Finerenone | Acetyl CoA Carboxyla Signal

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 end- organ 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 tis- sues. 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 epler- enone 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 ™ 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 Metab-
olism 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

myocardial apoptosis, and myocardial ﬁbrosis and thus con- tributes 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, ﬁbro- blasts, vascular endothelial, and smooth muscle cells.4 Con- sequently, 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 hyper- kalemia, current steroidal MR antagonists (MRAs) are the least frequently prescribed medications among all recommen- ded pharmacological treatments for chronic heart failure8 and if prescribed, MRAs are not dosed to their full organ pro- tective 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 conse- quence, the similar structural and physicochemical properties of the steroidal MRAs determine the resulting pharmacolog- ical 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 ligand- speciﬁc 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 Finere- none 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 recep- tors 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

J Cardiovasc Pharmacol ™ ● Volume 64, Number 1, July 2014 www.jcvp.org | 69

Kolkhof et al J Cardiovasc Pharmacol ™ ● Volume 64, Number 1, July 2014

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

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

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 proto- cols.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 per- formed 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 epler- enone (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 Sys- tems, Bad Homburg, Germany) after a treatment period of 10 weeks. At the end of the experiment, hemodynamic func- tion was measured in the left ventricle by a Millar-Tip (2F)

catheter. At the time of sacriﬁce, rats were administered an overdose of anesthesia.
Chronic Myocardial Infarction Model in Rats
Male Wistar rats (250–300 g; Harlan-Winkelmann, Borchen, Germany) were used in a chronic myocardial infarc- tion (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 liga- tion was performed in sham rats. One week after surgery, ani- mals 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-/salt- treated rats were analyzed as previously published.13
Blood and Urinary Biomarker Analyses
Plasma concentrations of aldosterone and rat prohor- mone 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 in- structions. 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 beta- actin, NM_009263 osteopontin (SSP1, OPN), NM_031054

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

matrix metalloproteinase (MMP-2), NM_008871 plasminogen activator inhibitor-1 (Serpine1, PAI-1), and NM_031530.1 monocyte chemoattractant protein 1 (CCL2, MCP-1).
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 distri- bution 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 appli- cation of 3 mg/kg [14C]-labeled ﬁnerenone revealed compa- rable 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 concentra- tions 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 ﬁnere- none 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 gastro- intestinal contents, the bile-duct contents, the lymph, and in the interstitial ﬂuid. The latter was more or less blood equiv- alent 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

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 ﬁner- enone resulted in similar natriuretic responses as 10 and 30 mg/kg eplerenone, whereas 10 mg/kg ﬁnerenone had a com- parable 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

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 appli- cation. Mean 6 SEM; n = 7–8 per group; *P , 0.05, **P ,
0.01 versus placebo.

ﬁnerenone doses had no effect on urinary potassium excretion (Fig. 2C).
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 hyperaldosteronism- induced 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, respec- tively). A 10-week DOCA/salt treatment period resulted in

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 pres- sure 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 epler- enone 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

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.

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 dose- dependently decreased pro-BNP with a minimal effective dose of 1 mg/kg (Fig. 3C). Application of 100 mg/kg epler- enone appeared to have effects on pro-BNP that were com- parable to 1 mg/kg ﬁnerenone.
Ten weeks of DOCA/salt treatment resulted in signif- icant 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 dose- dependent 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). Protein- uria (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 signif- icantly 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 re- sulted in less pronounced therapeutic effects and did not dem- onstrate 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 cardiopro- tective 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 deteriora- tion 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 ﬁner- enone group, there was a trend toward LVEDP and tau reduc- tion (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 Sham Placebo Finerenone Finerenone Finerenone Eplerenone Eplerenone
Dose 0.1 mg/kg 1 mg/kg 10 mg/kg 30 mg/kg 100 mg/kg
n 11 7 11 11 11 10 10
Heart injury
Myocard degen 0 6 0 1.6 6 0.4 1.8 6 0.3 1.1 6 0.3 0.6 6 0.2* 0.7 6 0.2 0.7 6 0.3
Vasculopathy 0 6 0 1.4 6 0.3 1.5 6 0.2 1.3 6 0.3 0.3 6 0.1† 0.8 6 0.1* 0.7 6 0.2
Kidney injury
Glomerulopathy 0 6 0 2.3 6 0.5 2.0 6 0.4 1.3 6 0.3 0.3 6 0.1‡ 0.6 6 0.2† 0.8 6 0.3*
Tubular degen 0 6 0 3.0 6 0.4 3.0 6 0.4 1.9 6 0.3 0.7 6 0.3‡ 1.7 6 0.2† 1.9 6 0.4
Vasculopathy 0 6 0 1.1 6 0.7 0.8 6 0.3 0.4 6 0.3 0 6 0 0.1 6 0.1 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.

