Identification of sulfonylpyrimidines as novel selective aldosterone synthase (CYP11B2) inhibitors.

4-[(5-[2-Methyl-5-(methylsulfonyl)pentan-2-yl]sulfonylpyrimidin-4-yl)amino]benzonitrile 2 was identified as a novel potent aldosterone synthase inhibitor. Compound 2 was found to inhibit human CYP11B2 in the nanomolar range, and showed an aldosterone-lowering effect in a furosemide-treated cynomolgus monkey model. Although human CYP11B2 has the high homology sequence with human CYP11B1, compound 2 showed more than 80 times higher selectivity over human CYP11B1 in vitro.

[Pharmacological profile, clinical efficacy, and safety of esaxerenone (Minnebro® tablets 1.25†mg, 2.5†mg, 5†mg)].

Esaxerenone is a novel non-steroidal mineralocorticoid receptor antagonisit (MR blocker), whose unique binding to the MR-ligand domain yields a stronger MR antagonistic effect and higher selectivity than existing MR antagonisits. Esaxerenone was approved for the treatment of hypertension in Japan in January 2019. Esaxerenone suppresses the reduction of urinary Na+/K+ ratio in adrenalectomized rats and blood pressure increase, proteinuria, and renal tissue lesions in salt-sensitive hypertensive rats-all in a dose-dependent manner. Esaxerenone is rapidly absorbed and reaches intracellular targets because of its high membrane permeability, exhibits high bioavailability with small interindividual exposure variation, and is metabolized via several pathways (e.g., oxidation, glucuronidation, and hydrolysis), which is associated with low drug-drug interaction risk. As esaxerenone is slightly excreted into urine, its exposure is similar between elderly and non-elderly patients, and between patients with normal and moderately deteriorated renal function. Given its 19-hour half-life, once-daily administration would have a sustainable antihypertensive effect. The ESAX-HTN phase 3 study demonstrated the non-inferiority of esaxerenone's antihypertensive effect versus that of eplerenone in essential hypertension. Another study showed a stable antihypertensive effect for 52 weeks as monotherapy or combination therapy. In hypertensive patients with moderate impairment or both type 2 diabetes and albuminuria treated with a renin-angiotensin system inhibitor, esaxerenone elicited a stable antihypertensive effect and manageable hyperkalemia incidence with titration from a low dose and monitoring including serum potassium. Thus, with careful monitoring of serum potassium, esaxerenone can be administered to patients with moderate renal impairment or both diabetes and albuminuria.

Crystal structure of the mineralocorticoid receptor ligand-binding domain in complex with a potent and selective nonsteroidal blocker, esaxerenone (CS-3150).

Activation of the mineralocorticoid receptor (MR) has long been considered a risk factor for cardiovascular diseases. It has been reported that the novel MR blocker esaxerenone shows high potency and selectivity for MR in vitro as well as great antihypertensive and renoprotective effects in salt-sensitive hypertensive rats. Here, we determined the cocrystal structure of the MR ligand-binding domain (MR-LBD) with esaxerenone and found that esaxerenone binds to MR-LBD in a unique manner with large side-chain rearrangements, distinct from those of previously published MR antagonists. This structure also displays an antagonist form that has not been observed for MR previously. Such a unique binding mode of esaxerenone provides great insight into the novelty, potency, and selectivity of this novel antihypertensive drug.

Histone demethylase LSD1 deficiency and biological sex: impact on blood pressure and aldosterone production.

