DSR-71167, a novel mineralocorticoid receptor antagonist with carbonic anhydrase inhibitory activity, separates urinary sodium excretion and serum potassium elevation in rats.

Mineralocorticoid receptor (MR) antagonists, such as spironolactone (SPI) and eplerenone (EPL), are useful for treating hypertension and heart failure. However, these two agents have the serious side effect of hyperkalemia. We hypothesized that adding the ability to inhibit carbonic anhydrase (CA) would reduce the risk of hyperkalemia associated with MR antagonists. We investigated the profiles of DSR-71167 [2-([(2,2-difluoroethyl)amino]methyl)-2'-fluoro-N-(3-methoxy-4-sulfamoylphenyl)biphenyl-4-carboxamide hydrochloride; an MR antagonist with weak CA inhibitory activity] with regard to antimineralocorticoid actions by examining relationships between the urinary excretion of sodium (index of antimineralocorticoid action) in deoxycorticosterone acetate-treated rats and elevation of serum levels of potassium in potassium-loaded rats compared with a DSR-71167 derivative without CA inhibition (2-(hydroxymethyl)-N-[4-(methylsulfonyl)phenyl]-2'-(trifluoromethyl)biphenyl-4-carboxamide), SPI, and EPL. DSR-71167 dose-dependently increased urinary excretion of sodium in deoxycorticosterone acetate-treated rats without elevating serum levels of potassium in potassium-loaded rats. 2-(Hydroxymethyl)-N-[4-(methylsulfonyl)phenyl]-2'-(trifluoromethyl)biphenyl-4-carboxamide, SPI, and EPL elevated serum levels of potassium significantly in potassium-loaded rats at doses that increased MR inhibitory activity. We confirmed that DSR-71167 significantly increases urinary bicarbonate and decreases blood bicarbonate, as pharmacodynamic markers of CA inhibition, in intact rats. Chronic DSR-71167 administration showed antihypertensive effects in high salt-loaded Dahl hypertensive rats. These results demonstrate that DSR-71167 is a novel type of MR antagonist, with CA inhibitory activity, which is expected to become a safer MR antagonist with a low potential risk for hyperkalemia.

Three-dimensional rational approach to the discovery of potent substituted cyclopropyl urea soluble epoxide hydrolase inhibitors.

We have previously reported a series of cyclopropyl urea derivatives as potent orally available soluble epoxide hydrolase (sEH) inhibitors. Here, we designed and synthesized three substituted cyclopropane derivatives that occupy all available pockets of sEH catalytic domain. Compound 14 with a diphenyl substituted cyclopropyl moiety showed good sEH inhibitory activity. Co-crystal structure of this compound and human sEH hydrolase catalytic domain revealed enzyme pockets occupied by the phenoxypiperidine part and the diphenyl cyclopropyl moiety. Furthermore, investigation of the phenoxypiperidine part of compound 14 resulted in the discovery of compound 19, which showed potent sEH inhibitory activity (sub-nM sEH IC50 values).

Structure-based optimization of cyclopropyl urea derivatives as potent soluble epoxide hydrolase inhibitors for potential decrease of renal injury without hypotensive action.

Epoxyeicosatrienoic acids (EETs) are known to have beneficial pharmacological effects on various cardiovascular events. However, EETs are biologically metabolized by soluble epoxide hydrolase (sEH) to less active metabolites. In our search for potent sEH inhibitors, we optimized a series of cyclopropyl urea derivatives and identified compound 38 as a potent sEH inhibitor with minimal CYP inhibition and good oral absorption in rats. Administration of 38 to DOCA-salt rats suppressed urinary albumin and MCP-1 excretion without affecting systolic blood pressure.

SM-368229, a novel promising mineralocorticoid receptor antagonist, shows antihypertensive efficacy with minimal effect on serum potassium level in rats.