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. Rep- resentative 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 lev- els 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 dose- dependently decreased in hearts from rats treated with ﬁner- enone (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).

In summary, 1 mg$kg21$d21 of ﬁnerenone improved several hemodynamic parameters of systolic and diastolic dysfunction, decreased cardiac OPN expression and the clin- ically relevant plasma marker pro-BNP in animals with heart failure after 8 weeks of treatment. The steroidal MRA epler- enone did not exhibit protective effects despite a high natri- uretic 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

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 spar- ing) by a combination of physicochemical properties that inﬂu- ence 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 non- steroidal, 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 protec- tion of the end organs heart and kidney especially in a hyper- tension-driven model of heart failure. The determined functional parameters, relative organ weights, plasma pro- BNP levels, gene expression analyses, and histopathological analyses revealed a conclusive antihypertrophic activity of ﬁnerenone protecting heart and kidney from degenerative ac- tions 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

use of equinatriuretic doses, much higher doses (10- to 100- fold) of the steroidal drug eplerenone were needed to demon- strate 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 reduc- tion) 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 eplere- none monotherapy reduced LVEDP and tau but in contrast to trandolapril monotherapy eplerenone did not reduce plasma NT-proANP. Similarly, Fraccarollo et al25 demonstrated addi- tive 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 func- tional improvements when eplerenone was applied either in combination with an ACEI or at doses which are at least 30- fold higher in comparison to respective natriuretic doses.

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 nonste- roidal 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

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 intes- tines . 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

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 HW/BW, g/kg RV/BW, g/kg LVP, mm Hg HR, min21 LVEDP, mm Hg Tau, ms
Sham 14 2.47 6 0.04* 0.45 6 0.01† 110 6 2 341 6 5 9.1 6 0.5‡ 11 6 0.4‡
Placebo 10 2.83 6 0.16 0.63 6 0.07 99 6 2 336 6 7 17.3 6 2.9 15 6 1
Finerenone 0.1, mg$kg21$d21 11 2.83 6 0.07 0.64 6 0.07 101 6 4 340 6 10 18.0 6 2.2 17 6 3
Finerenone 0.3, mg$kg21$d21 13 2.76 6 0.06 0.63 6 0.06 100 6 3 345 6 11 17.4 6 2.6 17 6 2
Finerenone 1.0, mg$kg21$d21 10 2.60 6 0.07 0.50 6 0.02 100 6 2 345 6 8 11.3 6 0.9 13 6 1
Eplerenone 100, mg$kg21$d21 11 2.65 6 0.06 0.58 6 0.05 97 6 3 331 6 7 16.4 6 2.2 18 6 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 end- organ 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-angiotensin- aldosterone 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 thor- oughly 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 co- medication of MRAs and other RAAS blockers to capture the development of hyperkalemia more directly.
Finerenone was investigated in a multicenter, random- ized, double-blind, placebo-controlled, parallel-group clinical phase II study called “ARTS” (MinerAlocorticoid Receptor Antagonist Tolerability Study12) among patients with heart fail- ure 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 in- cidences 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

found that ﬁnerenone reduced functional and structural parameters more efﬁciently than eplerenone. Using quantita- tive 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 kid- ney 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 mel- litus 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 pre- sented at the Congress of the European Society of Cardiology in 2012.

REFERENCES
1. Swedberg K, Eneroth P, Kjekshus J, et al. Hormones regulating cardio- vascular 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 physiolog- ical 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.

