ORIGINAL ARTICLE

Finerenone, a Novel Selective Nonsteroidal Mineralocorticoid Receptor Antagonist Protects From Rat Cardiorenal Injury Peter Kolkhof, PhD,* Martina Delbeck, PhD,* Axel Kretschmer, PhD,† Wolfram Steinke, PhD,‡ Elke Hartmann, PhD,§ Lars Bärfacker, PhD,¶ Frank Eitner, MD,* Barbara Albrecht-Küpper, PhD,* and Stefan Schäfer, MD†

Abstract: Pharmacological blockade of the mineralocorticoid receptor (MR) ameliorates end-organ damage in chronic heart failure. However, the clinical use of available steroidal MR antagonists is restricted because of concomitant hyperkalemia especially in patients with diminished kidney function. We have recently identified a novel nonsteroidal MR antagonist, finerenone, which uniquely combines potency and selectivity toward MR. Here, we investigated the tissue distribution and chronic cardiorenal endorgan protection of finerenone in comparison to the steroidal MR antagonist, eplerenone, in 2 different preclinical rat disease models. Quantitative whole-body autoradiography revealed that [14C]labeled finerenone equally distributes into rat cardiac and renal tissues. Finerenone treatment prevented deoxycorticosterone acetate-/ salt-challenged rats from functional as well as structural heart and kidney damage at dosages not reducing systemic blood pressure. Finerenone reduced cardiac hypertrophy, plasma prohormone of brain natriuretic peptide, and proteinuria more efficiently than eplerenone when comparing equinatriuretic doses. In rats that developed chronic heart failure after coronary artery ligation, finerenone (1 mg$kg21$d21), but not eplerenone (100 mg$kg21$d21) improved systolic and diastolic left ventricular function and reduced plasma prohormone of brain natriuretic peptide levels. We conclude that finerenone may offer end-organ protection with a reduced risk of electrolyte disturbances. Key Words: heart failure, hypertrophy, remodeling, chronic kidney disease (J Cardiovasc Pharmacol Ô 2014;64:69–78)

INTRODUCTION In patients with heart failure, increased levels of aldosterone are correlated with increased morbidity and mortality.1,2 Experimental overactivation of aldosterone within the heart leads to coronary endothelial dysfunction, Received for publication January 24, 2014; accepted February 19, 2014. From the Institutes of *Cardiology Research; †Clinical Sciences; ‡Drug Metabolism and Pharmacokinetics; §Toxicology; and ¶Medicinal Chemistry, Bayer HealthCare, Wuppertal, Germany. All authors are employees of Bayer HealthCare. Reprints: Peter Kolkhof, PhD, Department of Cardiology Research, Global Drug Discovery, Bayer HealthCare, Aprather Weg 18a, 42096 Wuppertal, Germany (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins

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myocardial apoptosis, and myocardial fibrosis and thus contributes to cardiac pathophysiological remodeling.3 Aldosterone acts through its specific nuclear hormone receptor, the mineralocorticoid receptor (MR), which is predominantly expressed in kidney, colon, vessels, and the heart.4 MR contributes to volume, sodium, and potassium homeostasis through renal tubular epithelial cells located in the distal tubule and the collecting duct. Pathophysiological myocardial remodeling and renal tubulointerstitial fibrosis are mediated through MRs expressed in cardiomyocytes, fibroblasts, vascular endothelial, and smooth muscle cells.4 Consequently, blockade of the MR has proven clinical benefit in patients with heart failure and chronic kidney disease.5–7 However, because of the inherent risk of developing hyperkalemia, current steroidal MR antagonists (MRAs) are the least frequently prescribed medications among all recommended pharmacological treatments for chronic heart failure8 and if prescribed, MRAs are not dosed to their full organ protective effect in heart failure patients.9 Molecular pharmacological considerations suggest that the balance between the interstitial, antiremodeling effects, and the renal epithelial, natriuretic, and antikaliuretic effects of MR blockade can be modulated by the molecular structure of the pharmacological agent.10 The 2 currently available MRAs, spironolactone and eplerenone, have a steroidal chemical structure and are thus similar to the natural ligands of the MR receptor, aldosterone, and cortisol. As a consequence, the similar structural and physicochemical properties of the steroidal MRAs determine the resulting pharmacological action not only by their binding mode to MR but also by their transport and distribution into different tissues and recruitment or blockade of tissue selective and ligandspecific co-factors.10 In an effort to overcome these inherent limitations of the steroidal MR blockers, we have developed a novel nonsteroidal, potent, and selective MRA, finerenone (previous nomenclature BAY 94-8862), as a candidate for clinical studies.11,12 Finerenone is a potent antagonist at human MR in functional cellular transactivation assays with an IC50 of 18 nM and exhibits no activity at any other steroid hormone receptor and on 65 receptors and ion channels (IC50 . 10 mM).11 Thus, finerenone uniquely combines potency and extraordinary selectivity (at least 500-fold) toward MR.12 We hypothesize that treatment with finerenone because of its nonsteroidal chemical structure www.jcvp.org |

