Eplerenone: a selective aldosterone receptor antagonist (SARA).

Aldosterone, the final product of the renin-angiotensin-aldosterone system (RAAS), is a mineralocorticoid hormone that classically acts, via the mineralocorticoid (aldosterone) receptor, on epithelia of the kidneys, colon, and sweat glands to maintain electrolyte homeostasis. Aldosterone has also been shown to act at nonepithelial sites where it can contribute to cardiovascular disease such as hypertension, stroke, malignant nephrosclerosis, cardiac fibrosis, ventricular hypertrophy, and myocardial necrosis. Although angiotensin-converting enzyme (ACE) inhibitors and angiotensin type 1 (AT(1)) receptor antagonists act to suppress the RAAS, these agents do not adequately control plasma aldosterone levels--a phenomenon termed "aldosterone synthesis escape." Spironolactone, a nonselective aldosterone receptor antagonist, is an effective agent to suppress the actions of aldosterone; its use is, however, associated with progestational and antiandrogenic side effects due to its promiscuous binding to other steroid receptors. For these reasons, eplerenone--the first agent of a new class of drugs known as the selective aldosterone receptor antagonists (SARAs)--is under development. In rodent models, eplerenone provides marked protection against vascular injury in the kidney and heart. In phase II clinical trials, eplerenone demonstrates 24-h control of blood pressure with once or twice daily dosing, and is safe and well tolerated in patients with heart failure when given with standard of care agents. Pharmacokinetic studies reveal that eplerenone has good bioavailability with low protein binding, good plasma exposure, and is highly metabolized to inactive metabolites and excreted principally in the bile. Eplerenone is well tolerated in acute and chronic safety pharmacology studies. Ongoing phase III trials of eplerenone in the treatment of hypertension and heart failure are underway. These studies will extend our understanding of selective aldosterone receptor antagonism in the treatment of chronic cardiovascular disease.

Effect of a selective aldosterone receptor antagonist in myocardial infarction.

Myocardial infarction (MI) initiates adaptive tissue remodeling, which is essential for heart function (such as infarct healing) but is also important for maladaptive remodeling (for example, reactive fibrosis and left ventricular dilation). The effect of aldosterone receptor antagonism on these processes was evaluated in Sprague-Dawley rats using eplerenone, a selective aldosterone receptor antagonist. Infarct healing and left ventricular remodeling were evaluated at 3, 7, and 28 days after MI by determination of the diastolic pressure-volume relationship of the left ventricle, the infarct-thinning ratio, and the collagen-volume fraction. Eplerenone did not affect reparative collagen deposition as was evidenced by a similar collagen volume fraction in the infarcted myocardium between eplerenone and vehicle-treated groups at 7 and 28 days post-MI. In addition, the thinning ratio, which is an index of infarct expansion, was comparable between the eplerenone and vehicle-treated animals at 7 and 28 days post-MI. A protective effect of eplerenone was demonstrated at 28 days post-MI, where reactive fibrosis in the viable myocardium was reduced in eplerenone-treated animals compared with vehicle-treated animals. Thus aldosterone receptor antagonism does not retard infarct healing but rather protects against maladaptive responses after MI.

Tissue-specific corticosteroidogenesis in the rat.

The steroid aldosterone plays a major role in the maintenance of total body sodium homeostasis and also contributes to cardiovascular pathophysiology by mediating cardiac hypertrophy and fibrosis. In addition to classical adrenal production of aldosterone, endogenous tissue production of aldosterone has been observed in various organs; aldosterone biosynthesis in cardiac tissues, however, remains highly controversial. The current study provides a comprehensive evaluation of steroid hormone biosynthethic capabilities in multiple tissues from two distinct rat strains under unstimulated and stimulated conditions. Panels of tissues from Wistar and Sprague-Dawley rats were probed for 11 beta-hydroxylase (P45011beta) and aldosterone synthase (P450aldo) by reverse transcriptase-polymerase chain reaction (RT-PCR). Under unstimulated conditions, cardiac P45011beta and P450aldo were detected only in Wistar rats. Angiotensin II (100 microg/day) stimulated myocardial expression of both enzymes in both strains. Cerebral cortex and mesenteric artery message levels in both strains was reduced by angiotensin II. These data demonstrate the potential for local steroid synthesis in vascular, cardiac, renal, and neuronal tissues, and that biosynthesis of non-adrenal aldosterone may be differentially regulated between strains. This variability may thus resolve in part or whole the current controversy over the existence of non-adrenal steroidogenic systems.

Mineralocorticoid receptor antagonists: the evolution of utility and pharmacology.

