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Comparison Of Agents That Affect Aldosterone Action Juan Tamargo, Anna Solini, Luis M Ruilope
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1 COMPARISON OF AGENTS THAT AFFECT ALDOSTERONE ACTION Juan Tamargo1, Anna Solini2, Luis M Ruilope3
1- Department of Pharmacology, School of Medicine, University Complutense, Madrid. 2- Department of Clinical and experimental Medicine, University of Pisa 3- Research Institute, Hypertension Unit, Hospital 12 de Octubre & Department of Public Health and Preventive Medicine, University Autonoma, Madrid
KEY WORDS: Aldosterone, aldosterone receptor antagonists, spironolactone, eplerenone, canrenone, aldosterone synthase inhibitors, hypertension, hyperaldosteronism, heart failure Address of autor to maintain correspondence: Luis M Ruilope Instituto de Investigación Unidad de Hipertensión Hospital 12 de Octubre 28041, Madrid SPAIN Acknowledgements: This work was supported by Instituto de Salud Carlos III (Red RIC, and PI11/01030) and Comunidad de Madrid (S2010/BMD-2374). INTRODUCTION For many years aldosterone was considered as a hormone devoted to the control of renal excretion of minerals, which explains the term mineralocorticoid used to describe this hormone. In two clinical situations, primary and secondary hyperaldosteronism were initially considered for the clinical use of the first aldosterone receptor blocker spironolactone as a diuretic. The discovery of many extra-renal sites of mineralocorticoid receptors (1,2) and the investigation of the pro-inflammatory and fibrogenic effects of the hormone (3) expanded the knowledge of the capacities of aldosterone to directly participate in the pathogenesis of arterial hypertension (4), heart failure (HF)(5), chronic kidney disease (CKD)(6) and metabolic syndrome (7). Eplerenone the second most widely used blocker of aldosterone receptors arrived when the role of these drugs as diuretics had been expanded to cardiorenal
2 and metabolic disease. Data with canrenone a third blocker of the receptor are scanty. Finally two other substances have been shown to antagonize the actions of aldosterone in the renal tubule, amiloride and triamterene and they continue to be used as diuretics. In this paper we will review the role of aldosterone antagonists in aldosteronism, cardiorenal disease and metabolic disease and we will refer in particular to the similarities and differences of the two main members of the class spironolactone and eplerenone. Finally we will comment on the new members of the class of aldosterone antagonists and their potential advantages. EFFECTS OF ALDOSTERONE Aldosterone, the final product of the renin-angiotensin-aldosterone system (RAAS), is a mineralocorticoid hormone secreted from the adrenal zona glomerulosa isolated and characterized in 1953 (8). Untill the mid-90s, it was thought that aldosterone acts primarily in the epithelial cells of the late renal distal convoluted tubule and the collecting duct, distal colon and salivary and sweat glands leading to an increase in the net reabsorption of Na+ and water and K+ excretion. The reabsorption of Na+ and water then elevates BP indirectly by expanding extracellular fluid volume. These effects are mediated by the binding of aldosterone to mineralocorticoid receptors (MR, NR3C2), a ligand-activated transcriptor factor belonging to the nuclear receptor superfamily (8). However, following the molecular cloning of the MR in 1987, both experimental and clinical evidence demonstrated that MRs are also present in a wide range of tissues, including endothelial and vascular smooth muscle cells, cardiac tissues (cardiomyocytes, fibroblasts and macrophages), kidney (mesangial cells and podocytes), adipocytes, monocytes and brain(2,3,9). Activation of MRs by aldosterone promotes multiple renal, cardiac and vascular deletereous effects, including endothelial dysfunction, hypertension, neurohumoral activation, cardiovascular (CV) and renal remodeling (hypertrophy, fibrosis, apoptosis), decreases arterial compliance, increases expression of cell adhesion molecules, platelet activation, plasminogen activator inhibitor type 1 (PAI-1) activity and oxidative stress [by both nicotinamide adenine dinucleotide phosphate, NADP(H), oxidase activity and mitochondria) and exerts proarrhythmic and proinflammatory effects (Figure 1)(1,3,5,10-12). Additionally, activation of central MRs increases central sympathetic tone to the kidneys, heart, and vascular smooth muscles and vasopressin release and decreases baroreceptor sensitivity. Moreover, aldosterone, via MR activation upregulates ACE expression in the cardiomyocytes, suggesting the existence of a positive feedback
3 pathway that activates the renin-angiotensin-aldosterone system (13). These effects are reported to be “genomic”, i.e. dependent on transcription and translation. Additionally, aldosterone produces rapid, translation- and transcription-independent effects (non-genomic effects) that may be mediated by G-protein-coupled receptor 30 (GPR30) and transactivation of the epithelial growth factor receptor (EGFR) (3). These effects have been described in vascular smooth muscle cells (VSMCs) and other tissues where aldosterone induces a rapid increase in Na+ influx (aldosterone increases Na+/K+/2Cl- cotransporter activity and Na+-H+ exchange, and inhibits Na+/K+ ATPase activity) and intracellular Ca2+ concentrations through an increase in Ca2+ entry through voltage-gated channels, and activates cAMP-protein kinase A, phospholipase C, phosphatidylinositol 3-kinase (PI3 kinase), diacylglycerol and protein kinase C (PKC) signaling pathways (3,14,15). Aldosterone also induces a rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) 1/2 kinase in VSMCs, endothelial and kidney cells, to promote a mitogenic and profibrotic phenotype, an effect that involves the transactivation of the EGFR (3,14,15). The non-genomic effects occur also independently of hemodynamic factors and play an important role in the mechanisms by which aldosterone contribute to endothelial dysfunction, vasoconstriction, resistant arterial hypertension, CV and renal remodeling, inflammation, heart failure, insulin resistance and chronic renal disease (CKD) (1,3,5,7,14-18). Interestingly, in VSMC, aldosterone activates both GPR30 and MR to mediate vasodilatation and apoptosis and to activate PI3 kinase, ERK, and myosin light chain phosphorylation (15). In contrast endothelial MR activation has been linked to enhanced vasoconstrictor and/or impaired vasodilator responses. The MR has highest homology to glucocorticoid receptor (GR), and both receptors are expressed together in aldosterone target cells. In epithelial cells (kidney, endothelial cells, colon), aldosterone selectivity is determined by the 11-β-hydroxysteroid dehydrogenase 2 (11β-HSD2), an enzyme colocalized with the MR which converts the active cortisol to the MR-inactive cortisone, but does not degradate aldosterone. However, other tissues (i.e., cardiomyocytes, some regions of the brain) lack 11β-HSD2, so that cortisol is the primary ligand for the MR (1). Thus, the tissue specificity of aldosterone is determined in part by the presence of 11β-HSD2. Although aldosterone and cortisol bind the MR has equal affinity, the aldosterone/MR complex is more stable, which in turn mediates a much stronger (200-fold) transactivation response than the cortisol/MR complex (19). Whether the deleterious events mediated via MR activation are mediated by aldosterone and/or cortisol at conditions of inappropriate salt or redox status is a matter of discussion (1).
4 All these results confirm the aldosterone via MR-dependent and MR-independent mechansims plays a pivotal role as a regulator of cellular and organ function, far beyond its effects on the kidney, and is directly involved in target organ damage in various CV and renal diseases [3,11]. This is the pharmacological rationale for the development of aldosterone antagonists for the treatment of CV diseases (Table 1). PHARMACOLOGICAL MODULATION OF ALDOSTERONE 1. MINERALOCORTICOID RECEPTOR ANTAGONISTS (MRAs) Three steroidal MRAs are presently in the market: spironolactone, eplerenone, and canrenoate (Table 1). Spironolactone and eplerenone have been widely studied in large randomized controlled trials (RCTs) (20-22), while potassium canrenoate (canrenone), an active metabolite of spironolactone, is available in some countries. Novel non-steroidal compounds are presently in preclinical and early clinical development. Spironolactone, the first MRA, was launched in 1960 as a K+-sparing diuretic with a complementary mode of action to that of the diuretics currently used for the management of edematous conditions, primary aldosteronism and essential hypertension. Spironolactone is a potent, competitive, non-selective MRA, as it also blocks androgen receptors and activates progesterone receptors causing progestogenic and antiandrogenic adverse effects limiting its use (23). This explains why its long-term use is associated with gynecomastia and other endocrine adverse effects, such as breast tenderness, impotence, loss of libido and menstrual irregularities (23-26). These adverse effects and the lack of selectivity for the MR stimulated the search for more selective and better-tolerated MRAs. Eplerenone, a 9,11-epoxy derivative of spironolactone, launched in 2002 for the treatment of congestive HF, represents the second generation of MRAs. It presents a higher selectivity for the MR and ~500 times lower affinity for androgen and progesterone receptors than spironolactone (27,28), leading to negligible sexual-related adverse effects (29). Thus, eplerenone represented an alternative in patients with spironolactone-induced sexual adverse effects (30,31). Despite its increased selectivity, in in vitro competition binding assays eplerenone exhibits a 10- to 20-fold lower affinity for MR compared with spironolactone, suggesting that eplerenone has 1 to 2 times the potency of spironolactone. The third generation of MRAs is represented by new non-steroidal, potent, selective compounds, while the fourth generation is based on third generation molecules with renal-sparing properties and in the near future should include tissue-specific MR antagonists (16).