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and physicochemical properties results in a different organ distribution and accordingly to a different balance of influences on renal electrolyte effects versus functional and structural endorgan protection in comparison to the steroidal MRA eplerenone.

catheter. At the time of sacrifice, rats were administered an overdose of anesthesia.

METHODS Animal Models Animals were housed with free access to chow and water and maintained on a light–dark cycle at 22–248C. All procedures conformed to European Community directives and national legislation (German law for the protection of animals) for the use of animals for scientific purposes and were approved by the competent regional authority.

Determination of Diuretic Effects in Conscious Rats Urine samples from male Wistar rats (300 g; Charles River, Sulzfeld, Germany) placed in metabolic cages were analyzed following a previously published protocol.11

Chronic Myocardial Infarction Model in Rats Male Wistar rats (250–300 g; Harlan-Winkelmann, Borchen, Germany) were used in a chronic myocardial infarction (MI) rat model. The left anterior descending coronary artery was permanently ligated under enflurane (4%–5%) anesthesia and buprenorphine (0.05 mg/kg) analgesia. Surgery without ligation was performed in sham rats. One week after surgery, animals were randomized to vehicle or treatment groups (0.1, 0.3, and 1 mg$kg21$d21 p.o. of finerenone, 100 mg$kg21$d21 p.o. of eplerenone; n = 10–14 per group) with a treatment period of 8 weeks. Invasive hemodynamic measurements were performed at the end of the experiment by LV catheterization under isoflurane (2%) anesthesia using the Powerlab Chart 5 software. Rats were killed by an overdose of anesthesia.

Histopathology Histopathology of kidneys and hearts of DOCA-/salttreated rats were analyzed as previously published.13

Blood and Urinary Biomarker Analyses Deoxycorticosterone Acetate-/Salt-induced Heart and Kidney Injury in Rats Male Crl:CD (Sprague-Dawley) rats (8 weeks old; Charles River) were used in the deoxycorticosterone acetate (DOCA)/salt study following previously published protocols.13 Rats were randomly divided into 7 groups (n = 7–12 per group): group 1 underwent sham operation, whereas groups 2–7 were uninephrectomized. Surgeries were performed under isoflurane (2.0%) and buprenorphine (0.03 mg/kg) anesthesia. One week after the operation, groups 2–7 received DOCA (30 mg/kg in sesame oil) once weekly subcutaneously for 10 weeks together with 1% NaCl added to drinking water, plus daily gavage of the following: group 2 received vehicle application (10% ethanol, 40% solutol, 50% water); groups 3–5 received finerenone (0.1, 1, and 10 mg$kg21$d21, respectively); groups 6 and 7 received eplerenone (30 and 100 mg$kg21$d21, respectively). Urine of animals was collected in metabolic cages, and systolic blood pressure was measured by the tail-cuff method (Noninvasive Blood Pressure Monitoring System, 9002-series; TSE Systems, Bad Homburg, Germany) after a treatment period of 10 weeks. At the end of the experiment, hemodynamic function was measured in the left ventricle by a Millar-Tip (2F)

Plasma concentrations of aldosterone and rat prohormone of brain natriuretic peptide (pro-BNP) (1–45) were measured with a commercially available radioimmunoassay kit (PhoenixPeptide, Belmont, CA), and urinary rat PAI-1 protein was quantified using a commercially available ELISA kit (Rat PAI-1 IMUCLONE No. 601; American Diagnostica, Pfungstadt, Germany) according to the manufacturer’s instructions. Creatinine was quantified in urine samples by the Jaffe method using the ADVIA 2400 Analyzer (Siemens Healthcare Diagnostics, Eschborn, Germany) according to the user manual, and total urinary protein was determined using the pyrogallol-molybdate method.