The purpose of this study was to evaluate the effects of SM-368229, a novel mineralocorticoid receptor (MR) antagonist with partial agonistic activity, and spironolactone (SPI) on systolic blood pressure (SBP) and serum potassium in spontaneously hypertensive rats. SM-368229 given for 2 weeks prevented the increase in SBP without serum potassium elevation, but the treatment with SPI prevented SBP increase with serum potassium elevation. To elucidate the contribution of partial agonistic activity of SM-368229 for MR in the mitigation of serum potassium elevation, we studied the relationships between sodium balance decrease, as an index of antimineralocorticoid action, and serum potassium elevation in adrenalectomized and/or potassium-loaded rats, using SM-368229 and its derivatives (DSR-11861 and DSR-14397) showing different partial agonist activities for MR (12%, 0%, and 36%, respectively). DSR-11861 and SPI reversed sodium balance and increased serum potassium. SM-368229 also reversed sodium balance but did not show apparent serum potassium increase. Although DSR-14397 did not show serum potassium increase, its antimineralocorticoid action was very weak. These findings indicate that serum potassium elevation is negatively related to partial agonistic activities for MR, and SM-368229 shows antihypertensive efficacy with minimal effect on serum potassium level, probably due to its partial agonistic property.

Antihypertensive and cardiorenal protective effects of SM-368229, a novel mineralocorticoid receptor antagonist, in aldosterone/salt-treated rats.

The purpose of this study was to evaluate the effects of SM-368229, a novel mineralocorticoid receptor (MR) antagonist, on the blood pressure and cardiorenal injury markers in aldosterone/salt-treated hypertensive rats, in comparison to those of spironolactone (SPI). Uninephrectomized rats, given 1% NaCl to drink, were infused with aldosterone (0.75 µg/h, s.c.). In experiment 1, SM-368229 (10, 30 mg/kg) or SPI (100 mg/kg) were administered for 14 days immediately after aldosterone/salt loading. In experiment 2, SM-368229 (10 mg/kg) or SPI (100 mg/kg) were administered for 10 days after 10 days of aldosterone/salt loading. In both experiments, SM-368229 prevented the increase in systolic blood pressure, heart/kidney weights, and urinary protein/N-acetyl-ß-D- glucosaminidase excretion caused by aldosterone infusion. In real-time polymerase chain reaction analysis, SM-368229 abolished aldosterone-induced gene expression levels for inflammatory, fibrosis and oxidative stress markers in hearts and kidneys. The antihypertensive effect of SM-368229 (30 mg/kg) was superior to that of SPI, and the antihypertensive and cardiorenal protective effects of SM-368229 (10 mg/kg) were similar to those of SPI (100 mg/kg) in both experiments. These results clearly demonstrated that SM-368229 strongly attenuated the progression of hypertension and exerted cardiorenal protection in aldosterone/salt-treated hypertensive rats.

SM-368229, a novel selective and potent non-steroidal mineralocorticoid receptor antagonist with strong urinary Na+ excretion activity.

Mineralocorticoid receptor (MR) antagonists, such as spironolactone (SPI) and eplerenone (EPL), are useful for the treatment of hypertension and heart failure. However, the use of these two agents has been limited due to endocrine disturbance (SPI) and poor drug action (EPL). In our search for safer and more effective MR antagonists, we identified SM-368229 as a novel non-steroidal MR antagonist. SM-368229 showed strong MR inhibitory activity with IC(50) values of 0.021 and 0.13 µM in the binding assay and reporter-gene assay, respectively. The selectivity of SM-368229 for MR was 18-fold higher than that for other steroid receptors, such as androgen, progesterone, and glucocorticoid receptors. SM-368229 dose-dependently increased urinary Na(+)/K(+) ratio with an ED(50) value of 5.6 mg/kg in adrenalectomized rats treated with deoxycorticosterone acetate, and its efficacy was superior to that of SPI (ED(50) = 14 mg/kg) or EPL (ED(50) = 147 mg/kg). Moreover, even at high doses of 100 and 300 mg/kg, SM-368229 showed very weak anti-androgenic effect in methyltestosterone-treated male rats and no progestagenic effect in estrus cycle synchronized female rats. These findings indicate that SM-368229 may offer a new promising therapeutic option for the treatment of hypertension and heart failure.