5 Although spironolactone and eplerenone are effective and improve clinical outcomes in patients with hypertension and HF, there are important differences in their mode of action, pharmacodynamic and pharmacokinetic profile, adverse effects and drug interactions which may be relevant for differentiating these drugs in a given patient (25,29,32,33). 1.1. Mechanism of action Aldosterone initiate its actions by binding to the ligand binding domain of the MR located in the cytoplasm, where MR are rendered transcriptionally inactive by several chaperone proteins like the 90-kDa heat shock protein. Aldosterone binding induces a conformational change in the MR resulting in dissociation of chaperone proteins, translocation of the aldosterone-MR complex to the nucleus and its binding to hormone response elements in the regulatory region of target gene promotors triggering the transcription of target genes (3,34). Spironolactone and eplerenone competitively inhibit aldosterone binding to the MR and render it transcriptionally inactive. As a consequence, they block the effects mediated via MR activation regardless of the ligand. However, spironolactone or eplerenone do not antagonize aldosterone-induced non-genomic renal and extrarenal effects (1,16). The MR consists of three principal domains: N-terminal, DNA-binding and ligand-binding (LBD) domains (8). The tertiary structure of the LBD is highly conserved and consists of 12 α-helices and 3 β strands folded into an anti-parallel three layer with a ligand-binding pocket in the lower third of the molecule (16). In the presence of aldosterone helix 12 is repositioned together with helices 3, 4 and 5 to form a hydrophobic groove on the surface of the LBD which allows binding of co-activator molecules with a leucine-rich LxxLL motif and subsequent MR activation (35). This function in the LBD is termed activation function 2 (AF-2) and is ligand dependent. Aldosterone-occupied MR recruits specific coactivator peptides that are needed to increase transcription and to stabilize the complex in its activated state (34). Coactivators and co-represors are cellular factors which interact with the MR to potentiate or attenuate transactivation, so that the ratio of coactivators to co-represors bound to the MR-ligand complex modulates the transcriptional sensitivity of the MR (36). There are important differences between spironolactone and eplerenone in the binding mode to MR and consequences for MR stability, nuclear translocation and co-factor recruitment. The mechanism by which antagonist ligands inactivate the MR is based on the instability of the antagonist-MR complexes, rather than on the ability of the antagonist to stabilize the MR in an inactive state, favoring the recruitment of co-repressors (37,38). However, the mechanisms of MR antagonism produced by spironolactone and eplerenone may occur
6 through different mechanisms. Spironolactone produces a “passive” antagonism. The drug binds and prevents MR from adopting the active conformation by failing to mediate hydrogen bonding to Asn770 and Thr945 and both helix 3 and the AF-2 helix are not arranged in the proper position to allow efficient binding of co-activators of transcription (35,39). Eplerenone binds to the LBD and antagonizes aldosterone-induced MR activation and dosedependently blocks the interaction of aldosterone with coactivator peptides (38). However, eplerenone does not actively recruit co-represors (possibly because it has no influence on the conformation of helix 12) or stabilizes a repressive conformation that allows corepressor binding; instead, it stabilizes a neutral conformation that is transcriptionally inert, perhaps similar to the unliganded MR conformation. 1.2. Pharmacodynamics Spironolactone was initially licensed as a potassium-sparing diuretic for the treatment of hypertension, primary hyperaldosteronism, volume-overload states and hypokalemia. In an early study in 182 men treated with spironolactone alone (mean dose 96.5 mg daily) during 23 months the reduction in SBP/DBP averaged 18/11 mm Hg (24). Nowadays, MRAs as antihypertensive drugs are preferentially indicated in the treatment of primary and secondary hyperaldosteronism, but their use can be contemplated in hypertensive patients with metabolic syndrome or target organ damage represented by left ventricular (LV) hypertrophy or albuminuria (17,18,40) or particularly as add-on therapy in patients with resistant hypertension where MRAs have shown positive results (41-43) On the other hand, MRAs are especifically indicated in patients with chronic congestive HF with diminished ejection fraction (5) and in post-MI as shown by the Ephesus (Epleronone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) trial (21). Spironolactone is also indicated as a diuretic in patients with cirrhosis and ascites, in whom secondary hyperaldosteronism is present, and hypokalemia is detrimental (44) 1.2.1. Primary hyperaldosteronism. Primary aldosteronism (PA) is characterized by arterial hypertension frequently accompanied by hypokalemia and probably constitutes the most frequent cause of secondary hypertension after chronic kidney disease (45). It ussually is accompanied by a high prevalence of cardiovascular and renal damage leading to an increased morbidity and mortality (46). Spironolactone is effective in the treatment of PA as short-term preoperative treatment or long-term treatment in patients with unilateral aldosterone-producing adenomas with high operative risks or who decline surgery and in patients with unilateral or bilateral (usually idiopathic adrenal hyperplasia) disease who are
7 unable or unwilling to undergo adrenalectomy (46,47) Spironolactone lowers BP to a similar extent in hypertensive patients with and without primary aldosteronism, although higher doses are required in those with PA (24,43,47) reduces CV and renal complications, decreases LV hypertrophy and urinary protein excretion and corrects hypokalemia and abnormalities of glucose metabolism (hypersinsulinemia and insulin resistance) (46,47). The long-term CV outcomes were studied in patients with PA after treatment with adrenalectomy or spironolactone (48). At baseline, the prevalence of cardiovascular events was greater in PA than in essential hypertension (35% vs 11%, P< .001). During a follow-up for a mean of 7.4 years, BP and CV outcomes were comparable in the two treatments, indicating that adrenalectomy and spironolactone are equally effective in preventing CV outcomes. Both treatments improve intrarenal hemodynamic pattern while protecting glomerular filtration rate and decreasing urinary albumin excretion (49,50). The incidence of LV hypertrophy in PA is higher than in essential hypertension and spironolactone and adrenalectomy exert similar positive influences facilitating the regression of LVH (33) but this is partly independent of changes in BP (51,52). The comparison of eplerenone and spironolactone in patients with PA led to contradictory results. In an open-label study, spironolactone (50 to 400 mg/day) and eplerenone (50-200 mg/day) produced a similar BP reduction BP (53), while in another double-blind study the decrease in SBP/DBP was greater in patients treated with spironolactone (75-225 mg/day) than with eplerenone (100-300 mg/day), although more patients randomized to spironolactone develop gynecomastia and mastodynia (54). In this latter study, despite a higher incidence of gynecomastia/mastodynia and hyperkalemia in the spironolactone group, the overall incidence of adverse events is comparable in both treatment groups. The natriuretic effects of spironolactone and eplerenone are greater in patients with hyperaldosteronism. 1.2.2. Arterial hypertension. Aldosterone acts as a downstream effector of the RAAS and an excessive aldosterone production is a significant cause of hypertension. In fact, an increase in plasma aldosterone accompanied by low renin levels predicts the development of arterial hypertension in middle-aged caucasian population (55,56). On the other hand, an increased aldosterone:renin ratio indicative of the existence of primary aldosteronism is found in 1020% of patients diagnosed with resistant hypertension (57). In these patients there is evidence of intravascular volume expansion, particularly in men, and the significant correlation between 24-hour urinary aldosterone levels and cortisol excretion suggests that a common stimulus, such as corticotropin, may underlie the aldosterone excess in patients with resistant
8 hypertension (57). MRAs are effective in mild-to-moderate hypertension (24,58) but are particularly effective in patients with low-renin or resistant hypertension (42,43,59). They are also effective in reducing BP in hypertensives treated with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) presenting elevated aldosterone plasma levels as a consequence of the aldosterone breakthrough that represents a situation of escape to the effect of the renin-angiotensin blockade after long-term therapy (60). This escape may contribute to the development of resistant hypertension and in fact beyond those cases with primary aldosteronism a high proportion of resistant hypertensives present with elevated plasma aldosterone levels (57). A Cochrane meta-analysis of 5 cross-over studies found that spironolactone significantly reduces SBP/SBP by 20/6.7 mmHg in patients with primary hypertension when doses of 100500 mg/day are given. One of the studies showed that spironolactone 25 mg/day did not significantly change BP as compared to placebo (61). Because of the possible increase of adverse effects at doses >50 mg/day, in patients with primary hypertension spironolactone is frequently prescribed at lower doses but in combination with a thiazide-like or loop diuretic (sequential nephron blockade) to prevent hypokalemia and/or hypomagnesemia and to produce additional antihypertensive and/or natriuretic effects (26). On the contrary, in resistant hypertension adding a low-dose of spironolactone (12.5-25 mg/d) to a multidrug regimen, that includes a diuretic and an ACEI or an ARB, in patients with resistant hypertension produces a mean decrease in SBP/DBP 21/10 mm Hg and 25/12 mm Hg at 6 weeks and 6 months treatment, respectively (62). A similar reduction in SBP/DBP (21.7/8.5 mm Hg) is observed in following the addition of spironolactone (25-50 mg) in patients with resistant hypertension receiving simultaneously an angiotensin-blocking drug (63). In the ASCOT (Anglo-Scandinavian Cardiac Outcomes Trial) trial spironolactone (mean dose of 25 mg/day) reduced SBP/DBP by 21.9/9.5 mm Hg in patients with uncontrolled hypertension and this reduction was unaffected by age, sex, and diabetic status (43). In this study, 6% of patients discontinue the drug due to adverse effects (gynecomastia and hyperkalemia). Eplerenone is also an effective antihypertensive drug (64). In hypertensive patients uncontrolled with an ACEI or an ARB, eplerenone (25, 50 and 200 mg bid) and spironolactone (50, 100, or 400 mg od) decrease dose-dependently SBP/DBP during the 24-h period (58). However, the mg-for-mg BP-lowering effect of eplerenone is lower than that of spironolactone, so that 200 mg eplerenone bid (or 400 mg od) produces a BP reduction comparable to that produced by 50 mg spironolactone bid (i.e., spironolactone produces 1.3– 2 times greater BP reduction). Eplerenone (50 mg daily) is more effective than losartan (50