Relative Messenger RNA Expression Analyses by Real-time Polymerase Chain Reaction Relative gene expression was determined by real-time quantitative polymerase chain reaction using the ABI Prism Sequence Detection System (ABI Prism 7700 Sequence Detector; Applied Biosystems, Life Technologies, Foster City, CA) as previously described.13 The primer probe sets were generated from the following sequences: NM_013226 ribosomal protein L32, NM_031144.2 betaactin, NM_009263 osteopontin (SSP1, OPN), NM_031054

FIGURE 1. Distribution of radioactivity in a male Wistar rat 1 hour after oral administration of 3 mg/kg [14C]labeled finerenone. Blue: lowest detectable concentration; red: highest detectable concentration of radioactivity; pink: above the upper detection limit. [14C]-radiation standards spiked with stated concentrations are given at the bottom.

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Finerenone Protects From Cardiorenal Injury

matrix metalloproteinase (MMP-2), NM_008871 plasminogen activator inhibitor-1 (Serpine1, PAI-1), and NM_031530.1 monocyte chemoattractant protein 1 (CCL2, MCP-1).

finerenone dose (100 mg/kg) after single dose application (Fig. 2A). Both MRAs dose-dependently induced urinary sodium excretion (Fig. 2B). The application of 1 mg/kg finerenone resulted in similar natriuretic responses as 10 and 30 mg/kg eplerenone, whereas 10 mg/kg finerenone had a comparable natriuretic efficacy as 100 mg/kg eplerenone. In the tested eplerenone doses, there was a trend toward decreased urinary potassium excretion, which reached significance (P = 0.05) at the highest dose of 100 mg/kg (Fig. 2C). The tested

Quantitative Whole-body Autoradiography Finerenone was radio-labeled with carbon-14 in the cyano moiety of the molecule. The specific radioactivity was 3.09 MBq/mg, and the radiochemical purity was 98.5%. [14C]-labeled finerenone was administered as a single oral dose of 3 mg/kg to male Wistar rats to investigate the distribution of radioactivity to organs and tissues by means of quantitative whole-body autoradiography according to the procedure described by Curtis et al.14 Sagittal whole-body sections of 50 mm thickness were cut at 2258C using a cryotome (Leica CM3600; Leica Biosystems, Nussloch, Germany) and freeze-dried for at least 24 hours. The radioactivity distribution in the dry whole-body sections was detected using the Fuji BAS 5000 phosphor imaging system (Fuji Photo Film Ltd., Tokyo, Japan). The quantitation of equivalent concentrations in organ and tissues was performed using the [14C]-spiked blood standards and the image analysis software AIDA (Raytest, Straubenhardt, Germany).

Statistics

Data were presented as means 6 SEM from 7 to 14 animals per group as indicated. Statistical analyses were performed using 1-way analysis of variance followed by Newman–Keuls Multiple Comparison Test or Student’s t test. P values of # 0.05 were considered significant.

RESULTS Finerenone Distributes Equally into Cardiac and Renal Tissues Determination of radioactivity 1 hour after oral application of 3 mg/kg [14C]-labeled finerenone revealed comparable concentrations in the heart (4409 mg-eq/L) and in the kidneys (3782 mg-eq/L). Consistently, the 24-hour AUC was similar in the heart (29,815 mg-eq$h$L21) and the kidneys (27,209 mg-eq$h$L21). Maximum radioactivity concentrations were reached 1 hour after application of finerenone. Figure 1 illustrates the radioactivity distribution in a male Wistar rat 1 hour after oral application. [14C]-labeled finerenone and possibly radioactive metabolites were mainly located in the vascular and interstitial spaces and in well-perfused organs such as heart, lung, liver, and kidney. Qualitatively, highest radioactivity was found in the gastrointestinal contents, the bile-duct contents, the lymph, and in the interstitial fluid. The latter was more or less blood equivalent suggesting a free and rapid exchange of the drug between blood and interstitial space.