Human risk allele carriers of lysine-specific demethylase 1 (LSD1) and LSD1-deficient mice have salt-sensitive hypertension for unclear reasons. We hypothesized that LSD1 deficiency causes dysregulation of aldosterone's response to salt intake resulting in increased cardiovascular risk factors (blood pressure and microalbumin). Furthermore, we determined the effect of biological sex on these potential abnormalities. To test our hypotheses, LSD1 male and female heterozygote-knockout (LSD1+/-) and WT mice were assigned to two age groups: 18 weeks and 36 weeks. Plasma aldosterone levels and aldosterone production from zona glomerulosa cells studied ex vivo were greater in both male and female LSD1+/- mice consuming a liberal salt diet as compared to WT mice consuming the same diet. However, salt-sensitive blood pressure elevation and increased microalbuminuria were only observed in male LSD1+/- mice. These data suggest that LSD1 interacts with aldosterone's secretory response to salt intake. Lack of LSD1 causes inappropriate aldosterone production on a liberal salt diet; males appear to be more sensitive to this aldosterone increase as males, but not females, develop salt sensitivity of blood pressure and increased microalbuminuria. The mechanism responsible for the cardiovascular protective effect in females is uncertain but may be related to estrogen modulating the effect of mineralocorticoid receptor activation.

Caloric restriction improves glucose homeostasis, yet increases cardiometabolic risk in caveolin-1-deficient mice.

BACKGROUND AND PURPOSE: The plasma membrane protein caveolin-1 (CAV-1) has been shown to be involved in modulating glucose homeostasis and the actions of the renin-angiotensin-aldosterone system (RAAS). Caloric restriction (CR) is widely accepted as an effective therapeutic approach to improve insulin sensitivity and reduce the severity of diabetes. Recent data indicate that polymorphisms of the CAV-1 gene are strongly associated with insulin resistance, hypertension and metabolic abnormalities in non-obese individuals. Therefore, we sought to determine whether CR improves the metabolic and cardiovascular (CV) risk factors in the lean CAV-1 KO mice. MATERIALS/METHODS: Twelve- to fourteen-week-old CAV-1 knockout (KO) and genetically matched wild-type (WT) male mice were randomized by genotype to one of two dietary regimens: ad libitum (ad lib) food intake or 40% CR for 4 weeks. Three weeks following the onset of dietary restriction, all groups were assessed for insulin sensitivity. At the end of the study, all groups were assessed for fasting glucose, insulin, HOMA-IR, lipids, corticosterone levels and blood pressure (BP). Aldosterone secretion was determined from acutely isolated Zona Glomerulosa cells. RESULTS: We confirmed that the CAV-1 KO mice on the ad lib diet display a phenotype consistent with the cardiometabolic syndrome, as shown by higher systolic BP (SBP), plasma glucose, HOMA-IR and aldosterone levels despite lower body weight compared with WT mice on the ad lib diet. CAV-1 KO mice maintained their body weight on the ad lib diet, but had substantially greater weight loss with CR, as compared to caloric restricted WT mice. CR-mediated changes in weight were associated with dramatic improvements in glucose and insulin tolerance in both genotypes. These responses to CR, however, were more robust in CAV-1KO vs. WT mice and were accompanied by reductions in plasma glucose, insulin and HOMA-IR in CAV-1KO but not WT mice. Surprisingly, in the CAV-1 KO, but not in WT mice, CR was associated with increased SBP and aldosterone levels, suggesting that in CAV-1 KO mice CR induced an increase in some CV risk factors. CONCLUSIONS: CR improved the metabolic phenotype in CAV-1 KO mice by increasing insulin sensitivity; nevertheless, this intervention also increased CV risk by inappropriate adaptive responses in the RAAS and BP.

Combined Salt and Caloric Restrictions: Potential Adverse Outcomes.

BACKGROUND: We hypothesized that caloric restriction (CR) and salt restriction (ResS) would have similar effects on reducing cardiovascular risk markers and that combining CR and ResS would be synergistic in modulating these markers. METHODS AND RESULTS: To test our hypothesis, rats were randomized into 2 groups: ad libitum liberal salt diet (ad libitum/high-sodium, 1.6% sodium) or ResS diet (ad libitum/ResS, 0.03% sodium). CR was initiated in half of the rats in each group by reducing caloric intake to 60% while maintaining sodium intake constant (CR/high-sodium, 2.7% sodium or CR/ResS, 0.05% sodium) for 4 weeks. CR in rats on a high-sodium diet improved metabolic parameters, renal transforming growth factor-ß and collagen-1α1 and increased plasma adiponectin and renal visfatin and NAD+ protein levels. Although CR produced some beneficial cardiovascular effects (increased sodium excretion and reduced blood pressure), it also was associated with potentially adverse cardiovascular effects. Adrenal zona glomerulosa cell responsiveness and aldosterone levels and activation were inappropriately increased for the volume state of the rodent. Like CR on HS, CR on a ResS diet also produced relative increased zona glomerulosa responsiveness and an increased blood pressure with no improvement in metabolic parameters. CONCLUSIONS: These results suggest that combining CR and ResS may decrease the beneficial effects of each alone. Furthermore, CR, regardless of dietary salt intake, inappropriately activates aldosterone production. Thus, caution should be used in combining ResS and CR because the combination may lead to increased cardiovascular risk.

