Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

Rationale GDF11 (Growth Differentiation Factor 11) is a member of the transforming growth factor β (TGFβ) super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH), and restoring GDF11 to normal levels in old mice rescued PCH. Objective To determine if and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results 24-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in-vitro. After treatment, rGDF11 levels were significantly increased but there was no significant effect on either heart weight (HW) or body weight (BW). HW/BW ratios of old mice were not different from 8 or 12 week-old animals, and the PCH marker ANP was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle treated animals at baseline and remained unchanged at 1, 2 and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes (NRVM), to explore the putative anti-hypertrophic effects of GDF11, showed that GDF11 did not reduce NRVM hypertrophy, but instead induced hypertrophy. Conclusions Our studies show that there is no age-related PCH in disease free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase-and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene. Soluble epoxide hydrolase (sEH)3 is a ubiquitous enzyme found in many tissues such as liver, kidney, heart, and ovary (1). sEH catalyzes the degradation of endogenous epoxy lipids such as epoxyeicosatrienoic acids (EETs) to their less active diols (dihydroxyeicosatrienoic acids, DHETs) and hence plays a critical role in the control of EET levels (2). These epoxy lipids are potent vasodilators, regulating cerebral and renal homodynamic and blood pressure (3-5). In addition, EETs inhibit platelet aggregation (6), promote fibrinolysis (7) and have antiinflammatory properties (8, 9). Whereas deletion of the sEH gene, Ephx2, has been reported to reduce blood pressure in male mice (10), the inhibition of endogenous EET hydrolysis may provide pharmacological benefit in hypertension and acute inflammation (11).The human Ephx2 gene encodes sEH and consists of 19 exons encoding 555 amino acids (12). There is 73% homology between the human and mouse sEH protein sequences (13), with 100% conservation in the catalytic residues (14). Each monomer of the homodimeric mouse sEH has two distinct domains (14,15). The N-terminal domain exhibits phosphatase activity, and the C-terminal domain is responsible for the epoxide hydrolase activities (...

Objective-Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) Ϫ/Ϫ and ϩ/ϩ mice. Methods and Results-EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N-adamantyl-NЈ-dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin/charybdotoxin (ChTx), but not by iberiotoxin, nor by the cytochrome P450 inhibitor PPOH. All four EETs (order of potency: 8,9-EET Ͼ14,15-EETϷ5,6-EET Ͼ11,12-EET) and all 4 dihydroxy derivatives (14,15-DHETϷ8,9-DHETϷ11,12-DHET Ͼ5,6-DHET) produced dose-dependent vasodilation. Endothelial removal or L-NAME blocked 8,9-EET and 14,15-DHET-dependent dilations. The effects of apamin/ChTx were minimal. 8,9-EET and 14,15-DHET induced NO production in endothelial cells. ADU (100 g/mL in drinking water) lowered blood pressure in angiotensin II-infused hypertension, but not in L-NAME-induced hypertension. Blood pressure and EDHF responses were similar in L-NAME-treated sEH ϩ/ϩ and Ϫ/Ϫ mice. Conclusions-Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release. Key Words: eicosanoids Ⅲ soluble epoxide hydrolase Ⅲ NO synthase Ⅲ L-NAME Ⅲ EDRF T he endothelium releases nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). 1,2 Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid (AA) that may be EDHFs. 3,4 Other candidates include K ϩ ions and hydrogen peroxide (H 2 O 2 ). [5][6][7] Endothelial cell hyperpolarization spreads to adjacent vascular smooth muscle cells (VSMCs) through myo-endothelial gap junctions. 8,9 Calcium-activated potassium channels, most probably the SK4 (IK Ca ) and SK3 (SK Ca ) expressed on the endothelium, are the end-cellular gateway mediating hyperpolarization, and subsequent EDHF relaxation. 2,4,10 -13 EETs convincingly cause hyperpolarization. 14 -16 They can induce vasodilation in certain vascular beds by increasing the open-state probability of calcium-activated potassium (BK) channels. 4,15,17 The soluble epoxide hydrolase (sEH) metabolizes EETs to dihydroxy derivatives (DHET). sEH inhibition could enhance EET activity. 18 Blood pressure decreased in spontaneously hypertensive rats (SHR) given an sEH inhibitor. 19 sEH inhibition also lowered blood pressure in rats given angiotensin II (Ang II). 20 Thus, sEH could contribute to Ang IIinduced hypertension 21 and salt-sensitivity. 22 Even desoxycorticosterone acetate (DOCA)-salt hypertension was ameliorated with sEH inhibition. 23 Finally, male sEH genedeleted (Ϫ/Ϫ) mice had lower blood pressures than sEH ϩ/ϩ mice. 24 EETs coul...