Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Evaluation of the Anticancer and Biological Activities of Istaroxime via Ex Vivo Analyses, Molecular Docking and Conceptual Density Functional Theory Computations.

Cancer is a disease that occurs as a result of abnormal or uncontrolled growth of cells due to DNA damage, among many other causes. Certain cancer treatments aim to increase the excess of DNA breaks to such an extent that they cannot escape from the general mechanism of cell checkpoints, leading to the apoptosis of mutant cells. In this study, one of the Sarco-endoplasmic reticulum Ca2+ATPase (SERCA2a) inhibitors, Istaroxime, was investigated. There has been very limited number of articles so far reporting Istaroxime's anticancer activity; thus, we aimed to evaluate the anticancer effects of Istaroxime by cell proliferation assay and revealed the cytotoxic activity of the compound. We further determined the interaction of Istaroxime with topoisomerase enzymes through enzyme activity tests and detailed molecular modeling analysis. Istaroxime exhibited an antiproliferative effect on A549, MCF7, and PC3 cell lines and inhibited Topoisomerase I, suggesting that Istaroxime can act as a Topoisomerase I inhibitor under in vitro conditions. Molecular docking analysis supported the experimental observations. A chemical reactivity analysis of the Istaroxime molecule was made in the light of Density Functional Theory computations. For this aim, important chemical reactivity descriptors such as hardness, electronegativity, and electrophilicity were computed and discussed as detailed.

Effect of thyroid hormones administration on urinary endogenous steroid profile of the athlete biological passport.

This work focused on the possible alterations of the markers of the steroidal module of the athlete biological passport, considering samples of athletes declaring and not-declaring the supplementation of thyroid hormones (TH) in the Doping Control Form (DCF). Concentrations of 5α-androstane-3α,17ß-diol (5α-Adiol), 5ß-androstane-3α,17ß-diol (5ß-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA), and 11ß-hydroxy-androsterone (OHA) were calculated using internal standards and external calibration by gas chromatography-tandem mass spectrometry. Also, ratios between the above biomarkers were also estimated. The data set was composed of samples from females and males declaring and not-declaring TH supplementation in the DCF. To corroborate these observations, a controlled urinary excretion study was carried out with multiple doses of sodium liothyronine (T3). Female data showed significant differences for the concentrations of 5α-Adiol, A, DHEA, E, OHA, and T and the ratio A/Etio between FD and FND groups, whereas the male groups only showed significant differences in OHA concentration. In both cases, males and females declaring the consumption of levothyroxine showed narrower data distribution and diminished percentiles from 17% to 67% with respect to the not-declaring corresponding groups (p < 0.05). Concentrations of 5α-metabolites showed a higher depression for the FND, and both FD and MD groups showed a peculiar behavior for the PD concentrations. The controlled study agreed with the observations, mainly for the female group with significant differences for concentrations of E, Etio, 5α-Adiol, and 5ß-Adiol after TH administration. The interpretation of the steroid markers of the ABP should consider TH administrations.

Safety and Efficacy of Istaroxime 1.0 and 1.5 µg/kg/min for Patients With Pre-Cardiogenic Shock.

INTRODUCTION: Istaroxime was shown, in a small study, to increase systolic blood pressure (SBP) in patients with pre-cardiogenic shock (CS) due to acute heart failure (AHF). OBJECTIVES: In the current analysis, we describe the effects of 2 doses of istaroxime 1.0 (Ista-1) and 1.5 µg/kg/min (Ista-1.5). METHODS: The target dose of istaroxime, administered in a double-blind, placebo-controlled fashion, was 1.5 µg/kg/min in the first cohort (n = 24), and it was reduced to 1.0 µg/kg/min in subsequent patients (n = 36). RESULTS: Ista-1 was associated with numerically larger effects on SBP area under the curve, with a 93.6% relative increase from baseline during the first 6 hours with Ista-1 vs 39.5% for Ista-1.5, and with a 49.4% and 24.3% relative increase, respectively, at 24 hours. Compared to placebo, Ista-1.5 had more worsening HF events until day 5 and fewer days alive out of hospital (DAOH) through day 30. Ista-1 had no worsening HF events, and DAOH to day 30 were significantly increased. Effects on echocardiographic measures were similar, although decreases in left ventricular end systolic and diastolic volumes were numerically larger in the Ista-1 group. Ista-1, but not Ista-1.5, showed numerically smaller creatinine increases and larger decreases in natriuretic peptides as compared to placebo. There were 5 serious adverse events in Ista-1.5 (4 of which were cardiac) but only 1 in Ista-1. CONCLUSIONS: In patients with pre-CS due to AHF, istaroxime 1.0 µg/kg/min induced beneficial effects on SBP and DAOH. Clinical benefits appear to be reached at dosages less than 1.5 ug/kg/min.