Diuretic Effects of Finerenone and Eplerenone To identify equiefficient natriuretic doses of the 2 MRAs eplerenone and finerenone for further chronic studies, we determined the natriuretic response of increasing doses of both MRAs in conscious rats. We found no influence on urinary volume with the exception of the highest tested Ó 2014 Lippincott Williams & Wilkins

FIGURE 2. Diuretic effects of 1, 3, 10, 30, and 100 mg/kg finerenone and 3, 10, 30, and 100 mg/kg eplerenone on (A) urinary volume, (B) urinary sodium, and (C) urinary potassium concentrations in conscious rats after single oral dose application. Mean 6 SEM; n = 7–8 per group; *P , 0.05, **P , 0.01 versus placebo. www.jcvp.org |

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finerenone doses had no effect on urinary potassium excretion (Fig. 2C).

a significant rise in systolic blood pressure compared with sham-treated animals (Fig. 3A). Finerenone reduced systolic blood pressure values after 10 weeks of DOCA/salt treatment only at the highest dose of 10 mg/kg but not at 1 mg/kg, whereas a significant hypotensive action was observed in both eplerenone groups (30 and 100 mg/kg). Systolic blood pressure in animals treated with 10 mg/kg finerenone was similar to respective values of control animals and significantly lower in comparison to respective values of the 100 mg/kg eplerenone group (Fig. 3A). The heart weight-to-body weight ratio is a sensitive parameter for cardiac hypertrophic processes that occur in the development of chronic heart failure. The

Finerenone Protects From Heart and Kidney Injury in the Rat DOCA/Salt Model

For further studying the in vivo efficacy of finerenone and eplerenone in a chronic model of hyperaldosteronisminduced end-organ injury, we decided to compare MRA doses that result in similar natriuretic efficacy (ie, the equiefficient natriuretic doses of 1 and 10 mg/kg of finerenone were compared with 30 and 100 mg/kg of eplerenone, respectively). A 10-week DOCA/salt treatment period resulted in

FIGURE 3. Effects of MRAs on (A) systolic blood pressure, (B) relative heart weights, and (C) plasma pro-BNP in the DOCA/salt rat model after 10 weeks of treatment. Mean 6 SEM; n = 7–12 per group; *P , 0.05 versus placebo; #P , 0.05 versus eplerenone. Representative histopathological features of hearts from DOCA-/salt-treated rats after 10 weeks of treatment: (D) control, (E) placebo, and (F) 10 mg/kg finerenone. DOCA/salt treatment results in moderate myocardial degeneration and fibrosis and focal vasculopathy (E) which is markedly attenuated by treatment with 10 mg/kg finerenone (F). Hematoxylin and eosin staining.

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heart weight-to-body weight ratio was significantly reduced with finerenone at 1 and 10 mg/kg compared with DOCA-/ salt-placebo rats at the end of the study (Fig. 3B). In both the eplerenone-treated groups, relative heart weight did not differ from the placebo control. Finerenone significantly and dosedependently decreased pro-BNP with a minimal effective dose of 1 mg/kg (Fig. 3C). Application of 100 mg/kg eplerenone appeared to have effects on pro-BNP that were comparable to 1 mg/kg finerenone. Ten weeks of DOCA/salt treatment resulted in significant structural heart damage in placebo-treated rats compared with sham rats that were not treated with DOCA/salt (Figs. 3D, E). Histological analyses revealed extensive myocardial degeneration and fibrosis as well as focal vasculopathy (Fig. 3E; Table 1). Rats treated with finerenone showed a dosedependent protection from structural heart injury, whereas 10 mg$kg21$d21 finerenone resulted in the most pronounced treatment effects (Fig. 3F, Table 1). Eplerenone treatment had some weaker effects on structural heart injury and did not demonstrate any dose-dependency in the analyzed dose range (Table 1). Treatment with finerenone additionally resulted in functional and structural protection from kidney injury in this model. Again, 1 mg/kg of finerenone significantly decreased relative kidney weights compared with DOCA-/ salt-placebo rats and 10 mg/kg of finerenone was significantly more effective than 100 mg/kg eplerenone (Fig. 4A). Proteinuria (measured as urinary protein to creatinine ratio) was dose-dependently reduced by finerenone and both eplerenone doses (Fig. 4B). The total amount of proteinuria was significantly lower in rats treated with 10 mg/kg of finerenone than in rats treated with the highest eplerenone dose tested (100 mg/kg). Parallel to the heart injury, DOCA-/salt-treated rats developed significant structural kidney damage. Histological analyses revealed extensive glomerular (glomerular sclerosis) and tubulointerstitial (tubular degeneration, tubular dilation, proteinuria casts) injury and focal vasculopathy (Fig. 4D; Table 1). Treatment with finerenone dose-dependently protected from glomerular, tubular, and vascular damage, while 10 mg$kg21$d21 finerenone resulted in the most pronounced