7. van den Meiracker AH, Baggen RG, Pauli S, et al. Spironolactone in type 2 diabetic nephropathy: effects on proteinuria, blood pressure and renal function. J Hypertens. 2006;24:2285–2292.
8. Samuel JL, Delcayre C. Heart failure: aldosterone antagonists are under- used by clinicians. Nat Rev Cardiol. 2010;7:125–127.
9. Funder JW. Mineralocorticoid-receptor blockade, hypertension and heart failure. Nat Clin Pract Endocrinol Metab. 2005;1:4–5.
10. Kolkhof P, Borden SA. Molecular pharmacology of the mineralocorticoid receptor: prospects for novel therapeutics. Mol Cell Endocrinol. 2012;350: 310–317.
11. Bärfacker L, Kuhl A, Hillisch A, et al. Discovery of BAY 94-8862: a nonsteroidal antagonist of the mineralocorticoid receptor for the treat- ment of cardiorenal diseases. ChemMedChem. 2012;7:1385–1403.
12. Pitt B, Filippatos G, Gheorghiade M, et al. Rationale and design of ARTS: a randomized, double-blind study of BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease. Eur J Heart Fail. 2012;14:668–675.
13. Schupp N, Kolkhof P, Queisser N, et al. Mineralocorticoid receptor- mediated DNA damage in kidneys of DOCA-salt hypertensive rats. FASEB J. 2011;25:968–978.
14. Curtis CG, Cross SAM, McCulloch RJ, et al. Whole-Body Autoradiog- raphy. London, New York: Academic Press; 1981.
15. Bauersachs J. The ARTS of third-generation mineralocorticoid receptor antagonists: achieving cardiovascular beneﬁt with minimized renal side effects? Eur Heart J. 2013;34:2426–2428.
16. Willnow TE, Nykjaer A. Cellular uptake of steroid carrier proteins– mechanisms and implications. Mol Cell Endocrinol. 2010;316:93–102.
17. de Gasparo M, Joss U, Ramjoue HP, et al. Three new epoxy-spirolactone derivatives: characterization in vivo and in vitro. J Pharmacol Exp Ther. 1987;240:650–656.
18. Kagawa CM. Blocking the renal electrolyte effects of mineralocorticoids with an orally active steroidal spirolactone. Endocrinology. 1960;67: 125–132.
19. Delyani JA, Robinson EL, Rudolph AE. Effect of a selective aldosterone receptor antagonist in myocardial infarction. Am J Physiol Heart Circ Physiol. 2001;281:H647–H654.
20. Fraccarollo D, Galuppo P, Hildemann S, et al. Additive improvement of left ventricular remodeling and neurohormonal activation by aldosterone

receptor blockade with eplerenone and ACE inhibition in rats with myo- cardial infarction. J Am Coll Cardiol. 2003;42:1666–1673.
21. Rocha R, Rudolph AE, Frierdich GE, et al. Aldosterone induces a vas- cular inﬂammatory phenotype in the rat heart. Am J Physiol Heart Circ Physiol. 2002;283:H1802–H1810.
22. Brandish PE, Forest TW, Chen H, et al. Eplerenone decreases inﬂamma- tory foci in spontaneously hypertensive rat hearts with minimal effects on blood pressure. J Cardiovasc Pharmacol. 2009;53:44–51.
23. Brandish PE, Chen H, Szczerba P, et al. Development of a simpliﬁed assay for determination of the antimineralocorticoid activity of com- pounds dosed in rats. J Pharmacol Toxicol Methods. 2008;57:155–160.
24. Schäfer A, Fraccarollo D, Hildemann SK, et al. Addition of the selective aldosterone receptor antagonist eplerenone to ACE inhibition in heart fail- ure: effect on endothelial dysfunction. Cardiovasc Res. 2003;58:655–662.
25. Fraccarollo D, Galuppo P, Schmidt I, et al. Additive amelioration of left ventricular remodeling and molecular alterations by combined aldoste- rone and angiotensin receptor blockade after myocardial infarction. Car- diovasc Res. 2005;67:97–105.
26. Orena S, Maurer TS, She L, et al. PF-03882845, a non-steroidal mineralo- corticoid receptor antagonist, prevents renal injury with reduced risk of hyper- kalemia in an animal model of nephropathy. Front Pharmacol. 2013;4:115.
27. Inspra Drug Approval Package, 2003. Pharmacological Review Inspra Drug Approval Package 21-437/S-002, FDA US Food and Drug Administration. http://www.accessdata.fda.gov/drugsatfda_docs/nda/ 2003/21-437S002_Inspra_Pharmr.pdf. Accessed March 2014.
28. Platt D, Pauli H. Studies on organ- and subcellular distribution of 3H- spironolactone in animals [in German]. Arzneimittelforschung. 1972;22: 1801–1802.
29. Hildebrand M, Krause W, Kühne G, et al. Pharmacokinetics and metab- olism of mespirenone, a new aldosterone antagonist, in rat and cynomol- gus monkey. Xenobiotica. 1987;17:623–634.
30. García NH, Baigorria ST, Juncos LI. Hyperkalemia, renal failure, and converting-enzyme inhibition: an overrated connection. Hypertension. 2001;38:639–644.
31. Pitt B, Kober L, Ponikowski P, et al. Safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial. Eur Heart J. 2013;34:2453–2463.