For more than 30 years after the discovery of aldosterone, scientists believed that its sole site of action was at epithelial tissues, most notably the kidney, where it mediated the transport of Na and K. It was soon recognized aldosterone contributed to several diseases by causing edema. Armed with this information, scientists set out more than 30 years ago to develop an antagonist of the mineralocorticoid receptor for the treatment of edematous states. From this effort, spironolactone (Aldactone was discovered. Spironolactone acts functionally as a competitive inhibitor of the mineralocorticoid (aldosterone) receptor, and although spironolactone is an effective mineralocorticoid receptor antagonist, it is not without limitations. These limitations include unwanted progestational and antiadrogenic side effects that limit its use in the chronic treatment of disease. In addition to its actions at the collecting tubule, aldosterone can participate in pathophysiology by actions at the heart, vasculature, and kidney, and it is likely that the most significant contributions to cardiovascular disease are due to actions at these sites rather than those related to Na and water retention. This is underscored by the recent clinical results from the RALES-004 Trial in which treatment with Aldactone demonstrated a significant benefit on mortality in patients with severe heart failure. The limited utility of spironolactone owing to the aforementioned steroid-related side effects has been especially frustrating, given the newly recognized role of aldosterone in cardiovascular disease. To obviate these limitations, eplerenone is currently being developed by Searle. Eplerenone is a competitive antagonist of the mineralocorticoid receptor that takes advantage of replacing the 17alpha-thoacetyl group of spironolactone with a carbomethoxy group, conferring excellent selectivity for the mineralocorticoid receptor over other steroid receptors. The pharmacological profile of eplerenone positions it to be an effective and selective mineralocorticoid receptor antagonist.

Anti-aldosterone therapy in the treatment of heart failure: new thoughts on an old hormone.

Activation of the renin-angiotensin-aldosterone system (RAAS) is a prominent feature of left ventricular dysfunction and plays an important role in the progression of chronic heart failure. Clinical and animal studies investigating agents that interrupt this hormonal system have focused primarily on the proximal constituents of the RAAS, namely angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists, and have largely neglected the possible pathological consequences of another hormone in the system, aldosterone. Clinical evidence indicates that aldosterone plays an important role in chronic heart failure, even when other RAAS inhibiting agents are employed. Moreover, animal studies have indicated that aldosterone, in addition to important renal effects, has direct cardiac and vascular effects. These data suggest that an anti-aldosterone therapeutic may provide important protection in chronic heart failure. Currently, only one therapeutic is available, spironolactone (Aldactone), and recent clinical studies support the contention that the addition of spironolactone to standard heart failure therapy provides additional benefit. A highly selective aldosterone receptor antagonist, eplerenone, is currently in clinical development. Data from this new agent should provide important evidence supporting the benefit of anti-aldosterone therapy in chronic heart failure, which may encourage physicians to include an anti-aldosterone agent in the armamentarium of therapeutics currently used to combat chronic heart failure.

S-nitrosylated tissue-type plasminogen activator protects against myocardial ischemia/reperfusion injury in cats: role of the endothelium.

S-Nitrosylated tissue plasminogen activator (tPA) is formed by S-nitrosylation of the clinically important agent tPA by nitric oxide, thus conferring nitric oxide donor properties to the molecule. Cats were subjected to 90 min of myocardial ischemia and 270 min of reperfusion and were treated with either tPA or S-nitrosylated tPA 10 min before reperfusion. S-Nitrosylated tPA-treated cats demonstrated marked attenuation of cardiac necrosis after myocardial ischemia/reperfusion, compared with cats receiving only tPA (13 +/- 3% vs. 28 +/- 3%, P < .01). Relaxation of ischemic/reperfused left anterior descending coronary artery rings in response to the endothelium-dependent dilators acetylcholine and A23187 was greater in the S-nitrosylated tPA-treated group, compared with the cats receiving only tPA, indicating that coronary vascular endothelial function was preserved by S-nitrosylated tPA. S-Nitrosylated tPA also resulted in markedly reduced adherence of neutrophils to the coronary vascular endothelium, compared with nonnitrosylated tPA (P < .01). Immunohistochemical localization of P-selectin in the ischemic region was also significantly reduced by S-nitrosylated tPA, compared with the control group (P < .01). These data indicate that S-nitrosylated tPA is a cardioprotective agent, likely exerting its effect by site-specific nitric oxide donation resulting in inhibition of neutrophil-endothelium interaction via a P-selectin-dependent mechanism.

Protection from myocardial reperfusion injury by acute administration of 17 beta-estradiol.