9 mg daily) in black patients that respond relatively poorly to ACEI or ARB, in the low-renin patients and in patients with differing baseline levels of aldosterone; however, eplerenone is as effective as losartan in white and high-renin patients (65). Eplerenone is more effective than losartan in reducing SBP/DBP in patients with low-renin hypertension; after 16 weeks of therapy, significantly fewer eplerenone-treated patients than losartan-treated patients (32.5% vs 55.6%) require the addition of hydrochlorothiazide (59). Eplerenone is as effective as amlodipine or enalapril in lowering SBP (3,66) and its combination with othere antihypertensive drugs provides an additional benefit in patients uncontrolled in monotherapy (67). In patients with hypertension and left ventricular hypertrophy, the 4E study demonstrated that eplerenone (200 mg/day) is as effective as enalapril (40 mg/day) in lowering SBP and LV hypertrophy (40) and their combination more effective than eplerenone alone. This trial supports that blockade of the MR is an effective method of reducing end-organ damage. The use of eplerenone in resistant hypertension at doses of 50 up-titrated to 100 mg/d showed a fall in office BP of -17.6/-7.9 mmHg and -12.2/-6.0 in ABPM values (68). However, the effects of spironolactone and eplerenone on clinical outcomes in patients with refractory hypertension are unknown. In a recent study, patients with resistant hypertension despite 4 weeks of treatment with triple therapy (irbesartan 300 mg/day, hydrochlorothiazide 12.5 mg/day and amlodipine 5 mg/day) were randomized to sequential nephron blockade (spironolactone 25 mg/day followed by furosemide 20 mg/day, furosemide 40 mg/day and amiloride 5 mg/day) or sequential RAAS blockade (ramipril 5 mg/day followed by ramipril 10 mg/day, bisoprolol 5 mg/day and bisoprolol 10 mg/day) (69). At week 12, the mean difference in daytime ambulatory BP was 10/4 mmHg in favour of the sequential nephron blockade. The BP goal (daytime ambulatory BP 50 mg/day), drugs producing hyperkalemia (Table 3) or K+ supplements (5,25,26,111,135). A small study identified that the polymorphism NR3C2 215G is associated with a higher risk of potassium increases >0.5 mEq/L when spironolactone is started in patients with HF (136). The
risk
of
hyperkalemia
decreases
by
ensuring
that
dietary
(http://www.ars.usda.gov/ba/bhnrc/ndl) and other pharmacologic sources of K+ (Table 3) are minimized. The combination of a MRA with a K+-losing diuretic may lower the baseline serum K+ value, but this combination should be used only if diuretic-related volume losses do not reduce the GFR, an effect that can significantly reduce urinary K+ excretion (25,26). A recent pilot study found that RLY5016, a non-absorbed, orally active, K+-binding polymer, prevents hyperkalemia and is relatively well tolerated in patients at risk of developing hyperkalemia, receiving standard therapy for HF and spironolactone (25–50 mg/day) (137). Thus, close monitoring of plasma K+ levels is recommended when spironolactone or eplerenone are added in patients treated with ACEIs/ARBs, particularly in patients with moderate-severe CKD (GFR 75 years (153). Furthermore, the reduction in SBP and the rise in serum aldosterone levels are smaller in patients receiving BAY 94-8862 than in patients receiving spironolactone. However, it remains to be proven that BAY 94-8862 is as effective as spironolactone or eplerenone in the prevention of major CV events. The ongoing phase IIb ARTS-HF trial (NCT01807221) compares the efficacy and safety of BAY94-8862 and
22 eplerenone in patients hospitalized with worsening chronic heart failure and LV systolic dysfunction and either type 2 diabetes with or without CKD or CKD alone. Thus, BAY 948862 may represent the first drug of the fourth generation of MRAs that can achieve a CV benefit without or at least with fewer renal adverse effects than spironolactone or eplerenone. We have already mentioned that the fourth generation of MRAs should present a renalsparing profile-. However, they should not completely spare renal effects as this might lead to a new condition characterized by hypokalemia (which can be more dangerous than hyperkalemia) and Na+ retention among patients with PA (1). Thus, a combined renal Na+ excretion and a mild K+ retention are clearly beneficial and demanded characteristics (130). A further step will be the development of tissue-selective MRAs, i.e., drugs with higher higher cardiovascular/renal ratio, as compared with available steroidal MRAs. The basis for this approach is the evidence that a heterogeneous group of non-receptor proteins, termed coregulators, are required to enhance or repress nuclear receptor-mediated transactivation of target genes (36). Certain coregulators would be expected to confer specificity to MRmediated responses because of their variable tissue expression and selectivity for different ligands. Thus, the interaction of novel MRAs with coregulator molecules that coordinate the specific interaction between the MR and their genes in a tissue-specific manner may allow modulation of MR activity in a ligand- and tissue-specific manner (while retaining normal MR function in other tissues) leading to the development of compounds with important overall pharmacodynamic differences (36,130). This tissue selectivity can also be reached by improving the physicochemical properties that influence plasma transport and tissue penetration of the new compounds in different tissues and the differential affinity of a MR– drug complex for tissue-specific coregulators. Whether this proposal is not an unrealistic idea or a pharmacological target available in the near future will depend on the coordinated efforts of academic, clinical and industrial research. 3. ALDOSTERONE SYNTHASE INHIBITORS Another approach to reduce aldosterone plasma and tissular levels and antagonize its effects mediated via MR-dependent and MR-independent pathways is to inhibit the enzyme aldosterone synthase (CYP11B2), a member of the cytochrome P450 family encoded by the CYP11B2 gene, that catalyses a rate-limiting step of aldosterone synthesis (154,155). This enzyme is activated by angiotensin II and K+ levels, and to a minor extent by adrenocorticotropin (ACTH), vasopressin, catecholamines, ET-1, serotonin, cytokines and Mg2+ (11,156)(Figure).
23 In contrast to MRAs, aldosterone synthase inhibitors (ASIs) prevent the reactive increase in aldosterone levels in response to ACEIs/ARBs and inhibit both the MR-dependent and MRindependent effects exerted by aldosterone in target organs (3,15). Thus, ASIs have emerged as a therapeutic alternative to MRAs, but their challenge is to demonstrate that they are as effective, but better tolerated, than MRAs for the treatment of hypertension, HF and renal disorders (157). However, because of the high sequence homology of CYP11B2 and CYP11B1, the 11-β-hydroxyase responsible for the final step in cortisol synthesis, ASIs might also suppress cortisol release. 3.1. FAD 286A, the enantiomer of the nonsteroidal aromatase inhibitor fadrozole, inhibits human recombinant CYP11B1 (IC50 9.9) and CYP11B2 (IC50 1.6 nM) and angiotensin IIstimulated aldosterone synthesis in NCI-H295R human adrenocortical carcinoma cells (155). FAD 286A decreases urinary free aldosterone levels in spontaneously hypertensive rats on a low sodium-high potassium (LS, high urinary aldosterone levels) or a high sodium-normal potassium (HS, low urinary aldosterone level) and increases plasma renin levels only in rats on LS (158). The combination of FAD 286A and spironolactone on the LS diet induces severe hypoaldosteronism, whereas the combination of FAD 286A and furosemide on the HS diet corrects the diuretic-induced hypokalemia. In rats overexpressing both the human renin and angiotensinogen genes (dTGR), FAD 286A inhibits CYP11B2, reduces circulating and cardiac aldosterone levels and improves endothelial function, cardiac and renal target-organ damage (decreases cardiac hypertrophy, albuminuria, cardiac and renal inflammation and matrix deposition) and survival. However, FAD 286A only slightly reduces BP, while losartan normalizes BP (155). In uninephrectomized rats on a HS diet angiotensin II causes albuminuria, azotemia, renovascular hypertrophy, glomerular injury and increases PAI-1 and osteopontin mRNA expression and tubulo-interstitial fibrosis in the kidney. FAD286 and spironolactone prevent these renal effects and attenuate cardiac hypertrophy and fibrosis (159). Furthermore, in a rat model of heart failure, FAD 286A and spironolactone improve LV hemodinamics, remodeling and function, but only FAD 286A normalizes LV oxidative stress and coronary endothelium-dependent vasodilatation without changes in BP (160). FAD 286A also reduces the severity of atherosclerotic plaques and the expression of proinflammatory markers in apolipoprotein E-deficient mice without affecting plasma aldosterone levels (161). These results indicate that ASIs might be a potential therapeutic stratagy for the treatment of HF probably by targeting MR-independent effects of aldosterone (15).
24 3.2. LCI699 is an orally active non-selective ASI structurally derived from FAD286 that also inhibits CYP11B1. In a Phase I study in healthy male subjects on a controlled salt diet, LCI699 causes a dose-dependent decrease in plasma and urinary aldosterone (up to 70 or 80%) without affecting basal cortisol levels compared with placebo, increases in plasma renin activity ansd is well tolerated (162). However, due to its short plasma half-life, the decrease in plasma aldosterone levels does not persist for 24 h. At the dose of 0.5 mg od, LCI699 selectively inhibits aldosterone synthesis, but not cortisol synthesis, even after ACTH stimulation, but CYP11B2 selectivity is lost at doses ≥1 mg od, and at doses ≥3 mg LCI699 blunts the cortisol response to and ACTH test. Four phase II trials study the efficacy and safety of LCI699 (163). The first study compared LCI699 (0.25, 0.5 and 1 mg od and 0.5 mg bid.) and eplerenone (50 mg bid) with placebo in hypertensive patients (164). All doses LCI699 significantly decreases 24-h ambulatory BP compared with placebo and the dose of 1 mg reduces mean sitting DBP to a similar extent to eplerenone. Paradoxically, LCI699 0.5 mg bid reduces BP less that the dose of 1 mg od, even though the 0.5 mg bid dose produces a greater inhibitory effect on aldosterone synthesis; this finding was attributed to increases in precursor mineralocorticoids. LCI699 is rapidly absorbed, reaches peak plasma concentrations after 1 h and presents an apparent half life of 3.8-5.5 h and does not accumulate following the administration of multiple doses up to 3 mg. As expected, LCI699 twice-daily regimen decreases, while eplerenone increases plasma aldosterone levels; however, both drugs increase plasma renin activity and serum K+, and decreases serum Na+. Although LCI699 has no effect on basal cortisol concentrations, it blunts ACTH stimulation of cortisol in ≈20% of subjects receiving 1.0 mg od. Another study analyzed the effects of LCI699 (0.5, 1 and 2 mg od or 1 mg bid) on cortisol in hypertensive patients (165). LCI699 produces a dose-dependent SBP/DBP reduction, reduces PALs (5080%) and suppresses ACTH-stimulated cortisol response. Another trial compared LCI699 (0.25 bid, 1 mg od and 0.5/1 mg bid) and eplerenone (50 mg bid) as an add-on therapy in patients with resistant hypertension. LCI699 lowers BP modestly in these patients, the BP response being smaller than with eplerenone and not statistically significant vs placebo (166). LCI699 suppresses plasma aldosterone levels in a dose-related manner, with corresponding dose-dependent increases in plasma renin activity and 11-deoxycorticosterone levels. The authors hypothesized that higher doses of CLI699 are necessary to reduce BP in patients with resistant hypertension, but such doses cannot be tested due to loss of steroidogenic target selectivity.
