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Thromboembolic diseases such as myocardial infarction, stroke, transient ischemic attacks and pulmonary embolism are major causes of morbidity and mortality worldwide. GPIIb/IIIa is the key receptor involved in platelet aggregation and is a validated target for therapeutic approaches and diagnostic imaging. The aim of this study was to develop and characterize a specific small molecule tracer for positron emission tomography (PET) imaging that binds with high affinity to GPIIb/IIIa receptors and has suitable pharmacokinetic properties to overcome limitations of previous approaches. Methods: Binding of 18F-GP1 to GPIIb/IIIa receptors was investigated in competition binding assays and autoradiography using a fresh cardiac thrombus from an explanted human heart. The clot-to-blood-ratio for 18F-GP1 was investigated by an in vitro blood flow model. Biodistribution and thrombus detection was investigated in cynomolgus monkeys after insertion of a roughened catheter into either the vena cava or aorta. Results:18F-GP1 is a novel fluorine-18 labeled small molecule for PET imaging of thrombi. The IC50 of 18F-GP1 to GPIIb/IIIa was determined to be 20nM. 18F-GP1 binds to thrombi with a mean clot-to-blood ratio of 95. Binding is specific and can be displaced by excess non-radioactive derivative. Binding is not effected by anticoagulants such as aspirin or heparin. 18F-GP1 shows rapid blood clearance and a low background after i.v. injection in cynomolgus monkeys. Small arterial, venous thrombi, thrombotic depositions on damaged endothelial surface and small cerebral emboli were detected in vivo by PET imaging. Conclusion:18F-GP1 binds specifically with high affinity to the GPIIb/IIIa receptor involved in platelet aggregation. Due to its favorable pre-clinical characteristics, 18F-GP1 is currently being investigated in a human clinical study.

Objectives The knowledge of chamber-specific gene expression in human atrial and ventricular myocardium is essential for the understanding of myocardial function and the basis for the identification of putative therapeutic targets in the treatment of cardiac arrhythmia and heart failure. In this study the gene expression pattern of human left atrial and ventricular myocardium was analyzed. Methods Global mRNA expression patterns with high-density oligonucleotide arrays between left atrial and left ventricular myocardium of 6 patients with heart failure undergoing heart transplantation were compared. Clustering of microarray data confirmed chamber-specific gene expression profiles. Genes similarly expressed in all patients were further analyzed, and data were confirmed by means of real-time polymerase chain reaction and Western blot analysis. Results Of 22,215 genes examined, 7115 transcripts were found to be expressed in all 12 human myocardial samples. One hundred twenty-five genes were differentially expressed between left atrial and left ventricular specimens in all patients examined. Novel genes preferentially expressed in human atria were identified. Interestingly, several potassium channels of subfamily K are more highly expressed in atria than in ventricles. Members of the potassium inwardly rectifying channel of subfamily J were found to be more highly expressed in human ventricular myocardium. Finally, chronic atrial fibrillation was associated with reduced atrial expression of the potassium channel TWIK-1, suggesting potential contribution of the corresponding current to electrical remodeling. Conclusions Human atria and ventricles show specific gene expression profiles. Our data provide the basis of a comprehensive understanding of chamber-specific gene expression in diseased human hearts and will support the identification of therapeutic targets in the treatment of arrhythmia and heart failure.

Material-methods The mRNA expression of PDE5 was determined in rat tissues of the lower urinary tract. To test the functional relevance of the PDE5 expression, sildenafil, vardenafil, and tadalafil were tested on pre-contracted isolated rat bladder, prostate and urethra strips. The in vivo efficacy was tested in a partial bladder outlet obstruction (BOO) model after acute and chronic treatment.

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.

Rivaroxaban (BAY 59-7939) is a novel, oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The antithrombotic activity of rivaroxaban has been demonstrated in several venous and arterial thrombosis models in rats, at doses that did not increase bleeding times. However, as with all anticoagulants, high doses of rivaroxaban cause prolongation of bleeding time. The aim of this study was to assess the ability of recombinant Factor VIIa (rFVIIa, NovoSeven®) to neutralize the anticoagulant effects of high-dose rivaroxaban. Animals were divided into four study groups receiving the following treatment and the appropriate placebo: rivaroxaban 2 mg/kg, rivaroxaban 2 mg/kg plus rFVIIa 100 or 400 μg/kg i.v, and rFVIIa 400 μg/kg i.v. Anesthetized rats were pretreated with rivaroxaban (or placebo); rFVIIa (or placebo) was then injected intravenously 1 minute after induction of bleeding. Mesenteric bleeding times were measured after cutting small branched arteries using microsurgery scissors, while the intestinal surface was superfused with 0.9% NaCl solution. Bleeding times were obtained from three control vessel incisions before treatment, and one after intravenous administration of rivaroxaban and/or rFVIIa (n=10). Prothrombin time (PT), thrombin generation (TG), and inhibition of FXa activity (activated by Russell’s viper venom) were measured in blood from animals that had received study medication (rivaroxaban 2 mg/kg and/or rFVIIa 400 μg/kg) or placebo, but had not been used for bleeding time determination (n=6). Administration of rFVIIa (400 μg/kg) reduced mesenteric bleeding time to 0.7-times baseline (Table). High-dose rivaroxaban increased bleeding time to 3.3-times baseline. The administration of rFVIIa to animals which had received rivaroxaban significantly reduced the bleeding times to 2.4- and 1.7-times baseline at doses of 100 μg/kg and 400 μg/kg, respectively. Administration of rFVIIa (400 μg/kg) reduced PT and the lag time of TG, but had no effect on endogenous thrombin potential (ETP: total amount of thrombin activity) in control animals. Rivaroxaban-induced prolongation of PT and lag time of TG were partially reversed by rFVIIa, as was the reduction in ETP. Rivaroxaban-induced inhibition of FXa activity was not affected by rFVIIa. These results demonstrate that rFVIIa partially reverses the anticoagulation effect (bleeding time, prolongation of PT, ETP, and lag time of TG) of rivaroxaban without affecting inhibition of FXa activity. Thus, these data indicate that rFVIIa has the potential to be used as an antidote to the oral, direct FXa inhibitor, rivaroxaban, if this was necessary. | Group | Change in bleeding time (x-fold) | PT (sec) | Thrombin generation (ETP) (nM x min) | Thrombin generation lag time (min) | Inhibition of FXa activity (%) | |:--------------------------------------:| -------------------------------- | -------- | ------------------------------------ | ---------------------------------- | ------------------------------ | | ETP: endogenous thrombin potential | | Placebo | 1 (baseline) | 15±0.4 | 516±16 | 1.2 ±0.04 | 0±3 | | rFVIIa 400 μg/kg | 0.7±0.1 | 7±0.2 | 507±19 | 0.7±0.01 | 1±4 | | Rivaroxaban 2 mg/kg | 3.3±0.5 | 97±5.6 | 363±14 | 2.4±0.2 | 77±4 | | Rivaroxaban 2 mg/kg + rFVIIa 400 μg/kg | 1.7±0.2 | 54±3.5 | 428±13 | 1.9±0.1 | 81±2