Sustained Activation of Guanylate Cyclase-A with TDT, a Natriuretic Peptide Derivative, Exhibits Cardiorenal Protection in Dahl Salt-Sensitive Hypertensive Rats.

Heart failure often presents with prognosis-relevant impaired renal function. To investigate whether the chronic activation of guanylate cyclase-A (GC-A) protects both heart and kidney, we examined the effects of TDT, a neprilysin (NEP)-resistant natriuretic peptide (NP) derivative, on cardiac and renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. Pretreatment with NEP or NEP inhibitor did not influence GC-A activation by TDT both in vitro and in vivo, resulting in a long-acting profile of TDT compared with native human atrial NP (hANP). The repeated administration of TDT to DS rats suppressed the progress of cardiac hypertrophy, systolic/diastolic dysfunction, and proteinuria in a dose-dependent manner. Compared with vehicle and hANP, salt diet-induced podocyte injury was reduced by TDT, as analyzed by urinary podocalyxin concentration, renal expression of nephrin mRNA, and glomerular expression of desmin protein. Since glomerular TRPC6 plays detrimental roles in podocyte homeostasis, we examined the renal expression of TRPC6 in DS rats and found that salt diet upregulated the expression of TRPC6. Importantly, TRPC6 induction was significantly decreased in TDT-treated rats, compared with vehicle and hANP. Consistently, in primary-culture podocytes from DS rats, TDT inhibited ATP-induced calcium influx, similar to TRPC inhibitor SKF96365. Finally, TDT-mediated protection of podocytes was abolished by protein kinase G inhibitor KT5823. In conclusion, TDT treatment attenuated heart and kidney dysfunction, accompanied by podocyte protection through inhibition of TRPC6. Thus, long-acting NPs could be a new avenue for treatment of heart failure.

4-Anilino-pyrimidine, novel aldosterone synthase (CYP11B2) inhibitors bearing pyrimidine structures.

2,2,2-Trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-yl}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol 1 was identified as a novel potent aldosterone synthase inhibitor. Despite large species differences, compound 1 inhibits both human and rodent CYP11B2 in a nano-molar range.

A novel aldosterone synthase inhibitor ameliorates mortality in pressure-overload mice with heart failure.

It has been elucidated that mineralocorticoid receptor antagonists reduce mortality in patients with congestive heart failure and post-acute myocardial infarction. A direct inhibition of aldosterone synthase (CYP11B2) is also expected to have therapeutic benefits equal in quality to mineralocorticoid receptor antagonists in terms of reducing mineralocorticoid receptor signaling. Therefore, we have screened our chemical libraries and identified a novel and potent aldosterone synthase inhibitor, 2,2,2-trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-y}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol (compound 1), by lead optimization. Pharmacological properties of compound 1 were examined in in vitro cell-based assays and an in vivo mouse model of pressure-overload hypertrophy by transverse aortic constriction (TAC). Compound 1 showed potent CYP11B2 inhibition against human and mouse enzymes (IC50; 0.003µM and 0.096µM, respectively) in a cell-based assay. The oral administration of 0.06% compound 1 in the food mixture of a mouse TAC model significantly reduced the plasma aldosterone level and ameliorated mortality rate. This study is the first to demonstrate that a CYP11B2 inhibitor improved survival rates of heart failure induced by pressure-overload in mice. The treatment of 0.06% compound 1 did not elevate plasma potassium level in this model, although further evaluation of hyperkalemia is needed. These results suggest that compound 1 can be developed as a promising oral CYP11B2 inhibitor for pharmaceutical applications. Compound 1 could also be a useful compound for clarifying the role of aldosterone in cardiac hypertrophy.