25 In patients with PA, LCI699 (0.5 mg b.i.d. for 2 weeks, force-titrated to 1 mg b.i.d. for 2 weeks) produces a modest decrease in BP (-4.1 mmHg), does not modify basal plasma cortisol levels, but decreases plasma and urinary aldosterone levels (∼70–80%), normalizes hypokalemia and increases plasma renin, ACTH (35%) and 11-deoxycorticosterone levels (> 700%) (167). The same group compared the effects of LCI699 (0.5-1 mg bid) and eplerenone (50 to 100 mg bid) in patients with PA (157). After 4 weeks of treatment, LCI699 was less effective in reducing BP than eplerenone, even though LCI699 markedly decreased (75%), whereas eplerenone increased plasma aldosterone levels (89%). In all these studies the clinical and biological safety and tolerability of LCI699 are similar to those of placebo and eplerenone and no patients require intervention for adrenal insufficiency. These studies reveal that LCI699 interferes with two endocrine feedback loops at the adrenal gland (163). First, inhibition of CYP11B2 reduces aldosterone levels, leading to the stimulation of the RAAS feedback axis, a slight decrease in plasma Na+ and an increase in plasma K+ levels and plasma renin levels and activity. Second, the increase in 11deoxycorticosterone levels in the presence of stable cortisol levels confirms the inhibitory effect of LCI699 of the 11β-hydroxylase in the adrenal gland and explains why the plasma cortisol response to an ACTH test is significantly blunted. The resultant stimulation of the hypothalamic-pituitary-adrenal feedback axis induces the compensatory increase of endogenous ACTH levels that stimulates adrenal steroidogenesis to compensate for the inhibited cortisol secretion and leads to a supraphysiological increase of 11deoxycorticosterone, the precursor of cortisol, consistent with the inhibition of the CYP11B1 gene product. This increase may explain the observed disappointing BP reductions achieved with the compound at higher doses and particularly upon twice-daily administration (163). The poor antihypertensive effect of LCI699 in patients with PA or resistant hypertension, its short half-life and the lack of selectivity for CYP11B2 that limit the use of a higher doses range indicate that LCI699 is not an attractive alternative to replace MRAs in patients with hypertension or other CV and renal disorders and support the development of secondgeneration compounds with much higher selectivity for CYP11B2. Very recently, several N(Pyridin-3-yl)benzamides derived from metapyrone were found to be potent and selective CYP11B2 inhibitors (IC50 values 53–166 nM) (168). Despite all these disadvantages, LCI699 opens new possibilities for treating patients with glucocorticoid excess, such as Cushing’s disease (168). Moreover, selective ASIs may be complementary or an alternative in patients with PA or hepatic cirrhosis that require high
26 doses of spironolactone (≥50 mg daily) that are associated with endocrine side-effects. Furthermore, the addition of a selective ASI in patients with hypertension or HF treated with low doses of spironolactone or eplerenone may allow a better dose-dependent normalization of plasma aldosterone and K+ levels. However, the development of new selective and potent ASIs presents several important problems because (130): 1) the degree of CYP11B2 inhibition required to neutralize the pathological, but not the physiological, effects of aldosterone is unknown; 2) they can produce an increase in 11-deoxycorticosterone that might act as a substitute for aldosterone on the MR; 3) the second generation of ASIs have to compete with the new potent and selective non-steroidal MRAs (147) and, 4) ASIs present some adverse events similar to those of MRAs, including electrolyte disorders, hypotension, renal insufficiency and hypoaldosteronism, and drug interactions with K+ retaining drugs. 3.3. There is evidence that some dihydropyridine Ca2+ channel blockers (i.e., azelnidipine, benidipine and efonidipine) exhibit inhibitory actions on adrenal aldosterone synthesis by decreasing the expression of CYP11B1 and CYP11B2, while cilnidipine suppresses Ang IIinduced CYP11B2 (but not CYP11B1) mRNA expression (157,164,169-171). These findings can be the basis for the development of new antihypertensive drugs that block L-type Ca2+ channels, inhibit CYP11B2 and antagonize MR. Drugs with dual or triple action on these targets might represent a novel approach to reduce BP and to prevent/treat hypertensive endorgan damage with less risk of hyperkalemia. 3.4. MicroRNAs (miRNAs) are endogenous, single-stranded non-protein-coding RNA molecules of ≈22 nucleotides that post-transcriptionally control gene expression, interfering with mRNA stability and/or translational rate, by base-pairing to the 3´UTR of proteincoding mRNAs. Screening of non-diseased human adrenal and aldosterone-producing adenoma samples yields to distinctive miRNA expression signatures for each tissue type. Bioinformatic analysis identifies putative binding sites for several miRNA in the 3' untranslated region of CYP11B1 and CYP11B2 mRNAs and in vitro manipulation of miR-24 confirms the ability of miR-24 to target both CYP11B1 and CYP11B2 mRNAs directly, decreasing aldosterone and cortisol production in adrenocortical cells (172). Thus, adrenal miRNA may represent a novel target for the therapeutic manipulation of mineralocorticoid biosynthesis. 4. EPITHELIAL NA+ CHANNEL BLOCKERS
27 Amiloride and triamterene block epithelial Na+ channels (EnaC) channels located in the luminal membrane of the principal cells of the distal tubule and collecting duct, perhaps by competing with Na+ for negative charges within the channel pore (173). The reduction in Na+ reabsorption hyperpolarizes the tubular apical membrane and reduces the electrochemical gradient K+ secretion from the principal cell and H+ secretion via the H+-ATPAse from the intercalated cell. Additionally, the reduction in K+ secretion decreases H+ secretion via K+/H+ ATPase. The net effect is an increase in Na+ excretion and a decrease in K+ and H+ excretion. However, in contrast to MRAs, this K+ retention is independent of aldosterone. Both drugs are filtered at the glomerulus and secreted by the organic cation secretory pathway into the proximal tubule. Amiloride presents low oral bioavailability, is not metabolized in the liver and is excreted unchanged in the urine, so that CKD prolongs its half-life, while hepatic insufficiency has little effect on its pharmacokinetics (173). Triamterene is extensively metabolized in the liver and the drug (50%) and its active metabolite (4-hydroxytriamterene sulfate) are excreted in the urine. The amount of metabolite reaching the tubule decreases in hepatic insufficiency. In patients with CKD both drugs have progressively limited access to their site of action and, therefore, became less effective. In monotherapy, amiloride and triamterene produce a weak natriuretic effect that increases in patients with primary or secondary hyperaldosteronism, but are relatively ineffective in lowering BP. Thus, they are co-administered with thiazide or loop diuretics to prevent K+ and Mg2+ excretion and/or to increase the diuresis in patients with low-renin hypertension, resistant hypertension, congestive HF or refractory edema (174). A recent study found that in patients with rosiglitazone-induced plasma volume retention and type 2 diabetes amiloride prevents protracted fluid retention, while hematocrit continues to decrease significantly patients treated with spironolactone, suggesting continued PV expansion (175). Amiloride is effective in patients with polyuria and polydipsia due to Li+-induced nephrogenic diabetes insipidus and in patients with Liddle syndrome and in patients with some ENaC mutations (176,177). In patients with chronic HF amiloride increases serum K+ levels (0.4 mmol/L), shortens the corrected QT interval and reduces QT dispersion and ventricular extrasystoles but, unlike spironolactone, amiloride does not improve endothelial dysfunction, heart rate variability or myocardial fibrosis (178).
28 Figure legends Figure 1. (A) Pathways for the synthesis of aldosterone in the adrenal cortex. Final steps of the synthesis involve the cytochrome P450 enzyme aldosterone synthase (CYP11B2). (B) Physiopathological effects of aldosterone. Abbreviations: ACTH, adrenocorticotropic hormone; Ang II, angiotensin II; ANP, atrial natriuretic peptide; ASI: aldosterone synthase inhibitors; CKD: chronic kidney disease; ET-1: endothelin-1; HRV: heart rate variability; MR: mineralocorticoid receptor; MRA: mineralocorticoid receptor antagonists; NE: norepinephrine; PAI-1: plasminogen activator inhibitor type 1; ROS: reactive oxygen species.