Early conversion of maternal androgens affects the embryo already in the first week of development.

Maternal androgen exposure has potent effects on offspring development. As substantial levels of maternal androgens are deposited in avian egg yolks, avian eggs are frequently used to study maternal effects, with a strong focus on post-natal development. However, the underlying pathways are largely unknown. Since the hormones are taken up during the embryonic phase, and these are rapidly metabolized by avian embryos into metabolites such as etiocholanolone, we studied the effects of yolk androgens (testosterone and androstenedione) and their metabolite etiocholanolone during the first few days of embryonic development. As embryonic heart rate is often used as an indicator of embryonic development, we measured the heart rate from day 3 to day 6 of incubation by using a shell-less culture technique in rock pigeon eggs (Columba livia). Increased androgen exposure increased heart rate, and increased etiocholanolone mimicked this effect, albeit in a small sample size. This indicates that exposure to maternal androgens increases embryonic overall metabolism which may account for the developmental outcomes found in previous studies such as increased growth. Moreover, etiocholanolone is likely to be an important metabolite in a non-genomic pathway underlying the androgen-mediated maternal effect.

Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Safety and efficacy of istaroxime in patients with acute heart failure-related pre-cardiogenic shock - a multicentre, randomized, double-blind, placebo-controlled, parallel group study (SEISMiC).

AIMS: We examined the effects of istaroxime in patients hospitalized for acute heart failure (AHF) related Society for Cardiovascular Angiography and Interventions (SCAI) stage B pre-cardiogenic shock (CS). METHODS AND RESULTS: Sixty patients with AHF without acute myocardial infarction with pre-CS, defined as systolic blood pressure (SBP) <90 mmHg without hypoperfusion, venous lactate ≥2 mmol/L and/or mechanical or inotropic support, were randomized to istaroxime 1.0-1.5 µg/kg/min or placebo for 24 h. The primary endpoint, the adjusted area under the curve (AUC) change in SBP from time of treatment to 6 h, was 53.1 (standard error [SE] 6.88) mmHg × hour versus 30.9 (SE 6.76) mmHg × hour with istaroxime versus placebo (p = 0.017). Adjusted SBP AUC at 24 h was 291.2 (SE 27.5) versus 208.7 (SE 27.0) mmHg × hour (p = 0.025). At 24 h, some echocardiographic measurements improved with istaroxime versus placebo including cardiac index (+0.21 L/min/m2 ; p = 0.016), left atrial area (-1.8 cm2 ; p = 0.008), and left ventricular end-systolic volume (-12.0 ml; p = 0.034). There were no significant differences in pulse pressure, laboratory measurements, serious adverse events or adverse events between the treatment groups except for more nausea, vomiting and infusion site pain in the istaroxime-treated patients. In a post-hoc analysis, patients receiving ≤1.0 µg/kg/min versus 1.5 µg/kg/min had similar increase in blood pressure, but a trend towards less adverse events. CONCLUSION: In a phase 2a study of patients with AHF related pre-CS, istaroxime improved blood pressure and some echocardiography measures related to heart failure and was well tolerated.

Investigations on the in vivo metabolism of 5α-androst-2-en-17-one.

