Objectives-Exercise increases fatty acid oxidation (FAO), improves serum high density lipoprotein cholesterol (HDLc) and triglycerides (TG), and upregulates skeletal muscle peroxisome proliferator activated receptor (PPAR)␦ expression. In parallel, PPAR␦ agonist-upregulated FAO would induce fatty-acid uptake (via peripheral lipolysis), and influence HDLc and TG-rich lipoprotein particle metabolism, as suggested in preclinical models. Methods and Results-Healthy volunteers were allocated placebo (nϭ6) or PPAR␦ agonist (GW501516) at 2.5 mg (nϭ9) or 10 mg (nϭ9), orally, once-daily for 2 weeks while hospitalized and sedentary. Standard lipid/lipoproteins were measured and in vivo fat feeding studies were conducted. Human skeletal muscle cells were treated with GW501516 in vitro and evaluated for lipid-related gene expression and FAO. Serum TG trended downwards (Pϭ0.08, 10 mg), whereas TG clearance post fat-feeding improved with drug (Pϭ0.02). HDLc was enhanced in both treatment groups (2.5 mg Pϭ0.004, 10 mg PϽ0.001) when compared with the decrease in the placebo group (Ϫ11.5Ϯ1.6%, Pϭ0.002). These findings complimented in vitro cell culture results whereby GW501516 induced FAO and upregulated CPT1 and CD36 expression, in addition to a 2-fold increase in ABCA1 (Pϭ0.002). However, LpL expression remained unchanged. Conclusions-This is the first report of a PPAR␦ agonist administered to man. In this small study, GW501516 significantly

On the basis of simulation results, adding dapagliflozin to currently available treatment options is projected to further decrease the CV and microvascular complications associated with T2DM.

We previously demonstrated that DNA-polylactic-polyglycolic acid (PLGA)-coated stents can deliver genes to the arterial wall with reporter expression involving 1% of neointimal cells. The present study investigated a novel formulation utilizing denatured collagen in DNA-stent coatings; denatured collagen was hypothesized to enhance gene transfer due to adhesion molecule interactions and actinrelated mechanisms. Arterial smooth muscle cells (SMCs) cultivated on denatured collagen had significantly greater plasmid DNA (b-galactosidase) transfection than SMC grown on native collagen (18.371.2 vs 1.070.1%, Po0.001). The denatured-collagen effect was completely blocked with antia v b 3 integrin antibody. SMCs cultivated on native collagen supplemented with tenascin-C (TN-C), a protein recognized by a v b 3 integrins, showed a 33-fold increase in transfection compared to control (Po0.001); this effect was also blocked with anti-a v b 3 antibody. We observed that cells grown on denatured collagen had marked F-actin-enriched stress fibers and intense perinuclear G actin, compared to those grown on native collagen, which demonstrated F-actinenriched focal adhesions without perinuclear G-actin localization. Cytochalasin-D, an F actin depolymerizing agent, caused significantly increased SMC transfection in cells cultivated on native collagen compared to control cells (18.071.8 vs 3.0270.9%, Po0.001) further supporting the view that actin-related cytoskeletal changes influence transfection. A denatured-collagen-PLGA composite vascular stent coating similarly resulted in increased plasmid DNA green fluorescent protein (GFP) expression compared to controls (Po0.001) in SMC cultures; the increased transfection was blocked by anti-a v b 3 antibody. Pig coronary studies comparing denatured-collagen-PLGA-coated stents containing plasmid DNA (encoding GFP) to coated stents without DNA demonstrated 10.8% of neointimal cells transfected; this level of expression was almost an order of magnitude greater than previously reported with a DNA delivery stent. It is concluded that denatured collagen incorporated into plasmid DNA-stent coating formulations may increase the level of gene expression in vitro and in vivo because of integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton.

Background Currently, there is no approved reversal agent for direct activated factor Xa (FXa) inhibitors; however, several agents are under investigation, including prothrombin complex concentrates (PCCs). Objective This open-label, randomized, placebo-controlled, three-period crossover study assessed the effect of two four-factor PCCs on apixaban pharmacodynamics and pharmacokinetics in 15 healthy subjects. Methods Subjects received apixaban 10 mg twice daily for 3 days. On day 4, 3 h after apixaban, subjects received a 30-min infusion of 50 IU kg Cofact, Beriplex P/N (Beriplex), or saline. Change in endogenous thrombin potential (ETP), measured with a thrombin generation assay (TGA), was the primary endpoint. Secondary endpoints included changes in other TGA parameters, prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time, anti-FXa activity, apixaban pharmacokinetics, and safety. Results Apixaban-related changes in ETP and several other pharmacodynamic measures occurred following apixaban administration. Both PCCs reversed apixaban's effect on ETP; the differences in adjusted mean change from pre-PCC baseline to end of infusion were 425 nm min (95% confidence interval [CI] 219.8-630.7 nm min; P < 0.001) for Cofact, and 91 nm min (95% CI - 31.3 to 212.4 nm min; P > 0.05) for Beriplex. Both PCCs returned ETP to pre-apixaban baseline levels 4 h after PCC infusion, versus 45 h for placebo. For both PCCs, mean ETP peaked 21 h after PCC initiation, and then slowly decreased over the following 48 h. Both PCCs reversed apixaban's effect on TGA peak height, PT, and INR. Apixaban pharmacokinetic and anti-FXa profiles were consistent across treatments. Conclusions Cofact and Beriplex reversed apixaban's steady-state effects on several coagulation assessments.