29
30 Table 1. Pharmacological modulation of aldosterone 1. Mineralocoticoid receptor antagonists (MRAs) • Steroidal compounds o First generation: spironolactone and canrenone o Second generation: eplerenone, prorenone*, mespirenone*, spirorenone*, drospirenone* • Non-steroidal compounds (third/fourth generation): BAY-94-8862, BR-4628, CS-3150, LY-2623091, MT-3995, PF-3882845, SM-368229 2. Aldosterone synthase (CYP11B2) inhibitors: Fadrozole, FAD286A, LCI699, some 1,4dihydropyridine Ca2+ channel blockers 3. Epithelial Na+ channel (ENaC) blockers: amiloride and triamterene
31 Table 2. Pharmacologic differences between the agents may be useful to inform drug selection for individual patients (IC50/EC50 values in nM) Paramater Chemical class Affinity for MR (μM)* AR PR GC Mode of antagonism Receptor conformation** Cofactor recruitment** Bioavailability (%) Onset of effect (h) Peak effects (h) Plasma protein binding (%) Vd (L/kg) Half-life (h) Duration of the effect (h) Metabolism
Drug interactions
Spironolactone
Eplerenone
BR-4628
Steroidal
Steroidal
24 77 740 2410
990 21240 31210 21980
Nonsteroidal 28 4440 9020 5470
Passive Partial stabilization of active MR conformation No recruitment of co-repressors 80-90 (100 with food) 24-48 48-72 > 90
Passive Stabilization of inert conformation
10 1.4 (active metabolites 1235) 72-120
0.6-1.3 4-6
Hepatic, multiple active metabolites)
70
Amilori de Pyrazin e
Triamte rene
15-25
50
Bulky Destabilizat or No cofactor recruitment 94
24 1.5 50
2 4-6 -
2-4 3 60-70
5-7 6-9
2.5
72 h
24
7-9
Hepatic, via CYP3A4, no active metabolites Multiple
No
8.5
Drugs and foods Drugs and foods increasing serum that raise serum K+ levels + K levels Renal excretion (%)
Comparison Of Agents That Affect Aldosterone Action Juan Tamargo, Anna Solini, Luis M Ruilope
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S0270-9295(14)00047-3 http://dx.doi.org/10.1016/j.semnephrol.2014.04.005 YSNEP50775
To appear in: Semin Nephrol
Cite this article as: Juan Tamargo, Anna Solini, Luis M Ruilope, Comparison Of Agents That Affect Aldosterone Action, Semin Nephrol , http://dx.doi.org/10.1016/j.semnephrol.2014.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 COMPARISON OF AGENTS THAT AFFECT ALDOSTERONE ACTION Juan Tamargo1, Anna Solini2, Luis M Ruilope3
1- Department of Pharmacology, School of Medicine, University Complutense, Madrid. 2- Department of Clinical and experimental Medicine, University of Pisa 3- Research Institute, Hypertension Unit, Hospital 12 de Octubre & Department of Public Health and Preventive Medicine, University Autonoma, Madrid
KEY WORDS: Aldosterone, aldosterone receptor antagonists, spironolactone, eplerenone, canrenone, aldosterone synthase inhibitors, hypertension, hyperaldosteronism, heart failure Address of autor to maintain correspondence: Luis M Ruilope Instituto de Investigación Unidad de Hipertensión Hospital 12 de Octubre 28041, Madrid SPAIN Acknowledgements: This work was supported by Instituto de Salud Carlos III (Red RIC, and PI11/01030) and Comunidad de Madrid (S2010/BMD-2374). INTRODUCTION For many years aldosterone was considered as a hormone devoted to the control of renal excretion of minerals, which explains the term mineralocorticoid used to describe this hormone. In two clinical situations, primary and secondary hyperaldosteronism were initially considered for the clinical use of the first aldosterone receptor blocker spironolactone as a diuretic. The discovery of many extra-renal sites of mineralocorticoid receptors (1,2) and the investigation of the pro-inflammatory and fibrogenic effects of the hormone (3) expanded the knowledge of the capacities of aldosterone to directly participate in the pathogenesis of arterial hypertension (4), heart failure (HF)(5), chronic kidney disease (CKD)(6) and metabolic syndrome (7). Eplerenone the second most widely used blocker of aldosterone receptors arrived when the role of these drugs as diuretics had been expanded to cardiorenal
2 and metabolic disease. Data with canrenone a third blocker of the receptor are scanty. Finally two other substances have been shown to antagonize the actions of aldosterone in the renal tubule, amiloride and triamterene and they continue to be used as diuretics. In this paper we will review the role of aldosterone antagonists in aldosteronism, cardiorenal disease and metabolic disease and we will refer in particular to the similarities and differences of the two main members of the class spironolactone and eplerenone. Finally we will comment on the new members of the class of aldosterone antagonists and their potential advantages. EFFECTS OF ALDOSTERONE Aldosterone, the final product of the renin-angiotensin-aldosterone system (RAAS), is a mineralocorticoid hormone secreted from the adrenal zona glomerulosa isolated and characterized in 1953 (8). Untill the mid-90s, it was thought that aldosterone acts primarily in the epithelial cells of the late renal distal convoluted tubule and the collecting duct, distal colon and salivary and sweat glands leading to an increase in the net reabsorption of Na+ and water and K+ excretion. The reabsorption of Na+ and water then elevates BP indirectly by expanding extracellular fluid volume. These effects are mediated by the binding of aldosterone to mineralocorticoid receptors (MR, NR3C2), a ligand-activated transcriptor factor belonging to the nuclear receptor superfamily (8). However, following the molecular cloning of the MR in 1987, both experimental and clinical evidence demonstrated that MRs are also present in a wide range of tissues, including endothelial and vascular smooth muscle cells, cardiac tissues (cardiomyocytes, fibroblasts and macrophages), kidney (mesangial cells and podocytes), adipocytes, monocytes and brain(2,3,9). Activation of MRs by aldosterone promotes multiple renal, cardiac and vascular deletereous effects, including endothelial dysfunction, hypertension, neurohumoral activation, cardiovascular (CV) and renal remodeling (hypertrophy, fibrosis, apoptosis), decreases arterial compliance, increases expression of cell adhesion molecules, platelet activation, plasminogen activator inhibitor type 1 (PAI-1) activity and oxidative stress [by both nicotinamide adenine dinucleotide phosphate, NADP(H), oxidase activity and mitochondria) and exerts proarrhythmic and proinflammatory effects (Figure 1)(1,3,5,10-12). Additionally, activation of central MRs increases central sympathetic tone to the kidneys, heart, and vascular smooth muscles and vasopressin release and decreases baroreceptor sensitivity. Moreover, aldosterone, via MR activation upregulates ACE expression in the cardiomyocytes, suggesting the existence of a positive feedback
3 pathway that activates the renin-angiotensin-aldosterone system (13). These effects are reported to be “genomic”, i.e. dependent on transcription and translation. Additionally, aldosterone produces rapid, translation- and transcription-independent effects (non-genomic effects) that may be mediated by G-protein-coupled receptor 30 (GPR30) and transactivation of the epithelial growth factor receptor (EGFR) (3). These effects have been described in vascular smooth muscle cells (VSMCs) and other tissues where aldosterone induces a rapid increase in Na+ influx (aldosterone increases Na+/K+/2Cl- cotransporter activity and Na+-H+ exchange, and inhibits Na+/K+ ATPase activity) and intracellular Ca2+ concentrations through an increase in Ca2+ entry through voltage-gated channels, and activates cAMP-protein kinase A, phospholipase C, phosphatidylinositol 3-kinase (PI3 kinase), diacylglycerol and protein kinase C (PKC) signaling pathways (3,14,15). Aldosterone also induces a rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) 1/2 kinase in VSMCs, endothelial and kidney cells, to promote a mitogenic and profibrotic phenotype, an effect that involves the transactivation of the EGFR (3,14,15). The non-genomic effects occur also independently of hemodynamic factors and play an important role in the mechanisms by which aldosterone contribute to endothelial dysfunction, vasoconstriction, resistant arterial hypertension, CV and renal remodeling, inflammation, heart failure, insulin resistance and chronic renal disease (CKD) (1,3,5,7,14-18). Interestingly, in VSMC, aldosterone activates both GPR30 and MR to mediate vasodilatation and apoptosis and to activate PI3 kinase, ERK, and myosin light chain phosphorylation (15). In contrast endothelial MR activation has been linked to enhanced vasoconstrictor and/or impaired vasodilator responses. The MR has highest homology to glucocorticoid receptor (GR), and both receptors are expressed together in aldosterone target cells. In epithelial cells (kidney, endothelial cells, colon), aldosterone selectivity is determined by the 11-β-hydroxysteroid dehydrogenase 2 (11β-HSD2), an enzyme colocalized with the MR which converts the active cortisol to the MR-inactive cortisone, but does not degradate aldosterone. However, other tissues (i.e., cardiomyocytes, some regions of the brain) lack 11β-HSD2, so that cortisol is the primary ligand for the MR (1). Thus, the tissue specificity of aldosterone is determined in part by the presence of 11β-HSD2. Although aldosterone and cortisol bind the MR has equal affinity, the aldosterone/MR complex is more stable, which in turn mediates a much stronger (200-fold) transactivation response than the cortisol/MR complex (19). Whether the deleterious events mediated via MR activation are mediated by aldosterone and/or cortisol at conditions of inappropriate salt or redox status is a matter of discussion (1).
4 All these results confirm the aldosterone via MR-dependent and MR-independent mechansims plays a pivotal role as a regulator of cellular and organ function, far beyond its effects on the kidney, and is directly involved in target organ damage in various CV and renal diseases [3,11]. This is the pharmacological rationale for the development of aldosterone antagonists for the treatment of CV diseases (Table 1). PHARMACOLOGICAL MODULATION OF ALDOSTERONE 1. MINERALOCORTICOID RECEPTOR ANTAGONISTS (MRAs) Three steroidal MRAs are presently in the market: spironolactone, eplerenone, and canrenoate (Table 1). Spironolactone and eplerenone have been widely studied in large randomized controlled trials (RCTs) (20-22), while potassium canrenoate (canrenone), an active metabolite of spironolactone, is available in some countries. Novel non-steroidal compounds are presently in preclinical and early clinical development. Spironolactone, the first MRA, was launched in 1960 as a K+-sparing diuretic with a complementary mode of action to that of the diuretics currently used for the management of edematous conditions, primary aldosteronism and essential hypertension. Spironolactone is a potent, competitive, non-selective MRA, as it also blocks androgen receptors and activates progesterone receptors causing progestogenic and antiandrogenic adverse effects limiting its use (23). This explains why its long-term use is associated with gynecomastia and other endocrine adverse effects, such as breast tenderness, impotence, loss of libido and menstrual irregularities (23-26). These adverse effects and the lack of selectivity for the MR stimulated the search for more selective and better-tolerated MRAs. Eplerenone, a 9,11-epoxy derivative of spironolactone, launched in 2002 for the treatment of congestive HF, represents the second generation of MRAs. It presents a higher selectivity for the MR and ~500 times lower affinity for androgen and progesterone receptors than spironolactone (27,28), leading to negligible sexual-related adverse effects (29). Thus, eplerenone represented an alternative in patients with spironolactone-induced sexual adverse effects (30,31). Despite its increased selectivity, in in vitro competition binding assays eplerenone exhibits a 10- to 20-fold lower affinity for MR compared with spironolactone, suggesting that eplerenone has 1 to 2 times the potency of spironolactone. The third generation of MRAs is represented by new non-steroidal, potent, selective compounds, while the fourth generation is based on third generation molecules with renal-sparing properties and in the near future should include tissue-specific MR antagonists (16).