treatment effects (Fig. 4E, Table 1). Eplerenone treatment resulted in less pronounced therapeutic effects and did not demonstrate any dose-dependency in the analyzed dose range (Table 1). For further validation of the kidney protective effects of finerenone and eplerenone in the 10-week DOCA/salt model, the expression of several pro-inflammatory and pro-fibrotic biomarker genes was analyzed by quantitative PCR using renal mRNA. It was found that finerenone dose-dependently reduced the induction of mRNA expression of important remodeling marker genes such as PAI-1, MCP-1, OPN, MMP-2 (Figs. 5A– D). The reduction in biomarker gene expression was at least equal or even more pronounced in comparison to the respective reduction in the 100 mg/kg eplerenone group. In summary, treatment with finerenone resulted in consistent protection from end-organ damage in hearts and kidneys in DOCA-/salt-treated rats.

Finerenone Protects From Heart Injury in the Rat Chronic Myocardial Infarction Model We subsequently investigated the potential cardioprotective effects of both MRAs in ischemia-driven heart failure, that is, post-MI. After observation of end-organ protection without significant influence on systemic blood pressure at 1 mg/kg finerenone in the DOCA/salt model, we used 1 mg/kg of finerenone as maximum dose in this second heart injury model. A decrease in contractility (dp/dtmax) and a deterioration of relaxation (dp/dtmin) was seen in the placebo group versus the sham-operated group 8 weeks after MI (Figs. 6A, B). These characteristic parameters of left ventricular systolic dysfunction in heart failure were significantly improved by finerenone (1 mg/kg) but not by eplerenone (100 mg/kg) in this study (Figs. 6A, B). Left ventricular end diastolic pressure (LVEDP) and left ventricular relaxation time constant (tau), both parameters for diastolic (dys-)function, were significantly increased in the placebo group (Table 2). In the 1 mg/kg finerenone group, there was a trend toward LVEDP and tau reduction (P = 0.06; Table 2). Left ventricular pressure and heart rate were not changed by finerenone or eplerenone in this model (Table 2). Determination of the heart weight-to-body weight ratio revealed no significant changes mediated by any MRA

TABLE 1. Histological Scoring of Heart and Kidney Injury in the Rat DOCA/salt Model Group Dose n Heart injury Myocard degen Vasculopathy Kidney injury Glomerulopathy Tubular degen Vasculopathy

Sham

Placebo 7

Finerenone 0.1 mg/kg 11

Finerenone 1 mg/kg 11

Finerenone 10 mg/kg 11

Eplerenone 30 mg/kg 10

Eplerenone 100 mg/kg 10

11 060 060

1.6 6 0.4 1.4 6 0.3

1.8 6 0.3 1.5 6 0.2

1.1 6 0.3 1.3 6 0.3

0.6 6 0.2* 0.3 6 0.1†

0.7 6 0.2 0.8 6 0.1*

0.7 6 0.3 0.7 6 0.2

060 060 060

2.3 6 0.5 3.0 6 0.4 1.1 6 0.7

2.0 6 0.4 3.0 6 0.4 0.8 6 0.3

1.3 6 0.3 1.9 6 0.3 0.4 6 0.3

0.3 6 0.1‡ 0.7 6 0.3‡ 060

0.6 6 0.2† 1.7 6 0.2† 0.1 6 0.1

0.8 6 0.3* 1.9 6 0.4 0.3 6 0.2

Group values are indicated as means 6 SEM (mg/kg = mg$kg21$d21). *P , 0.05 treatment groups vs. placebo. †P , 0.01 treatment groups vs. placebo. ‡P , 0.001 treatment groups vs. placebo.

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FIGURE 4. Effects of MRAs on (A) relative kidney weights and (B) the urinary protein/creatinine ratio in the DOCA/salt rat model after 10 weeks of treatment. Mean 6 SEM; n = 7–12 per group; *P , 0.05 versus placebo; #P , 0.05 versus eplerenone. Representative histopathological features of kidneys from DOCA-/salt-treated rats after 10 weeks of treatment: (C) control, (D) placebo, and (E) 10 mg/kg finerenone. DOCA/salt treatment results in significant glomerulopathy, tubular degeneration, and vasculopathy (D), which is markedly attenuated by treatment with 10 mg/kg finerenone (E). Hematoxylin and eosin staining.