RATIONALE: The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2ß,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. METHODS: The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. RESULTS: More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5ß-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5ß-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. CONCLUSIONS: The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5ß-configuration has not been reported so far and should be further investigated.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Testing the role of testosterone versus estrogens in mediating reproductive transitions in female rhesus macaques.

In male vertebrates, testosterone is generally known to coordinate reproductive trade-offs, in part by promoting the transition to the next reproduction at the expense of current parental care. The role of testosterone in reproductive transitions has been little tested in female vertebrates, especially in mammals. The present study sought to fill this gap, by first undertaking an experimental study, in which we identified DHT, androstenediol, and in particular etiocholanolone, as fecal androgen metabolites which reflect serum testosterone concentration in female rhesus macaques (Macaca mulatta). Using concentrations of fecal etiocholanolone as proxy for circulating testosterone, we then conducted a field study on 46 free-ranging rhesus macaques of Cayo Santiago, Puerto Rico, to test if testosterone mediates the trade-off between reproductive transition (a higher chance of reproducing in the next year) and current reproduction (providing more care to current offspring). While the evidence for testosterone was weak, the testing of fecal immunoreactive estrogen metabolites suggested a potential role of estrogen in reproductive trade-offs. We found large individual differences in fecal etiocholanolone concentrations during the early postpartum period that were unexplained even after accounting for sociodemographic factors such as age and dominance rank. Further investigation is needed to understand this variation. Our study suggests that the actions of testosterone in females may not have evolved to fulfil the same role in primate reproductive transitions as it does in males, and we encourage more studies to consider the function of testosterone in reproductive behaviors and life history transitions in females of mammalian taxa.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

AIMS: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. METHODS AND RESULTS: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. CONCLUSIONS: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Istaroxime treatment ameliorates calcium dysregulation in a zebrafish model of phospholamban R14del cardiomyopathy.

The heterozygous Phospholamban p.Arg14del mutation is found in patients with dilated or arrhythmogenic cardiomyopathy. This mutation triggers cardiac contractile dysfunction and arrhythmogenesis by affecting intracellular Ca2+ dynamics. Little is known about the physiological processes preceding induced cardiomyopathy, which is characterized by sub-epicardial accumulation of fibrofatty tissue, and a specific drug treatment is currently lacking. Here, we address these issues using a knock-in Phospholamban p.Arg14del zebrafish model. Hearts from adult zebrafish with this mutation display age-related remodeling with sub-epicardial inflammation and fibrosis. Echocardiography reveals contractile variations before overt structural changes occur, which correlates at the cellular level with action potential duration alternans. These functional alterations are preceded by diminished Ca2+ transient amplitudes in embryonic hearts as well as an increase in diastolic Ca2+ level, slower Ca2+ transient decay and longer Ca2+ transients in cells of adult hearts. We find that istaroxime treatment ameliorates the in vivo Ca2+ dysregulation, rescues the cellular action potential duration alternans, while it improves cardiac relaxation. Thus, we present insight into the pathophysiology of Phospholamban p.Arg14del cardiomyopathy.

Activation of the α1ß2γ2L GABAA Receptor by Physiological Agonists.

The Cl- permeable GABAA receptor is a major contributor to cellular inhibition in the brain. The receptor is normally activated by synaptically-released or ambient GABA but is sensitive to a number of physiological compounds such as ß-alanine, taurine, and neurosteroids that, to various degrees, activate the receptor and modulate responses either to the transmitter or to each other. Here, we describe α1ß2γ2L GABAA receptor activation and modulation by combinations of orthosteric and allosteric activators. The overall goal was to gain insight into how changes in the levels of endogenous agonists modulate receptor activity and influence cellular inhibition. Experimental observations and simulations are described in the framework of a cyclic concerted transition model. We also provide general analytical solutions for the analysis of electrophysiological data collected in the presence of combinations of active compounds.

Sensitive detection of testosterone and testosterone prohormone administrations based on urinary concentrations and carbon isotope ratios of androsterone and etiocholanolone.