CS-3150, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, Shows Preventive and Therapeutic Effects On Renal Injury in Deoxycorticosterone Acetate/Salt-Induced Hypertensive Rats.

The present study was designed to assess both preventive and therapeutic effects of (S)-1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidal mineralocorticoid receptor antagonist, on renal injury in deoxycorticosterone acetate (DOCA)/salt-induced hypertensive rats (DOCA rats). From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt diet. In experiment 1, CS-3150 (0.3-3 mg/kg) was orally administered once a day for 4 weeks coincident with DOCA administration. In experiment 2, after establishment of renal injury by 4 weeks of DOCA/salt loading, CS-3150 (3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. In experiment 1, DOCA/salt loading significantly increased systolic blood pressure (SBP), which was prevented by CS-3150 in a dose-dependent manner. Development of renal injury (proteinuria, renal hypertrophy, and histopathological changes in glomeruli and tubule) was also suppressed by CS-3150 with inhibition of mRNA expression of fibrosis, inflammation, and oxidative stress markers. In experiment 2, under continuous DOCA treatment, CS-3150 clearly ameliorated existing renal injury without lowering SBP, indicating that CS-3150 regressed renal injury independent of its antihypertensive action. Moreover, CS-3150 treatment in combination with withdrawal of DOCA showed further therapeutic effect on renal injury accompanied by reduction in SBP. These results demonstrate that CS-3150 not only prevents but also ameliorates hypertension and renal injury in DOCA rats. Therefore, CS-3150 could be a promising agent for the treatment of hypertension and renal disorders, and may have potential to promote regression of renal injury.

CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist, prevents hypertension and cardiorenal injury in Dahl salt-sensitive hypertensive rats.

The present study was designed to evaluate the antihypertensive and cardiorenal protective effects of CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist, in Dahl salt-sensitive hypertensive rats (DS rats), and to compare the effects with spironolactone and eplerenone. DS rats were fed a control diet (0.3% NaCl) or high salt diet (8% NaCl) from 7 weeks of age. CS-3150 (0.25-2mg/kg), spironolactone (10-100mg/kg) or eplerenone (10-100mg/kg) were orally administered once a day to DS rats fed a high salt diet for 7 weeks. The high salt diet significantly increased systolic blood pressure, which was prevented by treatment with CS-3150 in a dose-dependent manner with no hyperkalemia (>5.5mEq/L). The antihypertensive effect of CS-3150 (0.5mg/kg) was equivalent to that of spironolactone (100mg/kg) and eplerenone (100mg/kg). CS-3150 also suppressed proteinuria and renal hypertrophy induced by the high salt diet. Histopathological examination of kidneys showed that CS-3150 markedly ameliorated glomerulosclerosis, tubular injury and tubulointerstitial fibrosis. In addition, CS-3150 inhibited left ventricular hypertrophy and elevation of plasma brain natriuretic peptide level. In contrast, the cardiorenal protective effects of spironolactone or eplerenone were partial, and the dose-dependency was not clear, especially in eplerenone-treated rats. These results indicate that chronic treatment with CS-3150 exerts antihypertensive and cardiorenal protective effects in a DS hypertensive rat model, and its potency is much superior to that of spironolactone or eplerenone. Thus, CS-3150 could be a promising agent for the treatment of hypertension and cardiorenal disorders.

Pharmacological profile of CS-3150, a novel, highly potent and selective non-steroidal mineralocorticoid receptor antagonist.