(Table 2). In the 1 mg/kg finerenone, there was a trend toward decreased relative weight of the right ventricle (Table 2). Finerenone significantly reduced plasma pro-BNP levels at 1 mg/kg, whereas no benefit could be demonstrated with 100 mg/kg eplerenone on this parameter (Fig. 6C). Furthermore, the pro-inflammatory and pro-fibrotic marker osteopontin (measured as mRNA expression) was dosedependently decreased in hearts from rats treated with finerenone (Fig. 6D). We found no significant elevation of serum potassium by any used dose of both MRAs (data not shown). Plasma aldosterone levels revealed compensatory increases in the 0.3 mg/kg and 1 mg/kg finerenone groups as well as in the 100 mg/kg eplerenone group demonstrating in vivo MR blocking activities of both MRAs (Fig. 6E).

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In summary, 1 mg$kg21$d21 of finerenone improved several hemodynamic parameters of systolic and diastolic dysfunction, decreased cardiac OPN expression and the clinically relevant plasma marker pro-BNP in animals with heart failure after 8 weeks of treatment. The steroidal MRA eplerenone did not exhibit protective effects despite a high natriuretic dose (100 mg/kg) being used and a significant compensatory plasma aldosterone increase.

DISCUSSION The steroidal MRAs spironolactone and eplerenone have been proven to reduce mortality in patients with symptomatic heart failure.5,6,15 However, the broad application of these drugs Ó 2014 Lippincott Williams & Wilkins

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Finerenone Protects From Cardiorenal Injury

FIGURE 5. Effects of MRAs on renal mRNA expression of (A) PAI-1, (B) Osteopontin, (C) MCP-1, and (D) MMP-2 in the DOCA/salt rat model after 10 weeks of treatment. Mean 6 SEM; n = 7–12 per group; *P , 0.05 versus placebo.

has been significantly impaired by their side effect profiles in certain patient populations. Therefore, there is a demand for a next generation of nonsteroidal MRAs which should combine spironolactone’s potency with eplerenone’s selectivity and based on a nonsteroidal chemical structure may provide an improved cardiac versus renal activity ratio (relative renal sparing) by a combination of physicochemical properties that influence plasma transport and tissue penetration (cross-membrane transport) and differential affinity of a receptor–drug complex for tissue-specific co-factors.9,10 Important differences with respect to tissue distribution between synthetic derivatives of steroid hormones and novel nonsteroidal compounds may be the consequence of their function as substrates for specific transporter proteins. Recent studies demonstrated, for example, the existence of cellular uptake pathways for carrier-bound steroids [for a review see Ref. 16]. Finerenone was previously identified as a novel nonsteroidal, potent, and selective MRA.11,12 We compared in this study the efficacy of different doses of finerenone in 2 preclinical chronic models of heart failure. We found protection of the end organs heart and kidney especially in a hypertension-driven model of heart failure. The determined functional parameters, relative organ weights, plasma proBNP levels, gene expression analyses, and histopathological analyses revealed a conclusive antihypertrophic activity of finerenone protecting heart and kidney from degenerative actions in both the models. Importantly, we found that almost all parameters were improved with the dose of 1 mg/kg that had no significant influence on blood pressure. Despite the Ó 2014 Lippincott Williams & Wilkins

use of equinatriuretic doses, much higher doses (10- to 100fold) of the steroidal drug eplerenone were needed to demonstrate end-organ protection in these chronic models. In fact, the doses of the steroidal MRAs needed for the induction of natriuresis in acute studies in rats17,18 are at least 10-fold lower than cardiac protective (ie, antihypertrophic action as determined by relative heart weight reduction and BNP reduction) doses in similar chronic studies.19–22 A commonly used dose of eplerenone that protects from heart injury in rat MI or rat hypertension models is 100 mg$kg21$d21,22 whereas eplerenone already affects renal electrolyte handling at doses as low as 3 mg/kg.17,18,23 Eplerenone has been investigated in several preclinical rat models including the chronic MI model.19,20,24,25 Fraccarollo et al20 demonstrated that eplerenone monotherapy reduced LVEDP and tau but in contrast to trandolapril monotherapy eplerenone did not reduce plasma NT-proANP. Similarly, Fraccarollo et al25 demonstrated additive amelioration of left ventricular remodeling post-MI by combined eplerenone (100 mg$kg21$d21) and angiotensin receptor blockade with irbesartan (50 mg$kg21$d21). Delyani et al19 investigated the influence of a high eplerenone dose (300 mg$kg21$d21) on infarct healing and left ventricular remodeling up to 28 days after MI. They demonstrated that eplerenone did not affect reparative collagen deposition but reduced reactive fibrosis in the viable myocardium. Taken together, previous post-MI studies in rats demonstrated functional improvements when eplerenone was applied either in combination with an ACEI or at doses which are at least 30fold higher in comparison to respective natriuretic doses. www.jcvp.org |