Comparable Frel was observed for oral apixaban solution, tablet, NGT administration of solution flushed with D5W and infant formula, and NGT administration of crushed tablet suspension. Exposure was less when oral solution was administered via NGT with nutritional supplement. These results support several alternative methods of administering apixaban that may be useful in certain clinical situations. ClinicalTrials.gov identifiers: NCT02034565, NCT02034578, and NCT02034591.

The present studies investigated the cardiac potassium channel missense mutation, Q9E-hMiRP1, for potential use as a gene therapy construct for cardiac arrhythmias. This gene abnormality is one of a number of mutations that can cause the long QT syndrome (LQTS), a hereditary arrhythmia disorder that is associated with sudden death. However, individuals who carry the Q9E-hMiRP1 variant are predisposed to developing the LQTS only after clarithromycin administration. Because the electrophysiologic mechanism of action of Q9E-hMiRP1 (i.e., diminished potassium currents resulting in delayed myocardial repolarization) is comparable to that of class III antiarrhythmic agents, we examined Q9E-hMiRP1 as a candidate gene therapy construct for site-specific treatment of reentrant atrial cardiac arrhythmias. Our rationale was also based on the hypothetical safety of the atrial use of Q9E-hMiRP1 because LQTS characteristically causes ventricular but not atrial arrhythmias. Furthermore, the possible use of clarithromycin to control the conduction effects of overexpressed Q9E-hMiRP1 pharmacologically was another attractive feature. In our studies we investigated the use of two bicistronic plasmid DNA gene vectors with either hMiRP1 or Q9E-MiRP1 and green fluorescent protein (GFP), plus a C-terminus of the hMiRP1 or of the Q9E-hMiRP1 coding region for the FLAG (MDYKDDDDK) peptide. We generated two stable cell lines using HEK293 and SH-SY5Y (human cell lines), overexpressing the genes of interest, confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots. The expected plasma membrane localization of each overexpressed transgene was confirmed by immunofluorescent confocal fluorescent microscopy using anti-FLAG antibody. Patchclamp studies demonstrated that cells transfected with Q9E-hMiRP1 plasmid DNA exhibited significantly reduced potassium currents but only with clarithromycin administration. A novel plasmid DNA delivery system was formulated for use in our animal studies of the hMiRP1 vectors, which was composed of DNA-anti-DNA antibody-cationic lipid (DAC) heteroplexes. In vitro and in vivo studies using DAC heteroplexes containing anti-DNA antibodies with nuclear targeting capability demonstrated significantly increased transfection compared to naked DNA, and to DNA-cationic lipid complexes. Pig atrial myocardial injections of DAC heteroplexes demonstrated 16% of regional cardiac myocytes transfected using the Q9E-hMiRP1 plasmid, and 15% of cells with the hMiRP1 vector. It is concluded that the present studies support the view that site-specific gene therapy for atrial arrhythmias is feasible using plasmid vectors for overexpressing ion channel mutations that have electrophysiologic effects comparable to class III antiarrhythmic agents.

Abstract. The aim of the investigation was to evaluate alternatives to standard first-in-human (FIH) designs in order to optimize the information gained from such studies by employing novel agile trial designs. Agile designs combine adaptive and flexible elements to enable optimized use of prior information either before and/or during conduct of the study to seamlessly update the study design. A comparison of the traditional 6+2 (active+placebo) subjects per cohort design with alternative, reduced sample size, agile designs was performed by using discrete event simulation. Agile designs were evaluated for specific adverse event models and rates as well as dose-proportional, saturated, and steepaccumulation pharmacokinetic profiles. Alternative, reduced sample size (hereafter referred to as agile) designs are proposed for cases where prior knowledge about pharmacokinetics and/or adverse event relationships are available or appropriately assumed. Additionally, preferred alternatives are proposed for a general case when prior knowledge is limited or unavailable. Within the tested conditions and stated assumptions, some agile designs were found to be as efficient as traditional designs. Thus, simulations demonstrated that the agile design is a robust and feasible approach to FIH clinical trials, with no meaningful loss of relevant information, as it relates to PK and AE assumptions. In some circumstances, applying agile designs may decrease the duration and resources required for Phase I studies, increasing the efficiency of early clinical development. We highlight the value and importance of useful prior information when specifying key assumptions related to safety, tolerability, and PK.

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