) curve and can reduce ventricular fibrillation duration in failing ventricles. Methods and Results— We simultaneously mapped membrane potential and intracellular Ca (Ca i ) in 7 rabbit hearts with pacing-induced heart failure and in 7 normal hearts. A dynamic pacing protocol was used to determine APDR at baseline and after apamin (100 nmol/L) infusion. Apamin did not change APD 80 in normal ventricles, but prolonged APD 80 in failing ventricles at either long (≥300 ms) or short (≤170 ms) pacing cycle length, but not at intermediate pacing cycle length. The maximal slope of APDR curve was 2.03 (95% confidence interval, 1.73–2.32) in failing ventricles and 1.26 (95% confidence interval, 1.13–1.40) in normal ventricles at baseline ( P =0.002). After apamin administration, the maximal slope of APDR in failing ventricles decreased to 1.43 (95% confidence interval, 1.01–1.84; P =0.018), whereas no significant changes were observed in normal ventricles. During ventricular fibrillation in failing ventricles, the number of phase singularities (baseline versus apamin, 4.0 versus 2.5), dominant frequency (13.0 versus 10.0 Hz), and ventricular fibrillation duration (160 versus 80 s) were all significantly ( P Conclusions— Apamin prolongs APD at long and short, but not at intermediate pacing cycle length in failing ventricles. I KAS upregulation may be antiarrhythmic by preserving the repolarization reserve at slow heart rate, but is proarrhythmic by steepening the slope of APDR curve, which promotes the generation and maintenance of ventricular fibrillation.

The apamin-sensitive small-conductance calcium-activated potassium current (IKAS ) is increased in heart failure. It is unknown if myocardial infarction (MI) is also associated with an increase of IKAS .We performed Langendorff perfusion and optical mapping in 6 normal hearts and 10 hearts with chronic (5 weeks) MI. An additional 6 normal and 10 MI hearts were used for patch clamp studies. The infarct size was 25% (95% confidence interval, 20-31) and the left ventricular ejection fraction was 50 (46-54). The rabbits did not have symptoms of heart failure. The action potential duration measured to 80% repolarization (APD80 ) in the peri-infarct zone (PZ) was 150 (142-159) milliseconds, significantly (P = 0.01) shorter than that in the normal ventricles (167 [158-177] milliseconds. The intracellular Ca transient duration was also shorter in the PZ (148 [139-157] milliseconds) than that in normal ventricles (168 [157-180] milliseconds; P = 0.017). Apamin prolonged the APD80 in PZ by 9.8 (5.5-14.1)%, which is greater than that in normal ventricles (2.8 [1.3-4.3]%, P = 0.006). Significant shortening of APD80 was observed at the cessation of rapid pacing in MI but not in normal ventricles. Apamin prevented postpacing APD80 shortening. Patch clamp studies showed that IKAS was significantly higher in the PZ cells (2.51 [1.55-3.47] pA/pF, N = 17) than in the normal cells (1.08 [0.36-1.80] pA/pF, N = 15, P = 0.019).We conclude that IKAS is increased in MI ventricles and contributes significantly to ventricular repolarization especially during tachycardia.

Na channel blockers are effective in suppressing delayed afterdepolarizations (DADs) in isolated Purkinje fibers. However, in isolated mouse ventricular myocytes lacking calsequestrin, only those Na channel blockers that also inhibit type 2 ryanodine receptor channels were effective against spontaneous Ca elevation (SCaE) and DADs.To test the hypothesis that combined Na channel and type 2 ryanodine receptor channel blocker ((R)-propafenone) is more effective than a Na channel blocker (lidocaine) in suppressing SCaE and DADs in the intact rabbit ventricles.We compared (R)-propafenone (3 μmol/L) with lidocaine (50 μmol/L) on SCaE and DADs by using epicardial optical mapping of intracellular calcium (Ca(i)) and membrane voltage in Langendorff-perfused rabbit hearts. SCaE and DADs were induced by rapid pacing trains and isoproterenol (0.3 μmol/L) infusion. One arbitrary unit is equivalent to the Ca transient amplitude of paced beats.SCaEs were observed at the cessation of rapid pacing in all hearts at baseline. (R)-Propafenone nearly completely inhibited DADs and SCaE (0.04 arbitrary units [95% confidence interval 0.02-0.06] vs 0.23 arbitrary units [95% confidence interval 0.18-0.28] at baseline; n = 6 hearts; P.001). Lidocaine also significantly reduced the SCaE but was significantly (P.05) less effective than (R)-propafenone. Both drugs increased the rise time of action potential upstroke and reduced conduction velocity to a similar extent, suggesting a significant inhibition of I(Na).Both Na channel blockers significantly reduced tachycardia-induced SCaEs in the rabbit ventricles, but (R)-propafenone was significantly more effective than lidocaine. These data suggest that type 2 ryanodine receptor inhibition potentiates the activity of Na channel blockers against SCaE and DADs in the intact hearts.