5 Although spironolactone and eplerenone are effective and improve clinical outcomes in patients with hypertension and HF, there are important differences in their mode of action, pharmacodynamic and pharmacokinetic profile, adverse effects and drug interactions which may be relevant for differentiating these drugs in a given patient (25,29,32,33). 1.1. Mechanism of action Aldosterone initiate its actions by binding to the ligand binding domain of the MR located in the cytoplasm, where MR are rendered transcriptionally inactive by several chaperone proteins like the 90-kDa heat shock protein. Aldosterone binding induces a conformational change in the MR resulting in dissociation of chaperone proteins, translocation of the aldosterone-MR complex to the nucleus and its binding to hormone response elements in the regulatory region of target gene promotors triggering the transcription of target genes (3,34). Spironolactone and eplerenone competitively inhibit aldosterone binding to the MR and render it transcriptionally inactive. As a consequence, they block the effects mediated via MR activation regardless of the ligand. However, spironolactone or eplerenone do not antagonize aldosterone-induced non-genomic renal and extrarenal effects (1,16). The MR consists of three principal domains: N-terminal, DNA-binding and ligand-binding (LBD) domains (8). The tertiary structure of the LBD is highly conserved and consists of 12 α-helices and 3 β strands folded into an anti-parallel three layer with a ligand-binding pocket in the lower third of the molecule (16). In the presence of aldosterone helix 12 is repositioned together with helices 3, 4 and 5 to form a hydrophobic groove on the surface of the LBD which allows binding of co-activator molecules with a leucine-rich LxxLL motif and subsequent MR activation (35). This function in the LBD is termed activation function 2 (AF-2) and is ligand dependent. Aldosterone-occupied MR recruits specific coactivator peptides that are needed to increase transcription and to stabilize the complex in its activated state (34). Coactivators and co-represors are cellular factors which interact with the MR to potentiate or attenuate transactivation, so that the ratio of coactivators to co-represors bound to the MR-ligand complex modulates the transcriptional sensitivity of the MR (36). There are important differences between spironolactone and eplerenone in the binding mode to MR and consequences for MR stability, nuclear translocation and co-factor recruitment. The mechanism by which antagonist ligands inactivate the MR is based on the instability of the antagonist-MR complexes, rather than on the ability of the antagonist to stabilize the MR in an inactive state, favoring the recruitment of co-repressors (37,38). However, the mechanisms of MR antagonism produced by spironolactone and eplerenone may occur
6 through different mechanisms. Spironolactone produces a “passive” antagonism. The drug binds and prevents MR from adopting the active conformation by failing to mediate hydrogen bonding to Asn770 and Thr945 and both helix 3 and the AF-2 helix are not arranged in the proper position to allow efficient binding of co-activators of transcription (35,39). Eplerenone binds to the LBD and antagonizes aldosterone-induced MR activation and dosedependently blocks the interaction of aldosterone with coactivator peptides (38). However, eplerenone does not actively recruit co-represors (possibly because it has no influence on the conformation of helix 12) or stabilizes a repressive conformation that allows corepressor binding; instead, it stabilizes a neutral conformation that is transcriptionally inert, perhaps similar to the unliganded MR conformation. 1.2. Pharmacodynamics Spironolactone was initially licensed as a potassium-sparing diuretic for the treatment of hypertension, primary hyperaldosteronism, volume-overload states and hypokalemia. In an early study in 182 men treated with spironolactone alone (mean dose 96.5 mg daily) during 23 months the reduction in SBP/DBP averaged 18/11 mm Hg (24). Nowadays, MRAs as antihypertensive drugs are preferentially indicated in the treatment of primary and secondary hyperaldosteronism, but their use can be contemplated in hypertensive patients with metabolic syndrome or target organ damage represented by left ventricular (LV) hypertrophy or albuminuria (17,18,40) or particularly as add-on therapy in patients with resistant hypertension where MRAs have shown positive results (41-43) On the other hand, MRAs are especifically indicated in patients with chronic congestive HF with diminished ejection fraction (5) and in post-MI as shown by the Ephesus (Epleronone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) trial (21). Spironolactone is also indicated as a diuretic in patients with cirrhosis and ascites, in whom secondary hyperaldosteronism is present, and hypokalemia is detrimental (44) 1.2.1. Primary hyperaldosteronism. Primary aldosteronism (PA) is characterized by arterial hypertension frequently accompanied by hypokalemia and probably constitutes the most frequent cause of secondary hypertension after chronic kidney disease (45). It ussually is accompanied by a high prevalence of cardiovascular and renal damage leading to an increased morbidity and mortality (46). Spironolactone is effective in the treatment of PA as short-term preoperative treatment or long-term treatment in patients with unilateral aldosterone-producing adenomas with high operative risks or who decline surgery and in patients with unilateral or bilateral (usually idiopathic adrenal hyperplasia) disease who are
7 unable or unwilling to undergo adrenalectomy (46,47) Spironolactone lowers BP to a similar extent in hypertensive patients with and without primary aldosteronism, although higher doses are required in those with PA (24,43,47) reduces CV and renal complications, decreases LV hypertrophy and urinary protein excretion and corrects hypokalemia and abnormalities of glucose metabolism (hypersinsulinemia and insulin resistance) (46,47). The long-term CV outcomes were studied in patients with PA after treatment with adrenalectomy or spironolactone (48). At baseline, the prevalence of cardiovascular events was greater in PA than in essential hypertension (35% vs 11%, P< .001). During a follow-up for a mean of 7.4 years, BP and CV outcomes were comparable in the two treatments, indicating that adrenalectomy and spironolactone are equally effective in preventing CV outcomes. Both treatments improve intrarenal hemodynamic pattern while protecting glomerular filtration rate and decreasing urinary albumin excretion (49,50). The incidence of LV hypertrophy in PA is higher than in essential hypertension and spironolactone and adrenalectomy exert similar positive influences facilitating the regression of LVH (33) but this is partly independent of changes in BP (51,52). The comparison of eplerenone and spironolactone in patients with PA led to contradictory results. In an open-label study, spironolactone (50 to 400 mg/day) and eplerenone (50-200 mg/day) produced a similar BP reduction BP (53), while in another double-blind study the decrease in SBP/DBP was greater in patients treated with spironolactone (75-225 mg/day) than with eplerenone (100-300 mg/day), although more patients randomized to spironolactone develop gynecomastia and mastodynia (54). In this latter study, despite a higher incidence of gynecomastia/mastodynia and hyperkalemia in the spironolactone group, the overall incidence of adverse events is comparable in both treatment groups. The natriuretic effects of spironolactone and eplerenone are greater in patients with hyperaldosteronism. 1.2.2. Arterial hypertension. Aldosterone acts as a downstream effector of the RAAS and an excessive aldosterone production is a significant cause of hypertension. In fact, an increase in plasma aldosterone accompanied by low renin levels predicts the development of arterial hypertension in middle-aged caucasian population (55,56). On the other hand, an increased aldosterone:renin ratio indicative of the existence of primary aldosteronism is found in 1020% of patients diagnosed with resistant hypertension (57). In these patients there is evidence of intravascular volume expansion, particularly in men, and the significant correlation between 24-hour urinary aldosterone levels and cortisol excretion suggests that a common stimulus, such as corticotropin, may underlie the aldosterone excess in patients with resistant
8 hypertension (57). MRAs are effective in mild-to-moderate hypertension (24,58) but are particularly effective in patients with low-renin or resistant hypertension (42,43,59). They are also effective in reducing BP in hypertensives treated with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) presenting elevated aldosterone plasma levels as a consequence of the aldosterone breakthrough that represents a situation of escape to the effect of the renin-angiotensin blockade after long-term therapy (60). This escape may contribute to the development of resistant hypertension and in fact beyond those cases with primary aldosteronism a high proportion of resistant hypertensives present with elevated plasma aldosterone levels (57). A Cochrane meta-analysis of 5 cross-over studies found that spironolactone significantly reduces SBP/SBP by 20/6.7 mmHg in patients with primary hypertension when doses of 100500 mg/day are given. One of the studies showed that spironolactone 25 mg/day did not significantly change BP as compared to placebo (61). Because of the possible increase of adverse effects at doses >50 mg/day, in patients with primary hypertension spironolactone is frequently prescribed at lower doses but in combination with a thiazide-like or loop diuretic (sequential nephron blockade) to prevent hypokalemia and/or hypomagnesemia and to produce additional antihypertensive and/or natriuretic effects (26). On the contrary, in resistant hypertension adding a low-dose of spironolactone (12.5-25 mg/d) to a multidrug regimen, that includes a diuretic and an ACEI or an ARB, in patients with resistant hypertension produces a mean decrease in SBP/DBP 21/10 mm Hg and 25/12 mm Hg at 6 weeks and 6 months treatment, respectively (62). A similar reduction in SBP/DBP (21.7/8.5 mm Hg) is observed in following the addition of spironolactone (25-50 mg) in patients with resistant hypertension receiving simultaneously an angiotensin-blocking drug (63). In the ASCOT (Anglo-Scandinavian Cardiac Outcomes Trial) trial spironolactone (mean dose of 25 mg/day) reduced SBP/DBP by 21.9/9.5 mm Hg in patients with uncontrolled hypertension and this reduction was unaffected by age, sex, and diabetic status (43). In this study, 6% of patients discontinue the drug due to adverse effects (gynecomastia and hyperkalemia). Eplerenone is also an effective antihypertensive drug (64). In hypertensive patients uncontrolled with an ACEI or an ARB, eplerenone (25, 50 and 200 mg bid) and spironolactone (50, 100, or 400 mg od) decrease dose-dependently SBP/DBP during the 24-h period (58). However, the mg-for-mg BP-lowering effect of eplerenone is lower than that of spironolactone, so that 200 mg eplerenone bid (or 400 mg od) produces a BP reduction comparable to that produced by 50 mg spironolactone bid (i.e., spironolactone produces 1.3– 2 times greater BP reduction). Eplerenone (50 mg daily) is more effective than losartan (50