Application of Zwitterionic Polymer Hydrogels to Optical Tissue Clearing for 3D Fluorescence Imaging.

Zwitterionic polymers have both anion and cation groups in the side chain and have been used in various biomedical applications because of the unique properties. In this study, zwitterionic polymer hydrogels are applied to optical tissue clearing for 3D fluorescence imaging. Polyacrylamide hydrogels have been employed in Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue-hYdrogel method. Zwitterionic polymer hydrogels are produced using zwitterionic monomers, such as 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate (DAPS) and 2-methacryloyloxyethyl phosphorylcholine (MPC), and crosslinkers. The hydrogels made from poly(DAPS-co-acrylamide) and MPC homopolymers afford the most transparent tumor tissues. However, the tissues cleared using DAPS copolymers-containing hydrogels became turbid in a refractive index-matching solution, which are unable to obtain clear 3D fluorescence images. In contrast, the 3D fluorescence imaging is achieved in the MPC polymer-treated 2-mm-thick brain slices after immunostaining. The 3D fluorescence imaging of lung metastasis that is cleared by the MPC hydrogel to demonstrate the possible application to cancer diagnosis is performed. The results indicate the increased potentials of zwitterionic polymer hydrogels, especially MPC polymer hydrogels, in biomedical applications.

Atrogin-1 ubiquitin ligase is upregulated by doxorubicin via p38-MAP kinase in cardiac myocytes.

AIMS: Doxorubicin (DOX) is one of the most effective anti-neoplastic agents; however, its clinical use is limited by drug-induced cardiomyopathy. The molecular mechanisms responsible for this toxicity remain to be fully addressed. In the present study, we investigated the involvement of atrogin-1, one of the muscle-specific ubiquitin ligases, in DOX-induced cardiotoxicity. METHODS AND RESULTS: This method involved intraperitoneal administration of DOX-induced atrogin-1 in the hearts and skeletal muscles of C57BL/6 mice. Consistently, atrogin-1 mRNA was upregulated with DOX treatment in cultured rat neonatal cardiomyocytes. Adenoviral transfer of atrogin-1 induced a reduction in cell size that was ameliorated by the ubiquitin proteasome inhibitor, MG-132. The transduction of constitutively active Akt (caAkt), a serine/threonine protein kinase, inhibited the DOX-mediated induction of atrogin-1. The phosphorylation status of Akt and its downstream target, FOXO, was not affected by DOX. DOX treatment did not activate the atrogin-1 promoter that contains FOXO-binding sites, suggesting that DOX induced atrogin-1 without modulating the Akt/FOXO pathway; importantly, DOX activated p38-mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Furthermore, pharmacological inhibition of p38-MAPK, but not JNK, abrogated DOX-mediated induction of atrogin-1. Finally, adenoviral transfer of caAkt inhibited the DOX-induced p38-MAPK activation. CONCLUSIONS: DOX induces atrogin-1 through a p38-MAPK-dependent pathway in cardiac myocytes. Constitutive activation of Akt negatively regulates DOX-mediated atrogin-1 induction by inhibiting p38-MAPK activity as a novel mechanism.

Platelet activating factor induces cytoskeletal reorganization through Rho family pathway in THP-1 macrophages.

In the process of atherosclerosis, platelet activating factor (PAF) promotes the infiltration of inflammatory cells into atherosclerotic plaque by modulating their cytoskeleton. Here, we examined whether Rho family proteins are involved in PAF-induced cytoskeletal reorganization in THP-1 macrophages. PAF stimulation rapidly induced cell elongation, accompanied by filopodia formation. The inhibition of Rho family proteins by the overexpression of Rho-GDI attenuated the PAF-mediated morphological changes. Both RhoA and Cdc42 were activated in response to PAF. Inhibition of RhoA or Cdc42 by dominant negative mutants abrogated morphological changes induced by PAF. Collectively, PAF regulates cytoarchitecture through Rho family proteins in macrophages.