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FIGURE 6. Effects of investigated compounds on (A) cardiac contractility (dp/dtmax) and (B) cardiac relaxation (dp/dtmin) 8 weeks after MI. Effects of investigated compounds on cardiac and plasma biomarkers: (C) plasma pro-BNP levels, (D) cardiac osteopontin mRNA expression, and (E) plasma aldosterone concentrations 8 weeks after MI. plac = placebo; doses are indicated in mg$kg21$d21; *P # 0.05 versus combined placebo, mean 6 SEM; n = 10–14 animals per group.

Interestingly, Orena et al26 determined the respective plasma drug concentrations of eplerenone and another novel nonsteroidal MRA (PF-03882845) that were necessary to decrease urinary albumin and to increase serum potassium in a rat kidney injury model. These authors calculated a therapeutic index, that is, the ratio of the EC50 for increasing serum potassium to the EC50 for urinary albumin lowering, of 1.47 for eplerenone and 83.8 for PF-03882845. These results indicate also a greater potency of a nonsteroidal MRA in reducing urinary albumin compared with eplerenone, leading to an improved therapeutic index against hyperkalemia. The distribution pattern within heart and kidneys differs significantly between what we have measured for finerenone and what had been reported for the 3 steroidal MRAs spironolactone, eplerenone, and mespirenone. Experiments

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using [14C]-labeled eplerenone demonstrated at least 3-fold higher accumulation of the drug equivalent concentration in the kidney compared with heart tissue in conscious rats.27 A similar study with [3H]-spironolactone revealed significant renal drug equivalent concentrations while radioactivity in cardiac tissue was below the detection limit.28 A third study with the potent antimineralocorticoid steroid [3H]-mespirenone in rats found the following semiquantitative ranking in equivalent concentrations: liver = upper parts of the intestines . gastric mucosa . kidney = lung . adipose tissue . muscle tissue . heart29 indicating relatively low distribution of steroidal MRAs into cardiac tissue in general. This may—at least in part—explain the pronounced effects of all known steroidal MRAs on kidney electrolyte transport in relation to their cardiac antiremodeling effects. In contrast, the Ó 2014 Lippincott Williams & Wilkins

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Finerenone Protects From Cardiorenal Injury

TABLE 2. Effects of Investigated MRAs on Relative Heart Weight, Relative Right Ventricular Weight, and the Hemodynamic Parameters LVP, HR, LVEDP, and Relaxation Time Tau 9 Weeks After MI n Sham Placebo Finerenone 0.1, mg$kg21$d21 Finerenone 0.3, mg$kg21$d21 Finerenone 1.0, mg$kg21$d21 Eplerenone 100, mg$kg21$d21

14 10 11 13 10 11

HW/BW, g/kg 2.47 2.83 2.83 2.76 2.60 2.65

6 6 6 6 6 6

0.04* 0.16 0.07 0.06 0.07 0.06

RV/BW, g/kg 0.45 0.63 0.64 0.63 0.50 0.58

6 6 6 6 6 6

0.01† 0.07 0.07 0.06 0.02 0.05

LVP, mm Hg 110 99 101 100 100 97

6 6 6 6 6 6

2 2 4 3 2 3

HR, min21 341 336 340 345 345 331

6 6 6 6 6 6

5 7 10 11 8 7

LVEDP, mm Hg 9.1 17.3 18.0 17.4 11.3 16.4

6 6 6 6 6 6

0.5‡ 2.9 2.2 2.6 0.9 2.2

Tau, ms 11 15 17 17 13 18

6 6 6 6 6 6

0.4‡ 1 3 2 1 4

Group values are indicated as means 6 SEM. *P , 0.05, versus placebo. †P , 0.01, versus placebo. ‡P , 0.005, versus placebo. HW, heart weight; BW, body weight; RV, right ventricle; LVP, left ventricular pressure; HR, heart rate; LVEDP, Left ventricular end diastolic pressure; Tau, left ventricular relaxation time constant.

