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Rationale: Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes., Methods: Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation., Results: We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group., Conclusions: GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Phosphodiesterase type 5 (PDE5) is a multidomain protein that plays a crucial role in regulating cellular cyclic guanosine monophosphate (cGMP), a key signaling molecule involved in various physiological processes. Dysregulation of PDE5 and cGMP signaling is associated with a range of vasodysfunctional disorders, necessitating the development of effective therapeutic interventions. This study adopts comprehensive approach, combining virtual screening and molecular dynamics (MD) simulations, to repurpose FDA-approved drugs as potential PDE5 inhibitors. The initial focus involves selecting compounds based on their binding affinity. Shortlisted compounds undergo a meticulous analysis for their drug profiling and biological significance, followed by the activity evaluation and interaction analysis. Notably, based on binding potential and drug profiling, two molecules, Dutasteride and Spironolactone, demonstrate strong potential as PDE5 inhibitors. Furthermore, all atom MD simulations were employed (500 ns) to explore dynamic behavior of Dutasteride and Spironolactone in complexes with PDE5. Principal components analysis (PCA) and free energy landscape (FEL) analyses are further leveraged to decipher that the binding of Dutasteride and Spironolactone stabilizes the structure of PDE5 with minimal conformational changes. In summary, Dutasteride and Spironolactone exhibit remarkable affinity for PDE5 and possess characteristics that suggest their potential as therapeutic agents for conditions associated with PDE5 dysfunction., (© 2023 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Background: Immunoassays are important for routine clinical testing and medical diagnosis. However, they are limited by cross-reactivity especially at low analyte concentrations. There is a critical need to investigate compounds that can interfere with immunoassays. Herein, we describe the identification of canrenone, a spironolactone metabolite that falsely increases progesterone concentrations on the Abbott Architect i2000 Immunoassay., Methods: Serum samples and assay diluents were spiked with spironolactone or canrenone and progesterone concentrations were measured on the Architect i2000 and Immulite XPi immunoassay platforms. Blood samples from patients taking spironolactone were analyzed with liquid chromatography-tandem mass spectrometry to evaluate the intrinsic response of progesterone concentrations to the presence of canrenone., Results: We measured approximately 10-fold higher progesterone concentrations on the Abbott Architect i2000 compared to reference immunoassay analyzers (Siemens Immulite XPi and Roche Cobas e601/602), suggesting an analytical error which is unique to the Architect i2000 antibody and/or assay conditions. By measuring serum progesterone after addition of spironolactone or canrenone to serum samples, we found that canrenone falsely increased progesterone on the Architect i2000 immunoassay. However, this interference was more pronounced at low serum progesterone concentrations. Moreover, a strong positive correlation was seen between canrenone and measured serum progesterone concentrations., Conclusions: Our investigations are important for individuals who require progesterone measurements using the Architect i2000 immunoassay, especially because it is unlikely for clinicians to order canrenone measurements alongside progesterone measurements for individuals taking spironolactone. Further research is needed to determine whether canrenone can influence progesterone measurements on other immunoassay systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The glucocorticoid cortisol is the end product of the hypothalamic-pituitary-adrenal (HPA) axis and crucial for the stress response in humans. Cortisol regulates numerous biological functions by binding to two different types of receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Both receptors are found in the brain where they are crucially involved in various mental functions and in feedback inhibition of cortisol release. The precise role of both receptors in the human stress response is not completely understood. In this study, we examined the effects of pharmacological blockade of the MR or the GR on stress-induced cortisol release in a sample of 318 healthy young men (M = 25.42, SD = 5.01). Participants received the MR antagonist spironolactone (300 mg), the GR antagonist mifepristone (600 mg), or a placebo and were subjected 90 min later to a social-evaluative stressor (Trier Social Stress Test) or a non-stressful control condition. We found significantly higher stress-induced cortisol release in the spironolactone group, whereas participants after mifepristone administration did not differ from the control groups. These results suggest that MR blockade results in attenuated fast negative feedback processes and emphasize the important role of the MR during the early phase of the stress response., Competing Interests: Declaration of Competing Interest No conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Background: Alzheimer's disease (AD) is a chronic neurodegenerative disease needing effective therapeutics urgently. Sildenafil, one of the approved phosphodiesterase-5 inhibitors, has been implicated as having potential effect in AD., Objective: To investigate the potential therapeutic benefit of sildenafil on AD., Methods: We performed real-world patient data analysis using the MarketScan® Medicare Supplemental and the Clinformatics® databases. We conducted propensity score-stratified analyses after adjusting confounding factors (i.e., sex, age, race, and comorbidities). We used both familial and sporadic AD patient induced pluripotent stem cells (iPSC) derived neurons to evaluate the sildenafil's mechanism-of-action., Results: We showed that sildenafil usage is associated with reduced likelihood of AD across four new drug compactor cohorts, including bumetanide, furosemide, spironolactone, and nifedipine. For instance, sildenafil usage is associated with a 54% reduced incidence of AD in MarketScan® (hazard ratio [HR] = 0.46, 95% CI 0.32- 0.66) and a 30% reduced prevalence of AD in Clinformatics® (HR = 0.70, 95% CI 0.49- 1.00) compared to spironolactone. We found that sildenafil treatment reduced tau hyperphosphorylation (pTau181 and pTau205) in a dose-dependent manner in both familial and sporadic AD patient iPSC-derived neurons. RNA-sequencing data analysis of sildenafil-treated AD patient iPSC-derived neurons reveals that sildenafil specifically target AD related genes and pathobiological pathways, mechanistically supporting the beneficial effect of sildenafil in AD., Conclusions: These real-world patient data validation and mechanistic observations from patient iPSC-derived neurons further suggested that sildenafil is a potential repurposable drug for AD. Yet, randomized clinical trials are warranted to validate the causal treatment effects of sildenafil in AD.

Aim: Irrespective of the cause, albumin/proteinuria induces tubulointerstitial damage and accelerates the progression of kidney diseases. Our series of studies demonstrated that proteinuria, an independent prognostic factor for chronic kidney disease (CKD), is correlated with urinary basigin/CD147 (Bsg) levels. We examined the morphology and origin of Bsg in the tubular lumen through the effects of filtered glucose and protein solutes on the tubules., Methods: Diabetic kidney disease (DKD) patients (N = 50) were treated with spironolactone 25 mg for 4 weeks or by conservative treatment. The associations between urinary Bsg values and clinical indicators were examined. Primary-cultured proximal tubular epithelial cells (PTECs) from human adult kidneys were exposed to high glucose or bovine serum albumin (BSA)., Results: In patients with early phase DKD, urinary Bsg levels were closely correlated with proteinuria but not HbA1c. Full-length Bsg on extracellular vesicles (EVs) was investigated primarily in urine collected from DKD patients. EVs obtained from the urine of DKD patients included Bsg and SGLT2 proteins. Notably, spironolactone treatment concomitantly suppressed the release of Bsg-bearing EVs in correlation with decreased albuminuria. Exposure of PTECs to BSA (but not high glucose) enhanced the storage of supernatant Bsg in EVs despite the absence of exposure-specific changes in Bsg transcription., Conclusion: Proteinuria induces the release of Bsg-bearing EVs derived from PTECs into the tubular lumen., (© 2023 Asian Pacific Society of Nephrology.)

Liver cirrhosis is associated to circulatory abnormalities leading to hypovolemia and stimulation of the renin-angiotensin-aldosterone system (RAAS). Advanced stages of the disease cause renal failure, impairing K + and Na + homeostasis. It has been proposed that the distal colon undergoes functional remodeling during renal failure, in particular by aldosterone-driven increased K + excretion. In this study, we compared the transcriptional response of aldosterone target genes in the rat distal colon under two models of increased circulating aldosterone (one with concomitant RAAS activation) and in a model of secondary hyperaldosteronism induced by cirrhosis. The expression of a subset of these genes was also tested in distal colon biopsies from control subjects or patients with cirrhosis with varying levels of disease progression and treated or not with mineralocorticoid receptor inhibitor spironolactone. We examined known aldosterone-regulated transcripts involved in corticosteroid signaling and transepithelial ion transport. In addition, we included aldosterone-regulated genes related to cell proliferation. Our comparison revealed multiple aldosterone target genes upregulated in the rat distal colon during decompensated cirrhosis. Epithelial Na + channel β and γ subunit expression correlated positively with plasma aldosterone concentration and negatively with glomerular filtration rate. Patients with cirrhosis showed increased expression of 11-β-hydroxysteroid-dehydrogenase 2 (11βHSD2), which was reverted by spironolactone treatment, suggesting a sensitization of the distal colon to aldosterone action. In summary, our data show that decaying kidney function during cirrhosis progression toward a decompensated state with hypovolemia correlates with remodeling of distal colon ion transporter expression, supporting a role for aldosterone in the process. NEW & NOTEWORTHY Liver cirrhosis progression significantly alters ion transporter subunit expression in the rat distal colon, a change that correlated well with declining kidney function and the severity of the disease. Our data suggest that the steroid hormone aldosterone participates in this homeostatic response to maintain electrolyte balance.

Low serum concentrations of the amino acid homoarginine (HA) are associated with increased cardiovascular mortality by incompletely understood mechanisms. This study sought to assess the influence of HA on cardiac remodeling in rats undergoing either transaortic banding or inhibition of nitric oxide synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Male Wistar rats (n = 136) underwent sham operation (SH) or aortic banding (AB). Both groups were equally divided into 14 subgroups, receiving different doses of HA alone or in combination with lisinopril, spironolactone, or L-NAME for 4 weeks. HA treatment in AB animals resulted in a dose-dependent improvement of cardiac function up to a concentration of 800 mg·kg -1 ·day -1 . Combining 800 mg·kg -1 ·day -1 HA with spironolactone or lisinopril yielded additional effects, showing a positive correlation with LV ejection fraction (+33%, p = 0.0002) and fractional shortening (+41%, p = 0.0014). An inverse association was observed with collagen area fraction (-41%, p < 0.0001), myocyte cross-sectional area (-22%, p < 0.0001) and the molecular markers atrial natriuretic factor (-74%, p = 0.0091), brain natriuretic peptide (-42%, p = 0.0298), beta-myosin heavy chain (-46%, p = 0.0411), and collagen type V alpha 1 chain (-73%, p = 0.0257) compared to placebo-treated AB animals. Co-administration of HA and L-NAME was found to attenuate cardiac remodeling and prevent NO-deficient hypertension following AB. HA treatment has led to a dose-dependent improvement of myocardial function and marked histological and molecular changes in cardiac remodeling following AB. Combining HA with standard heart failure medication resulted in additional beneficial effects boosting its direct impact on heart failure pathophysiology., (© 2022 The Authors. Fundamental & Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of Société Française de Pharmacologie et de Thérapeutique.)

The gastrointestinal system of fish reacts rapidly to food deprivation. The relative masses of digestive organs and activities of digestive enzymes decrease within days of fasting. This is believed to be an energy-conserving strategy as the metabolic cost of maintaining digestive capacity is high. Cortisol is known for its role in energy mobilization following stress exposure, and prolonged elevated cortisol levels have been shown to reduce growth rates in fish. Fish experiencing chronic cortisol elevations show structural changes to their digestive tissues and overall reductions in relative digestive tissue masses. In fish fasting for prolonged periods, circulating cortisol levels have been reported to be downregulated, upregulated, or unchanged compared to feeding fish. This study aimed to investigate if RU486 and spironolactone, antagonists of the glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), respectively, alone or in combination affect circulating cortisol levels during prolonged starvation. In addition, we tested the effects of blocking GR and MR, on the down-regulation of relative digestive tissue mass during starvation, and its effects on weight loss. Three treatment groups of rainbow trout were intraperitoneally implanted with either GR, MR, or GR and MR blockers. A fourth group was implanted with cortisol, while a fifth group served as a control. All treatment groups were sampled over a course of four weeks of food deprivation and compared against each other and fed control fish at day 0 of the trial. Starvation for 2 weeks and longer significantly increased circulating cortisol levels in all groups except for the group implanted with GR and MR antagonists. Loss of body mass occurred most rapidly during the first week of starvation. Spironolactone treatment resulted in significantly reduced loss of mass during the first week, however, over the following weeks, no differences in mass loss were observed in the groups implanted with blockers, while cortisol-treated fish showed the highest decrease in body mass over time. Relative digestive tissue mass decreased in all groups but apparently, the fasting-induced elevation in plasma cortisol levels did not affect the relative weight loss of digestive tissues as no differences were observed between control fish and GR + MR antagonist treated fish. Very high cortisol levels caused by cortisol treatment however caused a faster decrease in the relative mass of some digestive organs, particularly the stomach., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Mineralocorticoid receptor (MR) activation plays an important role in hepatic insulin resistance. However, the precise mechanisms by which MR activation promotes hepatic insulin resistance remains unclear. Therefore, we sought to investigate the roles and mechanisms by which MR activation promotes Western diet (WD)-induced hepatic steatosis and insulin resistance. Six-week-old C57BL6J mice were fed either mouse chow or a WD, high in saturated fat and refined carbohydrates, with or without the MR antagonist spironolactone (1 mg/kg/day) for 16 wk. WD feeding resulted in systemic insulin resistance at 8 and 16 wk. WD also induced impaired hepatic insulin metabolic signaling via phosphoinositide 3-kinases/protein kinase B pathways, which was associated with increased hepatic CD36, fatty acid transport proteins, fatty acid-binding protein-1, and hepatic steatosis. Meanwhile, consumption of a WD-induced hepatic mitochondria dysfunction, oxidative stress, and inflammatory responses. These abnormalities occurring in response to WD feeding were blunted with spironolactone treatment. Moreover, spironolactone promoted white adipose tissue browning and hepatic glucose transporter type 4 expression. These data suggest that enhanced hepatic MR signaling mediates diet-induced hepatic steatosis and dysregulation of adipose tissue browning, and subsequent hepatic mitochondria dysfunction, oxidative stress, inflammation, as well as hepatic insulin resistance.

Background: Nephropathy diabetes is one of the important causes of death and a more prevalent cause of end-stage renal disease., Objective: The present study investigated the effect of applying spironolactone and captopril and their combination on some renal performance indices and cholesterol-efflux-related gene expression in nephropathy diabetic rats., Methods: Intraperitoneal injection of streptozotocin was used to induce diabetes in rats. FBS, creatinine, and BUN were assayed using the calorimetry technique; also, urine microalbumin was assayed by ELISA. Hepatic gene expressions of ABCA1, ABCG1, and miR-33 were evaluated by the real-time PCR method., Results: FBS levels in the captopril-treated group were significantly decreased compared with the untreated diabetic group. BUN levels of treated groups with captopril and a combination of captopril + spironolactone were significantly increased. GFR of both treated diabetic groups with captopril and spironolactone was significantly lower than an untreated diabetic group. ABCA1 gene expression in hepatic cells of the combination of spironolactone + captopril treated group was significantly increased compared to other treated and untreated diabetic groups. The hepatic expression of the ABCG1 gene in the treated and untreated diabetic groups was significantly lower than in the control group. Treatment of the diabetic group with only combination therapy decreased the hepatic gene expression of miR-33 significantly., Conclusion: Obtained results suggest that S+C combination therapy can improve nephropathy and diabetes disorders by targeting the ABCA1 and miR-33 gene expression. It is suggested that miR-33 and ABCA1 genes evaluation could be a new therapeutic strategy for nephropathy diabetes remediation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target., Competing Interests: The authors declare no conflict of interest.

Glucocorticoids are a mainstay for the treatment of immune-mediated conditions and inflammatory diseases. However, their chronic use causes numerous side-effects including delays in corneal and cutaneous wound healing. This is attributed to off-target agonism of the mineralocorticoid receptor, which can be reduced by co-administration of a mineralocorticoid receptor antagonist such as spironolactone. The aim of this study was to develop a fast, selective and sensitive UHPLC-ESI-MS method for the simultaneous quantification of spironolactone, its active metabolites (7α-thiomethylspironolactone and canrenone), the latter's water-soluble prodrug potassium canrenoate and the synthetic glucocorticoid, dexamethasone, in corneal samples (17α-methyltestosterone served as an internal standard). A one-step extraction procedure using MeOH-H 2 O (1:1) was validated and employed to recover the analytes from the corneal tissue. Extracts were centrifuged and the supernatant analyzed under isocratic conditions. Compounds were detected using selected ion recording mode. The method satisfied US Food and Drug Administration guidelines with respect to selectivity, precision and accuracy and displayed linearity from 5 to 1000 ng/mL for all of the analytes. The lower limit of quantitation of the method was 5 ng/mL, making it sufficiently sensitive for quantification of the analytes in samples from in vivo studies., (Copyright © 2018 John Wiley & Sons, Ltd.)

Mesophilic fungi are well recognized as models of mammalian drug metabolism. Thermophilic fungi remained unexplored despite having a unique mechanism of growing at higher temperatures and performing wide diverse reactions. The present investigation is directed to isolate a promising thermophilic fungal strain capable of biotransformation using spironolactone as a model drug. Two-stage fermentation protocol was followed for the process. The transformation of spironolactone was identified by HPLC and structure elucidation of the metabolites was done with the help of LC-MS/MS analysis and previous reports. A strain of Thermomyces lanuginosus isolated from decomposed banana peel waste was found to be most promising in transforming spironolactone to 4 metabolites viz.7α-thiospironolactone (M1) canrenone (M2), 7α-thiomethylspironolactone (M3) and 6β-OH-7α-thiomethylspironolactone (M4), the major mammalian metabolites reported previously. The synthesis of metabolites of spironolactone by T. lanuginosus similar to mammals clearly states that this fungus possess enzyme system similar to mammals. Hence, this fungus has the potential to use as a model organism for studying drug metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Stiffening of the vasculature with aging is a strong predictor of adverse cardiovascular events, independent of all other risk factors including blood pressure, yet no therapies target this process. MRs (mineralocorticoid receptors) in smooth muscle cells (SMCs) have been implicated in the regulation of vascular fibrosis but have not been explored in vascular aging. Comparing SMC-MR-deleted male mice to MR-intact littermates at 3, 12, and 18 months of age, we demonstrated that aging-associated vascular stiffening and fibrosis are mitigated by MR deletion in SMCs. Progression of cardiac stiffness and fibrosis and the decline in exercise capacity with aging were also mitigated by MR deletion in SMC. Vascular gene expression profiling analysis revealed that MR deletion in SMC is associated with recruitment of a distinct antifibrotic vascular gene expression program with aging. Moreover, long-term pharmacological inhibition of MR in aged mice prevented the progression of vascular fibrosis and stiffness and induced a similar antifibrotic vascular gene program. Finally, in a small trial in elderly male humans, short-term MR antagonism produced an antifibrotic signature of circulating biomarkers similar to that observed in the vasculature of SMC-MR-deleted mice. These findings suggest that SMC-MR contributes to vascular stiffening with aging and is a potential therapeutic target to prevent the progression of aging-associated vascular fibrosis and stiffness., (© 2018 American Heart Association, Inc.)

Objective: Uncontrolled rapid release of drugs can reduce their therapeutic efficacy and cause undesirable toxicity; however, controlled release from reservoir materials helps overcome this issue. The aims of this study were to determine the release profiles of ketoprofen and spironolactone from a pH-responsive self-assembling DPA-MPC-DPA triblock copolymer gel and elucidate underlying physiochemical properties., Methods: Drug release profiles from DPA 50 -MPC 250 -DPA 50 gel (pH 7.5), over 32 h (37 °C), were determined using UV-Vis spectroscopy. Nanoparticle size was measured by dynamic light scattering (DLS) and critical micelle concentration (CMC) by pyrene fluorescence. Polymer gel viscosity was examined via rheology, nanoparticle morphology investigated using scanning transmission electron microscopy (STEM) and the gel matrix observed using cryo-scanning electron microscopy (Cryo-SEM)., Key Findings: DPA 50 -MPC 250 -DPA 50 copolymer (15% w/v) formed a free-standing gel (pH 7.5) that controlled drug release relative to free drugs. The copolymer possessed a low CMC, nanoparticle size increased with copolymer concentration, and DLS data were consistent with STEM. The gel displayed thermostable viscosity at physiological temperatures, and the gel matrix was a nanostructured aggregation of smaller nanoparticles., Conclusions: The DPA 50 -MPC 250 -DPA 50 copolymer gel could be used as a drug delivery system to provide the controlled drug release of ketoprofen and spironolactone., (© 2017 Royal Pharmaceutical Society.)

The present study involved the design and development of oral bioadhesive pellets of eplerenone. A solid dispersion of eplerenone was developed with a hydrophilic carrier, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus ® ). Bioadhesive pellets were prepared from this solid dispersion using a combination of HPMC K4M and Carbopol 934P. Both the solid dispersion and the pellets were evaluated for various physicochemical properties such as solubility, entrapment efficiency, drug content, surface morphology, mucoadhesion and swelling behavior. Analysis carried out using FT-IR, DSC and XRD found no interaction between the eplerenone and excipients. The solid dispersion had irregular-shaped smooth-surfaced particles of diameter 265 ± 105.5 μm. In TEM analysis, eplerenone particles of size 79-120 nm were found. The solubility and dissolution of eplerenone in the Soluplus ® -based solid dispersion were 5.26 and 2.50 times greater, respectively. Investigation of the swelling behavior of the pellets showed that the thickness of the gel layer increased continuously over the duration of the study. Moreover, a correlation was observed between the thickness and strength of the gel layer and the percentage release. The mechanism of drug release was found to be non-Fickian (anomalous), with the release kinetics approaching first-order kinetics. The bioavailability of the eplerenone bioadhesive pellet formulation was studied using Wistar rats and was found to be improved. An in vivo mucoadhesion study showed that the pellets are retained for 24 h in rabbits. It was concluded that Soluplus ® had a positive effect on the solubility and dissolution of pellets without affecting the bioadhesion.

This paper describes an LC-MS/MS method to determine the concentration of spironolactone and its metabolites 7-alpha-methylthiospironolactone and canrenone in blood plasma samples. The resulting assay is simple (using protein precipitation for sample preparation) and sensitive (the lower limit of quantification is close to 0.5 ng/ml) while requiring only 50 μl of plasma, making it especially suitable for analyzing samples obtained from pediatric and neonatal patients where sample sizes are limited. The sensitivity is achieved by using ammonium fluoride as an eluent additive, which in our case amplifies the signal from our analytes in the plasma solution on average about 70 times. The method is fully validated according to the European Medicines Agency's guideline and used for the measurement of pediatric patients' samples in clinical trials for evaluating oral spironolactone's and its metabolites' pharmacokinetics in children up to 2 years of age.

Spironolactone is a well-known multi-target drug and is specifically used for the treatment of high blood pressure and heart failure. It is also used for the treatment of edema, cirrhosis of the liver, malignant, pediatric, nephrosis and primary hyperaldosteronism. Spironolactone in association with thiazide diuretics treats hypertension and in association with furosemide treats bronchopulmonary dyspepsia. The therapeutic mechanism of action of spironolactone involves binding to intracellular mineralocorticoids receptors (MRs) in kidney epithelial cells, thereby inhibiting the binding of aldosterone. Since its first synthesis in 1957 there are several synthetic approaches have been reported throughout the years, Synthetic community has devoted efforts to improve the synthesis of spironolactone and to synthesize its analogues and derivatives. This review aims to provide comprehensive insight for the synthetic endeavors devoted towards the synthesis of a versatile drug spironolactone and its analogues/derivatives., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Primary aldosteronism (PA), the most common form of secondary hypertension, causes relevant morbidity. The value of salivary measurements of aldosterone in clinical routine in PA so far has not been assessed. First, we analyzed salivary and plasma aldosterone concentrations of 42 patients with PA and 37 hypertensive controls (HC) during a sodium infusion test prospectively. Second, morning salivary and plasma aldosterone concentrations as well as diurnal saliva aldosterone profiles were analyzed in 115 patients treated for PA (46 adrenalectomy, 56 spironolactone, 13 eplerenone). Salivary aldosterone was substantially elevated in PA patients compared to HC at baseline (106±119 vs. 40±21 ng/l, p=0.01), and after 4-h sodium infusion test (60±36 vs. 23±14, p=0.01). Positive correlation between salivary and plasma aldosterone levels was evident, with exception of concentrations in or below the lower normal range. Applying a salivary aldosterone cutoff of 51.2 ng/l, found by ROC curve analysis, rendered a sensitivity of 81% and a specificity of 73% for PA. The diurnal rhythm of aldosterone was preserved in untreated PA patients, but concentrations were higher in the context of PA, and normalized after surgery (118±57 vs. 31±18 ng/l, p<0.01). Taken together, salivary aldosterone measurements correlate with plasma levels, allowing simple and cost effective assessments of aldosterone secretion in an outpatient setting. Nevertheless, as this method alone cannot replace other plasma parameters, and as aldosterone profiling would not alter diagnostic or treatment strategies, salivary aldosterone measurements in routine practice are of limited clinical value., (© Georg Thieme Verlag KG Stuttgart · New York.)

The aim of this study was to develop a novel, sensitive, precise, simple, and rapid capillary zone electrophoresis method for the quality control of spironolactone in three different formulation types and a rapid simultaneous determination of the content of spironolactone and canrenone in urine samples using fluocinonide as an internal standard. After optimization of separation conditions, the electrolyte solution was the pH 5.5, 20 mM phosphate buffer containing 4.5 g/L sulfated-β-cyclodextrin, 15 kV of electric filed across the capillary applied at 25°C. A diode array detector was used, and the detection wavelength was 260 nm. Under optimum conditions, good linearity was achieved with correlation coefficients from 0.9976 to 0.9997. Detection limits were 0.56 and 0.20 μg/mL, and the quantitation limits were 1.87 and 0.67 μg/mL, respectively. Excellent accuracy and precision were obtained. Recoveries of the analytes varied from 100.8 to 103.1%. The results indicated that baseline separation of analytes was obtained and this method was suitable for quantitative determination of spironolactone in pharmaceutical preparations and rapid simultaneous determination of the content of spironolactone and its major metabolite canrenone in urine samples., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The first mineralocorticoid receptor (MR) antagonist, spironolactone, was developed almost 60 years ago to treat primary aldosteronism and pathological edema. Its use waned in part because of its lack of selectivity. Subsequently, knowledge of the scope of MR function was expanded along with clinical evidence of the therapeutic importance of MR antagonists to prevent the ravages of inappropriate MR activation. Forty-two years elapsed between the first and MR-selective second generation of MR antagonists. Fifteen years later, despite serious shortcomings of the existing antagonists, a third-generation antagonist has yet to be marketed. Progress has been slowed by the lack of appreciation of the large variety of cell types that express the MR and its diverse cell-type-specific actions, and also its unique complex interaction actions at the molecular level. New MR antagonists should preferentially target the inflammatory and fibrotic effects of MR and perhaps its excitatory effects on sympathetic nervous system, but not the renal tubular epithelium or neurons of the cortex and hippocampus. This review briefly describes efforts to develop a third-generation MR antagonist and why fourth generation antagonists and selective agonists based on structural determinants of tissue and ligand-specific MR activation should be contemplated.

Prior work suggests blood pressure in African Americans is more sensitive to the effects of aldosterone than in Caucasians. This mechanism may relate to a negative response of the vascular endothelium to aldosterone, including reduced glucose-6-phosphate dehydrogenase (G6PD) activity. Thirty-three African Americans (11 hypertensives, 22 controls) without evidence of diabetes or metabolic syndrome completed the protocol. The protocol included measurement of in vivo microvascular endothelial function by digital pulse arterial tonometry and ex vivo measurement of endothelial function by videomicroscopy of arterioles obtained from these same subjects with and without exposure to aldosterone or spironolactone. Systemic and arteriolar G6PD activities were also measured. In vivo and ex vivo microvascular endothelial function were impaired in African Americans with hypertension. One-hour exposure with aldosterone impaired endothelium-dependent vasodilation in arterioles from normotensive subjects, while 1 hour of spironolactone exposure reversed endothelial dysfunction in arterioles from hypertensive subjects. G6PD activity was impaired in hypertensive arterioles. Aldosterone-related endothelial dysfunction may be responsible for at least a portion of the greater blood pressure sensitivity to aldosterone in African Americans. This may be in part related to vascular suppression of G6PD activity., (© The Author(s) 2015.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A cyclic boronate structure was incorporated into Si-rhodamine to design a pH-activatable near-infrared (NIR) probe based on the reversible ring-opening process triggered by H(+). The probe showed general suitability of specific labelling of acidic lysosomes and tracking their pH changes in living cells.

The aim of the current investigation was to prepare and evaluate the potential use of solid lipid nanoparticles for the dermal delivery of spironolactone (SP). The spironolactone loaded SLN (SP-SLN) was prepared by emulsion-solvent evaporation method followed by ultrasonication. The properties of obtained SLNs were characterized by photon correlation spectroscopy (PCS), scanning tunneling microscopy (STM) and differential scanning calorimetry. FT-IR was also used to investigate any interaction between SP and excipients in the molecular level during the preparation of SLNs. The performance of the formulations was investigated in terms of drug release, skin permeation and also the retention of drug by the skin. The SP-SLNs presented spherical shape with the mean diameter, zeta potential and entrapment efficiency of 88.9 nm, -23.9 mV and 59.86%, respectively. DSC study showed that SP alone encapsulated in SLNs was in the amorphous form. FT-IR analysis revealed that there were hydrogen bond interactions between the SP alone and SLN components. The dissolution results revealed that the drug release from SP-SLNs was at least 4.9 times faster than original SP within the first 30 min. The cumulative amount of SP penetrated through rat skin from SP-SLNs was almost twofold that of the SP alone in 24h after the administration. In vitro permeation studies indicated that SP-SLN may be a promising vector for use in the topical treatment. It can be concluded that SLNs provide good skin permeation for SP and may be a promising carrier for topical delivery of spironolactone offering the biphasic release pattern that might be interesting for topical application resulting in an effective treatment for skin disorders such as acne., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Spironolactone is often used to treat hypertension caused by hyperaldosteronism, and as a result, can form concentrically laminated electron dense spironolactone body inclusions within the adrenal gland. Spironolactone bodies have not been investigated in a contemporary cohort or in patients treated with the more recently approved aldosterone antagonist, eplerenone., Methods: Spironolactone bodies were retrospectively investigated in patients treated for hyperaldosteronism (n=15) from 2012-2013 that underwent a subsequent adrenalectomy., Results: Inclusions were identified in 33% of patients treated with aldosterone antagonists, far less than previously reported. Remarkably, 50% of patients treated with spironolactone had inclusions while no patients using eplerenone alone had inclusions. Two patients treated with spironolactone had bodies present longer than the duration described in prior studies. Inclusions unexpectedly persisted in 1 patient despite increased duration of discontinued pharmacological treatment. A spectrum of histologic and ultrastructural findings were encountered within an adrenal cortical adenoma from a patient treated with both spironolactone and eplerenone. Ultrastructural examination revealed laminated electron dense bodies with the appearance of classic spironolactone inclusions as well as electron dense bodies without laminations and laminated bodies without electron dense cores., Conclusions: Our incidence rate of spironolactone bodies was much lower than previously reported, with no inclusions seen in patients treated solely with the newer aldosterone antagonist, eplerenone. Pathologists should be aware of these infrequently encountered inclusions, particularly as the clinical history of hyperaldosteronism and pharmacologic treatment may not be provided., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4597918761268031.

Spironolactone is a medicinally important molecule that is clinically used in the treatment and management of many diseases such as oedema and ascites in cirrhosis of the liver, malignant ascites, nephrotic syndrome, chronic lung disease, resistant hypertension, congestive heart failure, and primary hyperaldosteronism. Microbial transformations of spironolactone by Cunninghamella elegans ATCC 9245 was carried out. Two new hydroxylated derivatives, 12β-hydroxy-spironolactone and 2α-hydroxy-spironolactone, were synthesized. Their structures were characterized on the basis of the spectroscopic data. The substrate can be efficiently converted into the products within 72 h after its addition to the fermentation broth of C. elegans ATCC 9245., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Eplerenone (Inspra) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of 5α-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (DHT); (3) competitive antagonism of the androgen receptor; and (4) inhibition of 5α-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.

Background/aims: The mammalian target of rapamycin (mTOR) is a serine kinase that regulates phosphorylation (p) of its target ribosomal S6 kinase (S6K1), whose activation can lead to glomerular and proximal tubular cell (PTC) injury and associated proteinuria. Increased mTOR/S6K1 signaling regulates signaling pathways that target fibrosis through adherens junctions. Recent data indicate aldosterone signaling through the mineralocorticoid receptor (MR) can activate the mTOR pathway. Further, antagonism of the MR has beneficial effects on proteinuria that occur independent of hemodynamics., Methods: Accordingly, hypertensive transgenic TG(mRen2)27 (Ren2) rats, with elevated serum aldosterone and proteinuria, and age-matched Sprague-Dawley rats were treated with either a low dose (1 mg/kg/day) or a conventional dose (30 mg/kg/day) of spironolactone (MR antagonist) or placebo for 3 weeks., Results: Ren2 rats displayed increases in urine levels of the PTC brush border lysosomal enzyme N-acetyl-β-aminoglycosidase (β-NAG) in conjunction with reductions in PTC megalin, the apical membrane adherens protein T-cadherin and basolateral α-(E)-catenin, and fibrosis. In concert with these abnormalities, Ren2 renal cortical tissue also displayed increased Ser2448 (p)/activation of mTOR and Thr389 (p)-S6K1 and increased 3-nitrotyrosine (3-NT) content, a marker for peroxynitrite. Low-dose spironolactone had no effect on blood pressure but decreased proteinuria and β-NAG comparable to a conventional dose of this MR antagonist. Both doses of spironolactone attenuated ultrastructural maladaptive alterations and led to comparable reductions in (p)-mTOR/(p)-S6K1, 3-NT, fibrosis, and increased expression of α-(E)-catenin, T- and N-cadherin., Conclusions: Thereby, MR antagonism improves proximal tubule integrity by targeting mTOR/S6K1 signaling and redox status independent of changes in blood pressure., (Copyright © 2011 S. Karger AG, Basel.)

Background: The inherent reproducibility of a bioanalytical approach is usually sustained through incurred sample reanalysis (ISR). Questions relating to the number of ISRs, the right moment for performing reanalysis, the way of performing an appropriate statistical refinement of experimental data and actions to be taken in the case of failure are frequently raised., Results: Data resulting from ISR following a bioequivalence study for spironolactone formulations are discussed. Reanalysis of samples was carried out twice: immediately after the end of the study and after a period that overcame the long-term stability study achieved during method validation. The Bland-Altman approach was used to assess experimental results. ISR was successful over the short reanalysis period for both compounds. Data produced through reanalysis after the long-term period indicated a systematic positive bias for the metabolite canrenone (although results supported reproducibility). The results obtained for spironolactone were affected by a strong negative systematic bias and failed to support reproducibility. The explanation deals with the continuous conversion of spironolactone to canrenone in plasma samples. However, reproducibility of the method may be sustained by comparing original and repeated differences between concentration values in samples by means of a paired t-test, Wilcoxon sign rank-sum test and linear regression., Conclusions: Different statistical approaches for making data comparisons are discussed and may be successfully applied during reanalysis of samples from a bioequivalence study. Results of the evaluations may differ in accordance with the statistical procedure being applied, thus a definitive conclusion requires consideration of all specific experimental circumstances arising during production of the processed data.

Solid-state (1)H/(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to two steroid compounds: dehydroepiandrosterone (DHEA) and spironolactone (SPI), to analyze their conformations at the atomic level. In the absence of lipid, the high-resolution (13)C CP/MAS NMR signals of DHEA and SPI in a powder form reveal multiple patterns, with splittings of 30-160 Hz, indicating the existence of multiple conformations. In the mimic lipid environment formed by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, the resulting DHEA and SPI spectra revealed mostly singlet patterns, suggesting that these steroids undergo a conformational change leading to a specific conformation in the lipid environment. Evidence from chemical shift isotropy and anisotropy analysis indicates that DHEA might adopt conformations subtly different from that seen in solution and in the powder form. In conclusion, we demonstrate by solid-state NMR that the structures of DHEA and SPI may adopt slightly different conformations in different chemical environments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spironolactone, which is metabolized to canrenone, is often used in combination with digoxin. Potassium canrenoate is a similar drug that is also metabolized to canrenone. As a result of reported interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with two relatively new digoxin assays for application on ARCHITECT clinical chemistry platforms (cDig, particle-enhanced turbidimetric inhibition immunoassay) and ARCHITECT immunoassay platforms (iDig, chemiluminescent microparticle immunoassay), both from Abbott Diagnostics. When aliquots of drug-free serum pool were supplemented with various amounts of spironolactone, potassium canrenoate, and canrenone, no apparent digoxin concentration was observed using cDig assay on ARCHITECT c4000, c8000, and c16000 or iDig assay on i1000SR and i2000SR analyzers. In addition, we observed no false increase in serum digoxin value when aliquots of a digoxin pool were further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone. We conclude that both the cDig and iDig assays on the ARCHITECT analyzers are free from interferences by spironolactone, potassium canrenoate, and canrenone.

Comparison between evolutionarily distant receptors can provide critical insights into both structure and function. Sequence comparison between the mineralocorticoid receptors (MR) of the zebrafish (zMR) and human (hMR) reveals a high degree of sequence conservation in the major functional domains. We isolated a zMR cDNA to contrast the transcriptional response to a range of ligands and to establish whether a teleost MR exhibits the amino/carboxyl-terminal interaction (N/C-interaction) previously reported for the hMR. Aldosterone, deoxycorticosterone (DOC) and cortisol induced zMR transcriptional activity with similar efficacy to that observed with the hMR. The hMR antagonist, spironolactone, acted as an agonist with the zMR. The zMR exhibited an N/C-interaction in response to aldosterone but, in contrast to the hMR, cortisol and DOC predominantly stimulated the interaction in the zMR. Conservation of the N/C-interaction between evolutionarily distant MR provides evidence of functional significance., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg[middle dot]kg-1[middle dot]d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg[middle dot]kg-1[middle dot]d-1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1[beta], p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.

Background: In an evaluation of androstenedione results from patient serum samples using the Siemens Immulite 2500 analyser and manual Coat-A-Count (CAC) methods, three outliers were evident with grossly elevated results in the CAC assay., Methods: The clinic notes of three patients with apparently high serum androstenedione concentrations by the CAC assay were checked for medications. The samples were all from patients with polycystic ovary syndrome taking 100-200 mg/d of a steroidal antiandrogen (spironolactone). Two other patients on 50 mg spironolactone per day had less markedly higher androstendione results with the CAC assay. In a further five patients who were selected since they were on spironolactone and had high androstenedione results by the CAC method, spironolactone was temporarily withdrawn and fresh blood samples obtained for analysis., Results: Spironolactone treatment was associated with higher androstenedione concentrations measured by the CAC assay that reverted to normal on treatment withdrawal. Based on a single test with spironolactone at 1000 ng/mL, the manufacturer reported only 0.109% interference in the CAC assay., Conclusions: Spironolactone (and/or its metabolites) may interfere in the Siemens CAC assay for androstenedione but not in the Immulite 2500 assay. This experience highlights the need for information from clinicians on drug treatment when laboratory investigations are requested. Drug interferences in immunoassay are common and need evaluation beyond tests performed to certify laboratory reagents.

We evaluated the effects of intracerebroventricular (icv) infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial Na(+) channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11beta-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF Na(+) concentration ([Na(+)]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. Na(+)-rich aCSF increased beta-ENaC mRNA and immunoreactivity in the choroid plexus and increased alpha- and beta-ENaC immunoreactivities in the ependyma. Na(+)-rich aCSF increased alpha- and beta-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in beta-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, Na(+)-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [Na(+)] in the control, but not Na(+)-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [Na(+)] by Na(+)-rich aCSF. These results suggest that CSF Na(+) upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of Na(+) homeostasis in the brain.