The present study was designed to characterize the pharmacological profile of CS-3150, a novel non-steroidal mineralocorticoid receptor antagonist. In the radioligand-binding assay, CS-3150 inhibited (3)H-aldosterone binding to mineralocorticoid receptor with an IC50 value of 9.4nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 36 and 713nM, respectively. CS-3150 also showed at least 1000-fold higher selectivity for mineralocorticoid receptor over other steroid hormone receptors, glucocorticoid receptor, androgen receptor and progesterone receptor. In the reporter gene assay, CS-3150 inhibited aldosterone-induced transcriptional activation of human mineralocorticoid receptor with an IC50 value of 3.7nM, and its potency was superior to that of spironolactone and eplerenone, whose IC50s were 66 and 970nM, respectively. CS-3150 had no agonistic effect on mineralocorticoid receptor and did not show any antagonistic or agonistic effect on glucocorticoid receptor, androgen receptor and progesterone receptor even at the high concentration of 5µM. In adrenalectomized rats, single oral administration of CS-3150 suppressed aldosterone-induced decrease in urinary Na(+)/K(+) ratio, an index of in vivo mineralocorticoid receptor activation, and this suppressive effect was more potent and longer-lasting than that of spironolactone and eplerenone. Chronic treatment with CS-3150 inhibited blood pressure elevation induced by deoxycorticosterone acetate (DOCA)/salt-loading to rats, and this antihypertensive effect was more potent than that of spironolactone and eplerenone. These findings indicate that CS-3150 is a selective and highly potent mineralocorticoid receptor antagonist with long-lasting oral activity. This agent could be useful for the treatment of hypertension, cardiovascular and renal disorders.

Spironolactone, but not eplerenone, impairs glucose tolerance in a rat model of metabolic syndrome.

Although some clinical studies have suggested that spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, appears to increase the blood glucose levels, experimental studies have not supported this notion. Here, we investigated the effect of SPL on blood glucose levels in SHR/NDmcr-cp(cp/cp) (ND) rats, an animal model of metabolic syndrome, in comparison with that of eplerenone (EPL), another MR antagonist. At the same dose of 100 mg/kg, SPL and EPL increased the urinary sodium-to-potassium ratio to a comparable extent, indicating that both agents have similar renal MR antagonistic efficacy in ND rats. Interestingly, SPL but not EPL significantly increased the level of blood glucose. The oral glucose tolerance test revealed that treatment with SPL led to glucose intolerance. The levels of serum insulin and adiponectin, regulators of the blood glucose level, were virtually unaffected by treatment with SPL. On the other hand, SPL induced a marked increase in the blood level of aldosterone, known to be a risk factor for insulin resistance. These results demonstrate that in comparison with EPL, SPL characteristically impairs glucose tolerance in an animal model of metabolic syndrome, in association with a higher blood level of aldosterone.

Activation of renal angiotensin type 1 receptor contributes to the pathogenesis of progressive renal injury in a rat model of chronic cardiorenal syndrome.

Although chronic cardiac dysfunction is known to progressively exacerbate renal injury, a condition known as type 2 cardiorenal syndrome (CRS), the mechanism responsible is largely unknown. The present study was undertaken to clarify the mechanism of renal injury in rats with both unilateral nephrectomy (NX) and surgically induced myocardial infarction (MI), corresponding to a model of type 2 CRS. Compared with a control group, rats with both MI and NX (MI+NX) exhibited progressive proteinuria during the experimental period (34 wk after MI surgery), whereas proteinuria was not observed in rats with MI alone and was moderate in rats with NX alone. The proteinuria in rats with MI+NX was associated with renal lesions such as glomerulosclerosis and infiltration of mononuclear cells and upregulation of the renal proinflammatory and -fibrotic cytokine and angiotensin II type 1a receptor (AT1aR) genes. In contrast, plasma renin activity was lowered in rats with MI+NX. Immunohistochemistry revealed that the increased AT1R protein was present mainly in renal interstitial mononuclear cells. Olmesartan medoxomil, an AT1R blocker, markedly reduced the proteinuria and infiltration of mononuclear cells, whereas spironolactone, a mineralocorticoid receptor blocker, did not. The present findings demonstrate the pathogenetic role of renal interstitial AT1R signaling in a model of type 2 CRS, providing evidence that AT1R blockade can be a useful therapeutic option for this syndrome.