Background:In cardiac surgery, the contact of blood with the artificial surfaces of the cardiopulmonary bypass results in activation of coagulation, fibrinolysis, and platelets, which is recognized as reason for increased bleeding tendency. Antifibrinolytics like tranexamic acid or the broad-spectrum protease inhibitor aprotinin attenuate this response. The marketing of aprotinin has been suspended after a recent clinical trial suggested increased risks associated with aprotinin. Moreover, aprotinin is a protein of animal origin and has antigenic properties. As a result, alternative antifibrinolytic compounds are desirable.Methods: This in vitro study compared the antifibrinolytic efficacy of the synthetic small molecule CU-2010 with aprotinin and tranexamic acid. Antifibrinolytic activity in plasma and whole blood of ten healthy volunteers was examined with a turbidometric method and with tissue factor-activated thromboelastometry (ROTEM ® ; Pentapharm, Munich, Germany). In addition, anticoagulant effects were assessed through measurement of plasma and whole blood clotting times and thrombin generation.Results: With its high affinity for plasmin (K i , 2 nM), CU-2010 inhibited fibrinolysis comparable to aprotinin (K i , 4 nM) and was ten times more potent than tranexamic acid. CU-2010 also inhibited plasma kallikrein (K i < 1 nM) and factors Xa (K i , 45 nM) and XIa (K i , 18 nM), which was reflected in prolongation of coagulation times and an attenuation of thrombin generation.Conclusion: These findings suggest that CU-2010 has similar antifibrinolytic potency compared to aprotinin, is more potent than tranexamic acid, and possesses some anticoagulant effects.

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Expression and secretion of apolipoprotein A-I (apoA-I) by cultured liver cells can be markedly stimulated by triazolodiazepines (TZDs). It has been shown previously that the thieno-TZD Ro 11-1464 increases plasma levels of apoA-I and in vivomacrophage reverse cholesterol transport in mice. However, these effects were only seen at high doses, at which the compound could act on central benzodiazepine (BZD) receptors or platelet activating factor (PAF) receptors, interfering with its potential utility. In this work, we describe 2 new thieno-TZDs MDCO-3770 and MDCO-3783, both derived from Ro 11-1464. These compounds display the same high efficacy on apoA-I production, metabolic stability, and lack of cytotoxicity in cultured hepatocytes as Ro 11-1464, but they do not bind to the central BZD receptor and PAF receptor. The quinazoline RVX-208 was less efficacious in stimulating apoA-I production and displayed signs of cytotoxicity. Certain TZDs stimulating apoA-I production are now known to be inhibitors of bromodomain (BRD) extra-terminal (BET) proteins BRDT, BRD2, BRD3, and BRD4, and this inhibition was inferred as a main molecular mechanism for their effect on apoA-I expression. We show here that the thieno-TZD (+)-JQ1, a potent BET inhibitor, strongly stimulated apoA-I production in Hep-G2 cells, but that its enantiomer (−)-JQ1, which has no BET inhibitor activity, also showed considerable effect on apoA-I production. MDCO-3770 and MDCO-3783 also inhibited BRD3 and BRD4 in vitro, with potency somewhat below that of (+)-JQ1. We conclude that the effect of thieno-TZDs on apoA-I expression is not due to inhibition of the BZD or PAF receptors and is not completely explained by transcriptional repression by BET proteins.

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A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-␣ (TNF-␣)]should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein ␣2-macroglobulin (A2M). The conjugate binds TNF-␣ as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-␣ from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 g/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.In the past several years, mortality in patients with sepsis syndrome has decreased somewhat, but in those patients with septic shock and multiple organ failure, mortality still exceeds 50% (Danai and Martin, 2005). This high mortality is observed despite ventilatory, hemodynamic, metabolic, and renal support. Some patients-in particular, patients with genetic polymorphisms associated with low or moderate TNF response-survive the ordeal more often, but it remains frustrating not being able to stop the downhill course leading to multiple organ failure and death in other patients that results from overwhelming inflammation. New therapies have been sought and tested, including those preventing the biological activity of pathogen-associated molecular patterns, such as endotoxin or the downhill host response, most notably TNF-␣. Based on a wealth of animal studies, anti-inflammatory strategies, such as neutralizing TNF-␣, have been advocated to provide adjunctive therapy to the patient who continues to deteriorate in the face of considerable support in the intensive care unit. Unfortunately, these anticytokine therapies have not dramatically reduced mortality in doubleblind, placebo-controlled trials involving thousands of patients, although there is a consistent but statistically non-