nonsteroidal [14C]-labeled finerenone seems to be almost equally distributed in cardiac and renal tissues, which supports the pharmacological observation that finerenone mediates endorgan protection at low natriuretic doses. Therefore, finerenone may protect from heart and kidney injury with a minimized risk of electrolyte disturbances, particularly hyperkalemia in clinical studies. One limitation of our studies is the absence of a direct evidence of increased serum potassium after MRA treatment. Unfortunately, unlike in humans hyperkalemia is rarely seen in rats after treatment with any inhibitor of the renin-angiotensinaldosterone system (RAAS). García et al30 found that serum potassium levels were maintained even in rats with 5/6 nephrectomy on a high potassium diet and spironolactone administration. Because urinary sodium excretion in rats is more comparable with humans, we decided to use natriuresis as surrogate of renal electrolyte homeostasis, determined thoroughly in acute experiments equiefficient natriuretic doses and used these doses in subsequent chronic models of end-organ damage. However, future preclinical studies may be conducted in models of reduced kidney function with possible comedication of MRAs and other RAAS blockers to capture the development of hyperkalemia more directly. Finerenone was investigated in a multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical phase II study called “ARTS” (MinerAlocorticoid Receptor Antagonist Tolerability Study12) among patients with heart failure with reduced left ventricular ejection fraction and chronic kidney disease. In these patients, 5 and 10 mg/d of finerenone was at least as effective as spironolactone 25 or 50 mg/d in decreasing BNP, NT-pro-BNP, and urinary albumin, but it was associated with lower increases in serum potassium, lower incidences of hyperkalaemia, and worsening of renal function.31

CONCLUSIONS In summary, we found end-organ protection by the novel nonsteroid MRA finerenone in 2 different rat models of heart failure independent of its etiology. When comparing equiefficient natriuretic doses as surrogate for electrolyte homeostasis in the disease models of end-organ damage, we Ó 2014 Lippincott Williams & Wilkins

found that finerenone reduced functional and structural parameters more efficiently than eplerenone. Using quantitative whole-body autoradiography, we found that [14C]labeled finerenone has a balanced distribution into rat cardiac and renal tissues. This is in contrast to the steroidal MRAs spironolactone and eplerenone, which demonstrated higher accumulation of the drug equivalent concentration in the kidney compared with heart tissue in conscious rats. Finerenone may, therefore, offer end-organ protection with a reduced risk of electrolyte disturbances. Finerenone is currently under investigation in 2 clinical phase IIb studies: (1) in patients with worsening chronic heart failure with additional type 2 diabetes mellitus and/or chronic kidney disease (ARTS-HF; NCT01807221) and (2) in patients with type 2 diabetes mellitus and diabetic nephropathy (ARTS-DN; NCT01874431).

ACKNOWLEDGMENTS The authors thank Dr. Raimund Kast for pro-BNP measurements and Dr. Diedrich Seidel for providing [14C]labeled finerenone. Portions of these results have been presented at the Congress of the European Society of Cardiology in 2012. REFERENCES 1. Swedberg K, Eneroth P, Kjekshus J, et al. Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. CONSENSUS Trial Study Group. Circulation. 1990;82:1730–1736. 2. Vantrimpont P, Rouleau JL, Ciampi A, et al. Two-year time course and significance of neurohumoral activation in the Survival and Ventricular Enlargement (SAVE) Study. Eur Heart J. 1998;19:1552–1563. 3. Delcayre C, Silvestre JS. Aldosterone and the heart: towards a physiological function? Cardiovasc Res. 1999;43:7–12. 4. Funder JW. Mineralocorticoid receptors: distribution and activation. Heart Fail Rev. 2005;10:15–22. 5. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999;341: 709–717. 6. Pitt B, Remme W, Zannad F, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med. 2003;348:1309–1321.

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Ó 2014 Lippincott Williams & Wilkins

Finerenone, a novel selective nonsteroidal mineralocorticoid receptor antagonist protects from rat cardiorenal injury.

Pharmacological blockade of the mineralocorticoid receptor (MR) ameliorates end-organ damage in chronic heart failure. However, the clinical use of av...
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