9 mg daily) in black patients that respond relatively poorly to ACEI or ARB, in the low-renin patients and in patients with differing baseline levels of aldosterone; however, eplerenone is as effective as losartan in white and high-renin patients (65). Eplerenone is more effective than losartan in reducing SBP/DBP in patients with low-renin hypertension; after 16 weeks of therapy, significantly fewer eplerenone-treated patients than losartan-treated patients (32.5% vs 55.6%) require the addition of hydrochlorothiazide (59). Eplerenone is as effective as amlodipine or enalapril in lowering SBP (3,66) and its combination with othere antihypertensive drugs provides an additional benefit in patients uncontrolled in monotherapy (67). In patients with hypertension and left ventricular hypertrophy, the 4E study demonstrated that eplerenone (200 mg/day) is as effective as enalapril (40 mg/day) in lowering SBP and LV hypertrophy (40) and their combination more effective than eplerenone alone. This trial supports that blockade of the MR is an effective method of reducing end-organ damage. The use of eplerenone in resistant hypertension at doses of 50 up-titrated to 100 mg/d showed a fall in office BP of -17.6/-7.9 mmHg and -12.2/-6.0 in ABPM values (68). However, the effects of spironolactone and eplerenone on clinical outcomes in patients with refractory hypertension are unknown. In a recent study, patients with resistant hypertension despite 4 weeks of treatment with triple therapy (irbesartan 300 mg/day, hydrochlorothiazide 12.5 mg/day and amlodipine 5 mg/day) were randomized to sequential nephron blockade (spironolactone 25 mg/day followed by furosemide 20 mg/day, furosemide 40 mg/day and amiloride 5 mg/day) or sequential RAAS blockade (ramipril 5 mg/day followed by ramipril 10 mg/day, bisoprolol 5 mg/day and bisoprolol 10 mg/day) (69). At week 12, the mean difference in daytime ambulatory BP was 10/4 mmHg in favour of the sequential nephron blockade. The BP goal (daytime ambulatory BP 50 mg/day), drugs producing hyperkalemia (Table 3) or K+ supplements (5,25,26,111,135). A small study identified that the polymorphism NR3C2 215G is associated with a higher risk of potassium increases >0.5 mEq/L when spironolactone is started in patients with HF (136). The
risk
of
hyperkalemia
decreases
by
ensuring
that
dietary
(http://www.ars.usda.gov/ba/bhnrc/ndl) and other pharmacologic sources of K+ (Table 3) are minimized. The combination of a MRA with a K+-losing diuretic may lower the baseline serum K+ value, but this combination should be used only if diuretic-related volume losses do not reduce the GFR, an effect that can significantly reduce urinary K+ excretion (25,26). A recent pilot study found that RLY5016, a non-absorbed, orally active, K+-binding polymer, prevents hyperkalemia and is relatively well tolerated in patients at risk of developing hyperkalemia, receiving standard therapy for HF and spironolactone (25–50 mg/day) (137). Thus, close monitoring of plasma K+ levels is recommended when spironolactone or eplerenone are added in patients treated with ACEIs/ARBs, particularly in patients with moderate-severe CKD (GFR 75 years (153). Furthermore, the reduction in SBP and the rise in serum aldosterone levels are smaller in patients receiving BAY 94-8862 than in patients receiving spironolactone. However, it remains to be proven that BAY 94-8862 is as effective as spironolactone or eplerenone in the prevention of major CV events. The ongoing phase IIb ARTS-HF trial (NCT01807221) compares the efficacy and safety of BAY94-8862 and
22 eplerenone in patients hospitalized with worsening chronic heart failure and LV systolic dysfunction and either type 2 diabetes with or without CKD or CKD alone. Thus, BAY 948862 may represent the first drug of the fourth generation of MRAs that can achieve a CV benefit without or at least with fewer renal adverse effects than spironolactone or eplerenone. We have already mentioned that the fourth generation of MRAs should present a renalsparing profile-. However, they should not completely spare renal effects as this might lead to a new condition characterized by hypokalemia (which can be more dangerous than hyperkalemia) and Na+ retention among patients with PA (1). Thus, a combined renal Na+ excretion and a mild K+ retention are clearly beneficial and demanded characteristics (130). A further step will be the development of tissue-selective MRAs, i.e., drugs with higher higher cardiovascular/renal ratio, as compared with available steroidal MRAs. The basis for this approach is the evidence that a heterogeneous group of non-receptor proteins, termed coregulators, are required to enhance or repress nuclear receptor-mediated transactivation of target genes (36). Certain coregulators would be expected to confer specificity to MRmediated responses because of their variable tissue expression and selectivity for different ligands. Thus, the interaction of novel MRAs with coregulator molecules that coordinate the specific interaction between the MR and their genes in a tissue-specific manner may allow modulation of MR activity in a ligand- and tissue-specific manner (while retaining normal MR function in other tissues) leading to the development of compounds with important overall pharmacodynamic differences (36,130). This tissue selectivity can also be reached by improving the physicochemical properties that influence plasma transport and tissue penetration of the new compounds in different tissues and the differential affinity of a MR– drug complex for tissue-specific coregulators. Whether this proposal is not an unrealistic idea or a pharmacological target available in the near future will depend on the coordinated efforts of academic, clinical and industrial research. 3. ALDOSTERONE SYNTHASE INHIBITORS Another approach to reduce aldosterone plasma and tissular levels and antagonize its effects mediated via MR-dependent and MR-independent pathways is to inhibit the enzyme aldosterone synthase (CYP11B2), a member of the cytochrome P450 family encoded by the CYP11B2 gene, that catalyses a rate-limiting step of aldosterone synthesis (154,155). This enzyme is activated by angiotensin II and K+ levels, and to a minor extent by adrenocorticotropin (ACTH), vasopressin, catecholamines, ET-1, serotonin, cytokines and Mg2+ (11,156)(Figure).
23 In contrast to MRAs, aldosterone synthase inhibitors (ASIs) prevent the reactive increase in aldosterone levels in response to ACEIs/ARBs and inhibit both the MR-dependent and MRindependent effects exerted by aldosterone in target organs (3,15). Thus, ASIs have emerged as a therapeutic alternative to MRAs, but their challenge is to demonstrate that they are as effective, but better tolerated, than MRAs for the treatment of hypertension, HF and renal disorders (157). However, because of the high sequence homology of CYP11B2 and CYP11B1, the 11-β-hydroxyase responsible for the final step in cortisol synthesis, ASIs might also suppress cortisol release. 3.1. FAD 286A, the enantiomer of the nonsteroidal aromatase inhibitor fadrozole, inhibits human recombinant CYP11B1 (IC50 9.9) and CYP11B2 (IC50 1.6 nM) and angiotensin IIstimulated aldosterone synthesis in NCI-H295R human adrenocortical carcinoma cells (155). FAD 286A decreases urinary free aldosterone levels in spontaneously hypertensive rats on a low sodium-high potassium (LS, high urinary aldosterone levels) or a high sodium-normal potassium (HS, low urinary aldosterone level) and increases plasma renin levels only in rats on LS (158). The combination of FAD 286A and spironolactone on the LS diet induces severe hypoaldosteronism, whereas the combination of FAD 286A and furosemide on the HS diet corrects the diuretic-induced hypokalemia. In rats overexpressing both the human renin and angiotensinogen genes (dTGR), FAD 286A inhibits CYP11B2, reduces circulating and cardiac aldosterone levels and improves endothelial function, cardiac and renal target-organ damage (decreases cardiac hypertrophy, albuminuria, cardiac and renal inflammation and matrix deposition) and survival. However, FAD 286A only slightly reduces BP, while losartan normalizes BP (155). In uninephrectomized rats on a HS diet angiotensin II causes albuminuria, azotemia, renovascular hypertrophy, glomerular injury and increases PAI-1 and osteopontin mRNA expression and tubulo-interstitial fibrosis in the kidney. FAD286 and spironolactone prevent these renal effects and attenuate cardiac hypertrophy and fibrosis (159). Furthermore, in a rat model of heart failure, FAD 286A and spironolactone improve LV hemodinamics, remodeling and function, but only FAD 286A normalizes LV oxidative stress and coronary endothelium-dependent vasodilatation without changes in BP (160). FAD 286A also reduces the severity of atherosclerotic plaques and the expression of proinflammatory markers in apolipoprotein E-deficient mice without affecting plasma aldosterone levels (161). These results indicate that ASIs might be a potential therapeutic stratagy for the treatment of HF probably by targeting MR-independent effects of aldosterone (15).
24 3.2. LCI699 is an orally active non-selective ASI structurally derived from FAD286 that also inhibits CYP11B1. In a Phase I study in healthy male subjects on a controlled salt diet, LCI699 causes a dose-dependent decrease in plasma and urinary aldosterone (up to 70 or 80%) without affecting basal cortisol levels compared with placebo, increases in plasma renin activity ansd is well tolerated (162). However, due to its short plasma half-life, the decrease in plasma aldosterone levels does not persist for 24 h. At the dose of 0.5 mg od, LCI699 selectively inhibits aldosterone synthesis, but not cortisol synthesis, even after ACTH stimulation, but CYP11B2 selectivity is lost at doses ≥1 mg od, and at doses ≥3 mg LCI699 blunts the cortisol response to and ACTH test. Four phase II trials study the efficacy and safety of LCI699 (163). The first study compared LCI699 (0.25, 0.5 and 1 mg od and 0.5 mg bid.) and eplerenone (50 mg bid) with placebo in hypertensive patients (164). All doses LCI699 significantly decreases 24-h ambulatory BP compared with placebo and the dose of 1 mg reduces mean sitting DBP to a similar extent to eplerenone. Paradoxically, LCI699 0.5 mg bid reduces BP less that the dose of 1 mg od, even though the 0.5 mg bid dose produces a greater inhibitory effect on aldosterone synthesis; this finding was attributed to increases in precursor mineralocorticoids. LCI699 is rapidly absorbed, reaches peak plasma concentrations after 1 h and presents an apparent half life of 3.8-5.5 h and does not accumulate following the administration of multiple doses up to 3 mg. As expected, LCI699 twice-daily regimen decreases, while eplerenone increases plasma aldosterone levels; however, both drugs increase plasma renin activity and serum K+, and decreases serum Na+. Although LCI699 has no effect on basal cortisol concentrations, it blunts ACTH stimulation of cortisol in ≈20% of subjects receiving 1.0 mg od. Another study analyzed the effects of LCI699 (0.5, 1 and 2 mg od or 1 mg bid) on cortisol in hypertensive patients (165). LCI699 produces a dose-dependent SBP/DBP reduction, reduces PALs (5080%) and suppresses ACTH-stimulated cortisol response. Another trial compared LCI699 (0.25 bid, 1 mg od and 0.5/1 mg bid) and eplerenone (50 mg bid) as an add-on therapy in patients with resistant hypertension. LCI699 lowers BP modestly in these patients, the BP response being smaller than with eplerenone and not statistically significant vs placebo (166). LCI699 suppresses plasma aldosterone levels in a dose-related manner, with corresponding dose-dependent increases in plasma renin activity and 11-deoxycorticosterone levels. The authors hypothesized that higher doses of CLI699 are necessary to reduce BP in patients with resistant hypertension, but such doses cannot be tested due to loss of steroidogenic target selectivity.