The testing strategy for the detection of testosterone (T) or T-prohormones is based on the longitudinal evaluation of urinary steroid concentrations accompanied by subsequent isotope ratio mass spectrometry (IRMS)-based confirmation of samples showing atypical concentrations or concentration ratios. In recent years, the IRMS methodology focussed more and more on T itself and on the metabolites of T, 5α- and 5ß-androstanediol. These target analytes showed the best sensitivity and retrospectivity, but their use has occasionally been challenging due to their comparably low urinary concentrations. Conversely, the carbon isotope ratios (CIR) of the main urinary metabolites of T, androsterone (A) and etiocholanolone (EITO), can readily be measured even from low urine volumes; those however, commonly offer a lower sensitivity and shorter retrospectivity in uncovering T misuse. Within this study, the CIRs of A and ETIO were combined with their urinary concentrations, resulting in a single parameter referred to as 'difference from weighted mean' (DWM). Both glucuronidated and sulfated steroids were investigated, encompassing a reference population (n = 110), longitudinal studies on three individuals, influence of ethanol in two individuals, and re-analysis of several administration studies including T, dihydrotestosterone, androstenedione, epiandrosterone, dehydroepiandrosterone, and T-gel. Especially DWM calculated for the sulfoconjugated steroids significantly prolonged the detection time of steroid hormone administrations when individual reference ranges were applied. Administration studies employing T encompassing CIR common for Europe (-23.8‰ and -24.4‰) were investigated and, even though for a significantly shorter time period and less pronounced, DWM could demonstrate the exogenous source of T metabolites.

Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime.

Background Istaroxime is an inhibitor of Na+/K+ ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban-dependent inhibition of the sarcoplasmic reticulum Ca2+ ATPase. We have previously shown that pharmacologic Na+/K+ ATPase inhibition promotes calcium/calmodulin-dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na+/K+ ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi-inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin-dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin-dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca2+ spark and wave frequency but increased the proportion of aborted Ca2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime-dependent relief on phospholamban-dependent sarcoplasmic reticulum Ca2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin-dependent kinase II-dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca2+ handling.

Prospective evaluation of urinary steroids and prostate carcinoma-induced deviation: preliminary results.

BACKGROUND: The serum prostate-specific antigen is the most widespread biomarker for prostate disease. Its low specificity for prostatic malignancies is a matter of concern and the reason why new biomarkers for screening purposes are needed. The correlation between altered production of the main steroids and prostate carcinoma (PCa) occurrence is historically known. The purpose of this study is to evaluate the modifications of a comprehensive urinary endogenous steroidal profile (USP) induced by PCa, by multivariate statistical methods. METHODS: A total of 283 Italian subjects were included in the study, 139 controls and 144 PCa-affected patients. The USP, including 17 steroids and five urinary steroidal ratios, was quantitatively evaluated using gas chromatography coupled with single quadrupole mass spectrometry (GC-MS). The data were interpreted using a chemometric, multivariate approach (intrinsically more sensible to alterations with respect to traditional statistics) and a model for the discrimination of cancer-affected profiles was built. RESULTS: Two multivariate classification models were calculated, the former including three steroids with the highest statistical significance (e.g. testosterone, etiocholanolone and 7ß-OH-DHEA) and PSA values, the latter considering the three steroids' levels only. Both models yielded high sensitivity and specificity scores near to 70%, resulting significantly higher than PSA alone. CONCLUSIONS: Three USP steroids resulted significantly altered in our PCa population. These preliminary results, combined with the simplicity and low-cost of the analysis, open to further investigation of the potential role of this restricted USP in PCa diagnosis.

Validation of steroid sulfates deconjugation for metabolic studies. Application to human urine samples.

BACKGROUND: Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected. METHODS: Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations. RESULTS: The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4ß-hydroxy-DHEA, 5-androstene-3ß,17ß-diol and 5α-androstane-3ß,17ß-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described. CONCLUSIONS: Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample.