Profiling gene expression dynamics underpinning conventional testing approaches to better inform pre-clinical evaluation of an age appropriate spironolactone formulation.

There is a need to accelerate paediatric formulation evaluation and enhance quality of early stage data in drug development to alleviate the information pinch point present between formulation development and clinical evaluation. This present work reports application of DNA microarrays as a high throughput screening tool identifying markers for prediction of bioavailability and formulation driven physiological responses. With a focus on enhancing paediatric medicine provision, an oral liquid spironolactone suspension was formulated addressing a paediatric target product profile. Caco-2 cells cultured on transwell inserts were implemented in transport assays in vitro and DNA microarrays were used to examine gene expression modulation. Wistar rats were used to derive in vivo bioavailability data. In vitro, genomic, and in vivo data sets were concurrently evaluated linking drug transport and the genomic fingerprint generated by spironolactone formulation exposure. Significant changes in gene expression are reported as a result of formulation exposure. These include genes coding for ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, cytochrome P450 (CYP) enzymes, and carboxylesterase enzymes. Genomic findings better inform pre-clinical understanding of pharmacokinetic and pharmacodynamic responses to spironolactone and its active metabolites than current in vitro drug transport assays alone.

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion.

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.

Epimeric spirolactone-type triterpenoids from Abies faxoniana Rehd.

Phytochemical investigation of Abies faxoniana Rehd. led to the isolation of two pairs of new epimeric spirolactone-type triterpenoids (1/1' and 2/2') and 11 known terpenoids (3-13). Compounds 1/1' and 2/2' were isolated as epimeric mixtures due to the C-23 ketal tautomerism in their spirolactone structures. The dynamic HPLC manifested that the C-23 epimeric mixtures interconverted into each other in solution. Structure determinations were based on extensive NMR and HRESIMS spectroscopic analysis. Meanwhile, their cytotoxic activities were tested by MTT method. Compound 5 showed cytotoxicities against MCF-7 and A549 cells with IC50 values of 6.5 and 5.7µM, respectively. Compounds 1/1' had IC50 values of 10.0 and 12.3µM for Huh7 and SMMC7721 cells, respectively.

Pharmacokinetic study of eplerenone in rats after long-term coadministration with buckwheat tea.

The aim of this study was to investigate the effect of long-term intake of Tartary buckwheat tea on the pharmacokinetics (PK) of eplerenone in rats. A validated high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was established to determine the eplerenone in plasma, and the portal vein absorption model was applied to conduct the pharmacokinetic study. Two groups of animals-buckwheat tea group and control group-were involved in this study. Plasma samples were obtained at different time points after administration, and were separated on Shimadzu HPLC-MS 2020 instruments. The method showed good linearity (r=0.9988) over a wide dynamic range (0.20-50 µg/mL). Within- and between-batch precisions ranged from 2.13% to 7.90%. The extraction recovery rates ranged from 91.96% to 94.96%. The data showed that in the Tartarian buckwheat group the area under the curve and maximum concentration of eplerenone were reduced compared with those of the blank group (p<0.01), but the time to reach peak concentrations of eplerenone (p<0.01) was prolonged. The results suggested that long-term consumption of Tartary buckwheat tea might induce the activities of the hepatic drug metabolizing enzyme, which can accelerate the metabolism of eplerenone. According to the results, the dosage of eplerenone should be adjusted in hypertension treatment trials when administered with Tartary buckwheat or Tartary buckwheat-containing dietary supplements to avoid potential drug interactions.

Chemical labeling of electrochemically cleaved peptides.

RATIONALE: Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. METHODS: Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. RESULTS: Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. CONCLUSIONS: This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.

Clinically insignificant negative interferences of spironolactone, potassium canrenoate, and their common metabolite canrenone in new dimension vista LOCI digoxin immunoassay.

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, which is also metabolized to canrenone. Due to reported both positive and negative interference of spironolactone, potassium canrenoate, and their common metabolite canrenone with digoxin immunoassays, we investigated potential interference of these compounds with the new homogenous sequential chemiluminescent assay for digoxin based on the luminescent oxygen channeling technology (LOCI digoxin) for application on the Dimension and Vista platform. When 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 LOCI digoxin assay, we observed no detected value except when aliquots were supplemented with very high amounts of potassium canrenoate or canrenone. However, we observed that apparent digoxin concentrations were very low. When aliquots of a serum digoxin pool (prepared by pooling specimens from patients receiving digoxin), were further supplemented with various amounts of spironolactone, potassium canrenoate, or canrenone and serum digoxin concentrations were remeasured using the LOCIdigoxin assay, only statistically significant falsely lower digoxin values (negative interference) were observed in specimens containing very high amounts of canrenone or potassium canrenoate. However, such small bias may not have any clinical significance. We conclude that new Dimension Vista LOCI digoxin assay is virtually free from interferences of spironolactone, potassium canrenoate, and their common metabolite canrenone.

SM-368229, a novel selective and potent non-steroidal mineralocorticoid receptor antagonist with strong urinary Na+ excretion activity.

Mineralocorticoid receptor (MR) antagonists, such as spironolactone (SPI) and eplerenone (EPL), are useful for the treatment of hypertension and heart failure. However, the use of these two agents has been limited due to endocrine disturbance (SPI) and poor drug action (EPL). In our search for safer and more effective MR antagonists, we identified SM-368229 as a novel non-steroidal MR antagonist. SM-368229 showed strong MR inhibitory activity with IC(50) values of 0.021 and 0.13 µM in the binding assay and reporter-gene assay, respectively. The selectivity of SM-368229 for MR was 18-fold higher than that for other steroid receptors, such as androgen, progesterone, and glucocorticoid receptors. SM-368229 dose-dependently increased urinary Na(+)/K(+) ratio with an ED(50) value of 5.6 mg/kg in adrenalectomized rats treated with deoxycorticosterone acetate, and its efficacy was superior to that of SPI (ED(50) = 14 mg/kg) or EPL (ED(50) = 147 mg/kg). Moreover, even at high doses of 100 and 300 mg/kg, SM-368229 showed very weak anti-androgenic effect in methyltestosterone-treated male rats and no progestagenic effect in estrus cycle synchronized female rats. These findings indicate that SM-368229 may offer a new promising therapeutic option for the treatment of hypertension and heart failure.

Effect of spironolactone, potassium canrenoate, and their common metabolite canrenone on Dimension Vista Digoxin Assay.

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.

A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities.

Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.

A bioactive spirolactone iridoid and triterpenoids from Himatanthus sucuuba.

Himatanthus sucuuba is an Amazonian tree with abundant, yet conflicting ethnobotanical information. Investigation of the polar and non-polar constituents led to the isolation of plumericin, a bioactive spirolactone iridoid, and four known pentacylic triterpenes: lupeol acetate, lupeol cinnamate, lupeol beta-phenyl propionate, and alpha-amyrin cinnamate.

Aldosterone receptor antagonism normalizes vascular function in liquorice-induced hypertension.

The enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) provides mineralocorticoid receptor specificity for aldosterone by metabolizing glucocorticoids to their receptor-inactive 11-dehydro derivatives. The present study investigated the effects of the aldosterone receptor antagonists spironolactone and eplerenone on endothelial function in liquorice-induced hypertension. Glycyrrhizic acid (GA), a recognized inhibitor of 11beta-HSD2, was supplemented to the drinking water (3 g/L) of Wistar-Kyoto rats over a period of 21 days. From days 8 to 21, spironolactone (5.8+/-0.6 mg. kg(-1). d(-1)), eplerenone (182+/-13 mg. kg(-1). d(-1)), or placebo was added to the chow (n=7 animals per group). Endothelium-dependent or -independent vascular function was assessed as the relaxation of preconstricted aortic rings to acetylcholine or sodium nitroprusside, respectively. In addition, aortic endothelial nitric oxide synthase (eNOS) protein content, nitrate tissue levels, and endothelin-1 (ET-1) protein levels were determined. GA increased systolic blood pressure from 142+/-8 to 185+/-9 mm Hg (P<0.01). In the GA group, endothelium-dependent relaxation was impaired compared with that in controls (73+/-6% versus 99+/-5%), whereas endothelium-independent relaxation remained unchanged. In the aortas of 11beta-HSD2-deficient rats, eNOS protein content and nitrate tissue levels decreased (1114+/-128 versus 518+/-77 microgram/g protein, P<0.05). In contrast, aortic ET-1 protein levels were enhanced by GA (308+/-38 versus 497+/-47 pg/mg tissue, P<0.05). Both spironolactone and eplerenone normalized blood pressure in animals on GA (142+/-9 and 143+/-9 mm Hg, respectively, versus 189+/-8 mm Hg in the placebo group; P<0.01), restored endothelium-dependent relaxation (96+/-3% and 97+/-3%, respectively, P<0.01 versus placebo), blunted the decrease in vascular eNOS protein content and nitrate tissue levels, and normalized vascular ET-1 levels. This is the first study to demonstrate that aldosterone receptor antagonism normalizes blood pressure, prevents upregulation of vascular ET-1, restores NO-mediated endothelial dysfunction, and thus, may advance as a novel and specific therapeutic approach in 11beta-HSD2-deficient hypertension.

Variable mapping of structure-activity relationships: application to 17-spirolactone derivatives with mineralocorticoid activity.

Fifty-four steroid homologs, belonging to the series of 17-spirolactones, were modelled by molecular and quantum mechanics. We studied the affinity of these compounds for the cytosolic mineralocorticoid receptor by way of various parameters describing each structure and its molecular properties. After the failure of a classic preliminary QSAR study, demonstrating the nonlinear relationships between affinity and structural descriptors, we constructed a model allowing us to predict the affinity of new compounds. Our method is based on simple graphic tools coupled to a cluster significance analysis. A complementary study of the activity relating the prediction of the antagonist/agonist character of 37 high-affinity compounds was also carried out using the same methodology. The principal electronic and structural characteristics leading to a selective activity were revealed.