Although several studies have demonstrated that chronic exposure to estrogen appears to be cardioprotective, acute circulatory effects of estrogen are largely unknown. Therefore, we studied the effects of acute administration of 17 beta-estradiol in myocardial ischemia/reperfusion. Cats were subjected to 90 min of left anterior descending coronary artery (LAD) occlusion and 270 min of reperfusion (MI/R). Either the estrogenic steroid, 17 beta-estradiol or its non-estrogenic isomer, 17 alpha-estradiol was administered (i.v.) 30 min prior to reperfusion at 1 microgram/kg bolus followed by a constant infusion lasting the remaining duration of the protocol at 1 microgram/kg/h. Control cats were subjected to sham MI/R. Cats treated with 17 beta-estradiol demonstrated a marked reduction in cardiac necrosis following MI/R compared to cats receiving 17 alpha-estradiol or phosphate buffered saline (17 +/- 2% v 33 +/- 1% or 34 +/- 4% area of necrosis indexed to the area-at-risk, P < 0.01). In addition, cats receiving 17 beta-estradiol exhibited reduced myocardial PMN infiltration in necrotic tissue as compared to 17 alpha-estradiol treated cats. Moreover, 17 beta-estradiol administration attenuated neutrophil adherence to ex vivo coronary vascular endothelium compared to the two controls (44 +/- 8 PMNs/mm2 v 79 +/- 7 PMNs/mm2 or 86 +/- 7 PMNs/mm2 P < 0.01). These data indicate that 17 beta-estradiol protects against myocardial ischemia/reperfusion, in part, by attenuating PMN infiltration and subsequent injury due to PMN mediator release.

Blockade of platelet endothelial cell adhesion molecule-1 protects against myocardial ischemia and reperfusion injury in cats.

Polymorphonuclear leukocytes have been shown to play an important role in myocardial ischemia (MI) and reperfusion (R) injury. Since blockade of platelet-endothelial cell adhesion molecules (PECAM-1) inhibits neutrophil transmigration in vitro and in vivo, the effects of a polygonal Ab directed against PECAM-1 were examined in a feline model of Ml/R. We established cross-reactivity of our anti-human PECAM-1 Ab to cat coronary vasculature and neutrophils by immunohistochemistry and flow cytometry. The anti-PECAM-1 Ab markedly blocked leukocyte transmigration into the peritoneal cavity of cats after glycogen-induced peritonitis. Then, anti-PECAM-1 Ab (1 mg/kg) was tested to determine whether it attenuates MI/R injury in a well characterized feline model of Ml and R. Anti-PECAM-1 Ab administered 10 min before R significantly inhibited the myocardial necrosis seen 4.5 h post-R compared with that in MI/R cats treated with control isotype rabbit IgG (12 +/- 2 vs 29 +/- 4% of area at risk; p less than 0.01) and significantly attenuated the rise in plasma creatine kinase activity (p less than 0.05). The Ab did not prevent increases in cardiac myeloperoxidase activity within the affected regions and did not significantly inhibit autologous neutrophil adhesion to coronary endothelium after stimulation of either neutrophils (by leukotriene B4) or coronary endothelium (by thrombin) in vitro. These results indicate that in vivo blockade of PECAM-1 significantly attenuates MI/R injury, presumably by inhibiting transendothelial migration of neutrophils.

Novel recombinant serpin, LEX-032, attenuates myocardial reperfusion injury in cats.

We studied the potential cardioprotective effects of the novel recombinant serine protease inhibitor (serpin), LEX-032, which inhibits the serine proteases elastase and cathepsin G. LEX-032 is a recombinant construct of human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of human alpha 1-protease inhibitor. Cats were subjected to 90 min of left anterior descending coronary artery (LAD) occlusion and 270 min of reperfusion (MI/R). Either LEX-032 or its vehicle (i.e., phosphate-buffered saline) was administered intravenously 10 min before reperfusion. Control cats were subjected to sham MI/R. Cats treated with LEX-032 demonstrated a marked reduction in cardiac necrosis after MI/R compared with cats receiving only vehicle (10 +/- 3 vs. 31 +/- 3%, P < 0.01). In addition, relaxation of LAD rings to the endothelium-dependent dilators (e.g., acetylcholine and A23187) was greater in the LEX-032-treated group than in cats receiving vehicle (72 +/- 5 vs. 52 +/- 7%, P < 0.05, and 74 +/- 8 vs. 50 +/- 8%, P < 0.05, respectively), indicating that endothelial function was preserved by LEX-032. Moreover, LEX-032 administration resulted in a marked reduction of polymorphonuclear leukocyte (PMN) adherence to ex vivo coronary vascular endothelium compared with vehicle (33 +/- 4 vs. 86 +/- 7 PMNs/mm2, P < 0.01). These data indicate that LEX-032 is a significant cardioprotective agent exerting its protective effect by inhibition of PMN-mediated cellular injury, and this agent represents a novel means of attenuating PMN-mediated reperfusion injury.

Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin.

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 microgram/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P<0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P<0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-L-arginine-methyl ester (L-NAME)-stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the L-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or L-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.

Reducing lactate accumulation does not attenuate lethal ischemic injury in isolated perfused rat hearts.

The role of lactate accumulation in lethal ischemic myocardial cell injury was assessed by partially depleting hearts of glycogen before ischemia by using glucagon. Isolated adult rat hearts were perfused with glucose-free Krebs-Henseleit buffer containing acetate as substrate. After stabilization, treated hearts were perfused briefly (3 min) with buffer containing 2 micrograms/ml glucagon to reduce tissue glycogen stores, followed by 10 min of perfusion with control buffer, and 60 or 90 min of global ischemia. Before the onset of ischemia, glucagon-treated hearts contained 40% less glycogen than untreated hearts, but myocardial function and tissue levels of high-energy phosphates, lactate, and glucose 6-phosphate were similar. Lactate production during ischemia in the glucagon-treated hearts was 50% less than in untreated hearts. However, there was no decrease in the amount of creatine kinase release during reperfusion after either 60 or 90 min of ischemia. Thus although partial glycogen depletion reduced lactate accumulation during ischemia, this did not decrease the amount of lethal myocardial cell injury.

Cardioprotective effects of selective inhibition of the two complement activation pathways in myocardial ischemia and reperfusion injury.

The complement (C) system-mediated neutrophil activation, adhesion to the coronary endothelium and accumulation into cardiac tissue are key steps in the pathogenesis of myocardial ischemia-reperfusion (MI/R) injury. We examined the differential role of the classical and the alternative complement pathway in MI/R injury in vivo. Rats were subjected to 20 min of myocardial ischemia followed by 24 h of reperfusion. Either a classical pathway inhibitor [C1 esterase inhibitor (C1-INH) (15 mg/kg)] or an alternative pathway inhibitor soluble complement receptor 1 (sCR1)[des-LHR-A](15 mg/kg) or their vehicle were administered intravenously 1 min prior to reperfusion, and myocardial necrosis (creatine kinase loss) and neutrophil accumulation, cardiac myeloperoxidase activity, were examined. C1-INH significantly attenuated cardiac creatine kinase loss compared to MI/R rats given only vehicle (p < 0.05) 24 h after reperfusion. An alternative pathway inhibitor, sCR1 [des-LHR-A] attenuated myocardial injury to a lesser extent, although it was not significantly different from the value for C1-INH or vehicle. Besides cardiac myeloperoxidase activity, the ischemic cardiac tissue was significantly attenuated by both C1-INH and sCR1[desLHR-A] (p < 0.05 vs. vehicle). Both the classical and alternative pathways may contribute to MI/R injury via a neutrophil-dependent mechanism in vivo. Selective inhibition of the classical pathway of complement activation seems to be slightly more effective in limiting necrotic MI/R injury than the selective alternative pathway inhibition in this 24 h model of reperfusion injury, but equal doses of each inhibitor attenuated neutrophil accumulation.

Endocardial and epicardial interstitial purines and lactate during graded ischemia.

The purpose of this study was to compare interstitial fluid (ISF) levels of purine metabolites and lactate in the endocardium and the epicardium during graded regional myocardial ischemia and reperfusion. Anesthetized dogs were subjected to 60 min of regional myocardial ischemia induced by either partial or complete occlusion of the left anterior descending coronary artery (LAD), followed by 60 min of reperfusion. To sample ISF, cardiac microdialysis probes were implanted in the LAD-perfused myocardium; dialysate levels served as indexes of ISF concentrations. During severe ischemia, dialysate adenosine increased transiently in both the endocardium and epicardium, reaching maximal values at approximately 20 min of ischemia. Inosine, hypoxanthine, xanthine, and lactate increased most rapidly during the first 30 min of severe ischemia, after which the rate of increase was diminished. The ISF profiles of these metabolites were qualitatively similar during moderate ischemia, although the ISF levels achieved during ischemia were not as great. With both severe and moderate ischemia, ISF purines and lactate were greater in the endocardium than epicardium, consistent with a greater energy imbalance in the endocardium during ischemia. ISF total purines (the sum of the individual purine metabolites) were relatively stable until myocardial blood flow was reduced below 50 ml.min-1 x 100 g-1, after which ISF total purines increased in proportion to the severity of the blood flow deficit. These data suggest that functional and metabolic adaptations keep the myocardium in energy balance until blood flow is reduced below approximately 50% of control, and they attest to the usefulness of cardiac microdialysis for establishing transmural profiles of ISF metabolites.