Spironolactone, a potassium sparing diuretic metabolized to canrenone, is often used with digoxin to treat various conditions including congestive heart failure. Potassium canrenoate is a similar drug that is also metabolized to canrenone. Due to reported interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with Dimension Vista Digoxin immunoassay using Flex reagent cartridge. Aliquots of a drug-free serum pool were supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone and apparent digoxin values were measured using Dimension Vista digoxin assay, we observed none-detected value except when aliquots were supplemented with higher amounts of spironolactone or canrenone. Similarly, when aliquots of a serum digoxin pool (prepared by pooling specimens from patients receiving digoxin) where further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone, we observed moderately falsely elevated digoxin values only in specimens containing higher amounts of spironolactone or canrenone. We conclude that spironolactone and canrenone but not potassium canrenoate may cause modest interference with Dimension Vista digoxin assay but such interferences may not be clinically significant except with very high amounts of canrenone., (© 2010 Wiley-Liss, Inc.)

The ultra high performance liquid chromatography (UHPLC)-UV method for the simultaneous determination of furosemide, saluamine (furosemide metabolite), spironolactone, carnenone (spironolactone active metabolite), terbinafine, N-desmethylcarboxy terbinafine (terbinafine metabolite) and vancomycin in human plasma and urine is proposed. Good separation of the analytes was achieved with the gradient RP-UHPLC-UV with the mobile phase composed as acetonitrile and 0.1% formic acid. The determined substances were eluted from a Hypersil GOLD C(18)e (50 mm x 2.1 mm, 1.7 microm particles) column in 3.3 min. Good linear relationships were observed for all of the analytes (R(2) higher than 0.994). The limit of detection (LOD) values varied from 0.01 to 0.07 microg ml(-1), with vancomycin as an exception (0.11 microg ml(-1)). After protein precipitation and solid-phase extraction, samples of plasma and urine were analyzed. Thanks to the short analysis time and small quantities of urine or plasma needed, this method can be applied to routine clinical analysis.

This study aims to investigate hepatic pharmacokinetics of the four most common drugs (metoprolol, omeprazole, spironolactone, and furosemide) given to patients undergoing liver transplantation before surgery. The investigation was carried out in CCl(4)-induced fibrotic perfused rat livers and the results were compared to those in normal rat liver. Drug outflow fraction-time profiles were obtained after bolus injection into a single-pass-perfused normal or fibrotic rat liver. The pharmacokinetic parameters were estimated using previously developed barrier-limited and space-distributed models. The results showed a marked increase in the liver fibrosis index for CCl(4)-treated rats compared to controls (p<0.05). The extraction ratios (E) for all drugs were significantly lower (p<0.05) in fibrotic than in normal livers and the decrease in E was consistent with the decrease in intrinsic clearance and permeability-surface area product. In addition, other than for furosemide, the mean transit times for all drugs were significantly longer (p<0.01) in the fibrotic livers than in normal livers. Pharmacokinetic model and stepwise regression analyses suggest that these differences arise from a reduction in both the transport of drugs across the basolateral membrane and their metabolic clearance and were in a manner similar to those previously found for another group of drugs.

Unlabelled: Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown., Objective: Identify the function of estrogens and of different synthetic progestins on aldosterone salt-mediated renal disease., Methods: The role of 17beta-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment., Results: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (alpha-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels., Conclusion: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

Background and Purpose: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis., Experimental Approach: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques., Key Results: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone., Conclusion and Implications: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

Eplerenone is an aldosterone receptor antagonist indicated for the treatment of hypertension and congestive heart failure. Eplerenone contains an epoxy group, which offers greater mineralocorticoid receptor specificity. It is an effective antihypertensive that has been shown to reduce morbidity and mortality in individuals with left ventricular dysfunction post myocardial infarction. Studies are continuing to determine whether the benefit of mineralocorticoid receptor blockade in advanced congestive heart failure is also observed when eplerenone treatment is initiated in earlier stages of the disease. The most common side effect is hyperkalemia necessitating close monitoring in individuals with diabetes and proteinuria, heart failure or in those who are taking moderate CYP450 3A4 inhibitors. It is category B in pregnancy.

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. It has been well documented in the literature that spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with several digoxin immunoassays at concentrations expected after therapeutic usage of these drugs and falsely elevate or lower serum digoxin concentrations. Recently, Abbott Laboratories marketed a new Digoxin III immunoassay for application on the AxSYM analyzer. We studied the potential interference of these compounds with this new digoxin assay. The Tina-quant assay was used as the reference method because spironolactone, potassium canrenoate, and canrenone do not interfere with serum digoxin measurement using this assay. Aliquots of drug-free serum were supplemented with therapeutic and above therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin concentrations were measured using the Digoxin III assay and Tina-quant assay. Significant apparent digoxin concentrations were observed when the Digoxin III digoxin assay was used, but no apparent digoxin levels was observed using the Tina-quant assay. When serum pools prepared from patients receiving digoxin were further supplemented with these compounds in concentrations expected in sera of patients receiving these medications, falsely elevated digoxin levels were observed using Digoxin III assay, but no statistically significant change was observed using the Tina-quant assay. We conclude that spironolactone, potassium canrenoate, and their common metabolite canrenone interfere with the serum digoxin measurements using the new Digoxin III assay.

Steroid receptors belonging to the superfamily of nuclear receptors do not exist as single monomeric proteins but mediate their effects by the interaction with numerous other proteins, e.g., cofactors for transcription, but also other proteins involved in cellular signaling. This interaction may be ligand dependent, which explains the differential effects of receptor ligands. Whereas some receptors, e.g., the estrogen receptor, have been studied in great detail, much less is known about proteins interacting with the mineralocorticoid receptor (MR). In this study, we aimed to identify interacting proteins using a proteomics approach involving tagged receptor constructs. After affinity isolation of MR complexes, blue native electrophoresis revealed the presence of several populations of MR complexes differing in size and composition. During the identification of interacting proteins, various heat shock proteins but also several previously undescribed potential interactors were found, including 14-3-3-epsilon. We also demonstrate here that the cytosolic MR in the presence of detergent interacts in a ligand-selective manner with glucose-regulated protein 78 and propionyl-CoA carboxylase-beta precursor, which are found in the unliganded or aldosterone-containing complex but not with spironolactone.