Niemann-Pick type C1-like 1 (NPC1L1) protein is responsible for intestinal cholesterol absorption. The aim of the study was to identify genetic polymorphisms of the NPC1L1 gene as well as their functional significance. The method involved screening of promoter and coding regions of the NPC1L1 gene for genetic polymorphisms by direct DNA sequencing of genomic DNA from 50 individuals. Functional studies on promoter polymorphisms were performed using luciferase assay. Association between the polymorphisms and serum cholesterol levels were investigated in 224 individuals. The results showed that in total, 11 single nucleotide polymorphisms were identified. Among them, a promoter polymorphism, g.-762TC, and a synonymous polymorphism, g.1679CG, were common (34 and 36%, respectively). These two polymorphisms were highly linked (D' value=0.7459, P-value0.00001). For the g.-762TC promoter polymorphism, luciferase assay in HepG2 cell line demonstrated that the -762C allele had a significantly higher promoter activity than the -762T allele (1.30+/-0.22 vs 0.37+/-0.06, 3.5-fold, P0.05). We also showed that the NPC1L1 promoter activity was downregulated by cholesterol content in both genotypes. When association studies were performed, we found that -762C allele was associated with significantly higher serum total cholesterol and LDL-cholesterol content levels in a recessive model (LDL-cholesterol value=131.2+/-8.1 vs 116.4+/-2.2 mg dl(-1); total cholesterol value=214.7+/-9.0 mg dl(-1) vs 196.9+/-2.6, P-value0.05, n=224). In conclusion, the C allele at -762 position of the NPC1L1 gene was common in people of Chinese ethnicity. The -762C allele had a higher promoter activity and was associated with a higher serum total cholesterol and LDL-cholesterol level.

The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively), and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV). In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.

Background/Purpose Desmosome gene mutations have been reported in patients with arrhythmogenic right ventricular dysplasia (ARVD). However, there are hardly any genetic studies in Asians. We studied the clinical characteristics, cardiac manifestations and desmosome gene mutations in ARVD patients in Taiwan. Methods Medical records of five ARVD patients were reviewed and genomic DNA was obtained from peripheral blood samples. Mutation screening in desmoplakin (DSP), plakophilin-2, desmoglein-2 (DSG2) and desmocollin-2 genes was performed using polymerase chain reaction and DNA sequencing techniques. Results Among the five patients, three presented with palpitations followed by loss of consciousness, and the other two had palpitations or chest tightness without loss of consciousness. Electrocardiogram (ECG), magnetic resonance imaging and signal averaged ECG results were similar to those reported in Western countries. Mutations in the desmosome genes were identified in four of the five patients (three with a DSG2 mutation and one with a DSP mutation). Five gene mutations were noted in four patients and all mutations were novel (one patient had a DSG2 double mutation). The mutation types were missense in four and splicing mutation in one. Conclusion Patients with ARVD in Taiwan had similar clinical and cardiac manifestations as reported in the Western literature. More than half of the patients had desmosome gene mutations.

Metformin has been reported to reduce cardiovascular complications in diabetic patients. The purpose of the present study was to investigate the anti-inflammatory effects of metformin on endothelial cells and the related molecular mechanisms.Human umbilical vein endothelial cells (HUVEC) were used for the experiments. The effects of metformin on TNF-alpha-induced IL-6 production were investigated. Modulation of AMPK and related signal transduction pathways were also performed.TNF-alpha increased IL-6 secretion by HUVEC in a dose-dependent manner but inhibitors of NF-kappaB abolished the TNF-alpha-induced IL-6 production. Pre-treatment with metformin (100-1000 micromol/L) also inhibited TNF-alpha-induced IL-6 production, phosphorylation of IkappaB kinase (IKK) alpha/beta and IkappaB-alpha degradation. Metformin increased phosphorylation of AMP-activated kinase (AMPK) but wortmannin, a PI3K inhibitor, negated its effects on AMPK phosphorylation and TNF-alpha-induced IkappaB-alpha degradation. AICAR, a direct AMPK activator, had inhibitory effects on TNF-alpha-induced IL-6 production, similar to that of metformin. Transfection of siRNA against alpha1-AMPK eradicated the inhibitory effects of metformin on TNF-alpha-induced IL-6, implying the essential role of AMPK.Metformin had anti-inflammatory effects on endothelial cells and inhibited TNF-alpha-induced IKKalpha/beta phosphorylation, IkappaB-alpha degradation and IL-6 production in HUVEC. This effect was related to PI3K-dependent AMPK phosphorylation.