25 In patients with PA, LCI699 (0.5 mg b.i.d. for 2 weeks, force-titrated to 1 mg b.i.d. for 2 weeks) produces a modest decrease in BP (-4.1 mmHg), does not modify basal plasma cortisol levels, but decreases plasma and urinary aldosterone levels (∼70–80%), normalizes hypokalemia and increases plasma renin, ACTH (35%) and 11-deoxycorticosterone levels (> 700%) (167). The same group compared the effects of LCI699 (0.5-1 mg bid) and eplerenone (50 to 100 mg bid) in patients with PA (157). After 4 weeks of treatment, LCI699 was less effective in reducing BP than eplerenone, even though LCI699 markedly decreased (75%), whereas eplerenone increased plasma aldosterone levels (89%). In all these studies the clinical and biological safety and tolerability of LCI699 are similar to those of placebo and eplerenone and no patients require intervention for adrenal insufficiency. These studies reveal that LCI699 interferes with two endocrine feedback loops at the adrenal gland (163). First, inhibition of CYP11B2 reduces aldosterone levels, leading to the stimulation of the RAAS feedback axis, a slight decrease in plasma Na+ and an increase in plasma K+ levels and plasma renin levels and activity. Second, the increase in 11deoxycorticosterone levels in the presence of stable cortisol levels confirms the inhibitory effect of LCI699 of the 11β-hydroxylase in the adrenal gland and explains why the plasma cortisol response to an ACTH test is significantly blunted. The resultant stimulation of the hypothalamic-pituitary-adrenal feedback axis induces the compensatory increase of endogenous ACTH levels that stimulates adrenal steroidogenesis to compensate for the inhibited cortisol secretion and leads to a supraphysiological increase of 11deoxycorticosterone, the precursor of cortisol, consistent with the inhibition of the CYP11B1 gene product. This increase may explain the observed disappointing BP reductions achieved with the compound at higher doses and particularly upon twice-daily administration (163). The poor antihypertensive effect of LCI699 in patients with PA or resistant hypertension, its short half-life and the lack of selectivity for CYP11B2 that limit the use of a higher doses range indicate that LCI699 is not an attractive alternative to replace MRAs in patients with hypertension or other CV and renal disorders and support the development of secondgeneration compounds with much higher selectivity for CYP11B2. Very recently, several N(Pyridin-3-yl)benzamides derived from metapyrone were found to be potent and selective CYP11B2 inhibitors (IC50 values 53–166 nM) (168). Despite all these disadvantages, LCI699 opens new possibilities for treating patients with glucocorticoid excess, such as Cushing’s disease (168). Moreover, selective ASIs may be complementary or an alternative in patients with PA or hepatic cirrhosis that require high
26 doses of spironolactone (≥50 mg daily) that are associated with endocrine side-effects. Furthermore, the addition of a selective ASI in patients with hypertension or HF treated with low doses of spironolactone or eplerenone may allow a better dose-dependent normalization of plasma aldosterone and K+ levels. However, the development of new selective and potent ASIs presents several important problems because (130): 1) the degree of CYP11B2 inhibition required to neutralize the pathological, but not the physiological, effects of aldosterone is unknown; 2) they can produce an increase in 11-deoxycorticosterone that might act as a substitute for aldosterone on the MR; 3) the second generation of ASIs have to compete with the new potent and selective non-steroidal MRAs (147) and, 4) ASIs present some adverse events similar to those of MRAs, including electrolyte disorders, hypotension, renal insufficiency and hypoaldosteronism, and drug interactions with K+ retaining drugs. 3.3. There is evidence that some dihydropyridine Ca2+ channel blockers (i.e., azelnidipine, benidipine and efonidipine) exhibit inhibitory actions on adrenal aldosterone synthesis by decreasing the expression of CYP11B1 and CYP11B2, while cilnidipine suppresses Ang IIinduced CYP11B2 (but not CYP11B1) mRNA expression (157,164,169-171). These findings can be the basis for the development of new antihypertensive drugs that block L-type Ca2+ channels, inhibit CYP11B2 and antagonize MR. Drugs with dual or triple action on these targets might represent a novel approach to reduce BP and to prevent/treat hypertensive endorgan damage with less risk of hyperkalemia. 3.4. MicroRNAs (miRNAs) are endogenous, single-stranded non-protein-coding RNA molecules of ≈22 nucleotides that post-transcriptionally control gene expression, interfering with mRNA stability and/or translational rate, by base-pairing to the 3´UTR of proteincoding mRNAs. Screening of non-diseased human adrenal and aldosterone-producing adenoma samples yields to distinctive miRNA expression signatures for each tissue type. Bioinformatic analysis identifies putative binding sites for several miRNA in the 3' untranslated region of CYP11B1 and CYP11B2 mRNAs and in vitro manipulation of miR-24 confirms the ability of miR-24 to target both CYP11B1 and CYP11B2 mRNAs directly, decreasing aldosterone and cortisol production in adrenocortical cells (172). Thus, adrenal miRNA may represent a novel target for the therapeutic manipulation of mineralocorticoid biosynthesis. 4. EPITHELIAL NA+ CHANNEL BLOCKERS
27 Amiloride and triamterene block epithelial Na+ channels (EnaC) channels located in the luminal membrane of the principal cells of the distal tubule and collecting duct, perhaps by competing with Na+ for negative charges within the channel pore (173). The reduction in Na+ reabsorption hyperpolarizes the tubular apical membrane and reduces the electrochemical gradient K+ secretion from the principal cell and H+ secretion via the H+-ATPAse from the intercalated cell. Additionally, the reduction in K+ secretion decreases H+ secretion via K+/H+ ATPase. The net effect is an increase in Na+ excretion and a decrease in K+ and H+ excretion. However, in contrast to MRAs, this K+ retention is independent of aldosterone. Both drugs are filtered at the glomerulus and secreted by the organic cation secretory pathway into the proximal tubule. Amiloride presents low oral bioavailability, is not metabolized in the liver and is excreted unchanged in the urine, so that CKD prolongs its half-life, while hepatic insufficiency has little effect on its pharmacokinetics (173). Triamterene is extensively metabolized in the liver and the drug (50%) and its active metabolite (4-hydroxytriamterene sulfate) are excreted in the urine. The amount of metabolite reaching the tubule decreases in hepatic insufficiency. In patients with CKD both drugs have progressively limited access to their site of action and, therefore, became less effective. In monotherapy, amiloride and triamterene produce a weak natriuretic effect that increases in patients with primary or secondary hyperaldosteronism, but are relatively ineffective in lowering BP. Thus, they are co-administered with thiazide or loop diuretics to prevent K+ and Mg2+ excretion and/or to increase the diuresis in patients with low-renin hypertension, resistant hypertension, congestive HF or refractory edema (174). A recent study found that in patients with rosiglitazone-induced plasma volume retention and type 2 diabetes amiloride prevents protracted fluid retention, while hematocrit continues to decrease significantly patients treated with spironolactone, suggesting continued PV expansion (175). Amiloride is effective in patients with polyuria and polydipsia due to Li+-induced nephrogenic diabetes insipidus and in patients with Liddle syndrome and in patients with some ENaC mutations (176,177). In patients with chronic HF amiloride increases serum K+ levels (0.4 mmol/L), shortens the corrected QT interval and reduces QT dispersion and ventricular extrasystoles but, unlike spironolactone, amiloride does not improve endothelial dysfunction, heart rate variability or myocardial fibrosis (178).
28 Figure legends Figure 1. (A) Pathways for the synthesis of aldosterone in the adrenal cortex. Final steps of the synthesis involve the cytochrome P450 enzyme aldosterone synthase (CYP11B2). (B) Physiopathological effects of aldosterone. Abbreviations: ACTH, adrenocorticotropic hormone; Ang II, angiotensin II; ANP, atrial natriuretic peptide; ASI: aldosterone synthase inhibitors; CKD: chronic kidney disease; ET-1: endothelin-1; HRV: heart rate variability; MR: mineralocorticoid receptor; MRA: mineralocorticoid receptor antagonists; NE: norepinephrine; PAI-1: plasminogen activator inhibitor type 1; ROS: reactive oxygen species.
29
30 Table 1. Pharmacological modulation of aldosterone 1. Mineralocoticoid receptor antagonists (MRAs) • Steroidal compounds o First generation: spironolactone and canrenone o Second generation: eplerenone, prorenone*, mespirenone*, spirorenone*, drospirenone* • Non-steroidal compounds (third/fourth generation): BAY-94-8862, BR-4628, CS-3150, LY-2623091, MT-3995, PF-3882845, SM-368229 2. Aldosterone synthase (CYP11B2) inhibitors: Fadrozole, FAD286A, LCI699, some 1,4dihydropyridine Ca2+ channel blockers 3. Epithelial Na+ channel (ENaC) blockers: amiloride and triamterene
31 Table 2. Pharmacologic differences between the agents may be useful to inform drug selection for individual patients (IC50/EC50 values in nM) Paramater Chemical class Affinity for MR (μM)* AR PR GC Mode of antagonism Receptor conformation** Cofactor recruitment** Bioavailability (%) Onset of effect (h) Peak effects (h) Plasma protein binding (%) Vd (L/kg) Half-life (h) Duration of the effect (h) Metabolism
Drug interactions
Spironolactone
Eplerenone
BR-4628
Steroidal
Steroidal
24 77 740 2410
990 21240 31210 21980
Nonsteroidal 28 4440 9020 5470
Passive Partial stabilization of active MR conformation No recruitment of co-repressors 80-90 (100 with food) 24-48 48-72 > 90
Passive Stabilization of inert conformation
10 1.4 (active metabolites 1235) 72-120
0.6-1.3 4-6
Hepatic, multiple active metabolites)
70
Amilori de Pyrazin e
Triamte rene
15-25
50
Bulky Destabilizat or No cofactor recruitment 94
24 1.5 50
2 4-6 -
2-4 3 60-70
5-7 6-9
2.5
72 h
24
7-9
Hepatic, via CYP3A4, no active metabolites Multiple
No
8.5
Drugs and foods Drugs and foods increasing serum that raise serum K+ levels + K levels Renal excretion (%)
