Digoxin was one of the first identified RORγT receptor inverse agonists inhibiting the differentiation of Th17 cells. However, this compound exhibits inhibitory activity at relatively high concentrations that mediate cytotoxic effects. We previously identified several cardenolides that are structurally similar to digoxin that were able to induce RORγ/RORγT-dependent transcription. These observations encouraged us to reanalyze the effects of digoxin on RORγ/RORγT-dependent transcription at low, noncytotoxic concentrations. Digoxin induced RORγ/RORγT-dependent transcription in HepG2 and Th17 cells. Furthermore, analysis of the transcriptomes of Th17 cells cultured in the presence of digoxin revealed the induction of the expression of numerous Th17-specific genes, including IL17A/F, IL21, IL22, IL23R, CCR4, and CCR6. Thus, our study, which includes data obtained from intact cells, indicates that digoxin, similar to other cardenolides, is a potent RORγ/RORγT receptor activator and that its structure may serve as a starting point for the design of dedicated molecules that can be used in the development of adoptive cell therapy (ACT).

Na,K-ATPases are energy consuming ion pumps that are required for maintaining ion homeostasis in most cells. In the retina, Na,K-ATPases are especially important to sustain the dark current in photoreceptor cells needed for rapid hyperpolarization of rods and cones in light. Cardiac glycosides like digoxin inhibit the activity of Na,K-ATPases by targeting their catalytic alpha subunits. This leads to a disturbed ion balance, which can affect cellular function and survival. Here we show that the treatment of wild-type mice with digoxin leads to severe retinal degeneration and loss of vision. Digoxin induced cell death specifically in photoreceptor cells with no or only minor effects in other retinal cell types. Photoreceptor-specific cytotoxicity depended on the presence of bleachable rhodopsin. Photoreceptors of Rpe65 knockouts, which have no measurable rhodopsin and photoreceptors of Rpe65R91W mice that have <10% of the rhodopsin found in retinas of wild-type mice were not sensitive to digoxin treatment. Similarly, cones in the all-cone retina of Nrl knockout mice were also not affected. Digoxin induced expression of several genes involved in stress signaling and inflammation. It also activated proteins such as ERK1/2, AKT, STAT1, STAT3 and CASP1 during a period of up to 10 days after treatment. Activation of signaling genes and proteins, as well as the dependency on bleachable rhodopsin resembles mechanisms of light-induced photoreceptor degeneration. Digoxin-mediated photoreceptor cell death may thus be used as an inducible model system to study molecular mechanisms of retinal degeneration.

Adult male Wistar rats were treated with either 0.1 or 3 mg/kg body weight X day of digoxin for five days, then killed and stored at 4 degrees C for 12 h in an attempt to mimic the normal preautopsy procedures in our hospital. In rats treated with 0.1 mg/kg body weight X day, the antemortem serum digoxin concentrations (SDC) were 1.1 +/- 0.4 ng/mL while the 12-h postmortem concentration was markedly increased (16.3 +/- 5.9 ng/mL) (P less than 0.01). In rats treated with 3 mg/kg body weight X day, SDC was not changed significantly (11.2 +/- 4.8 ng/mL antemortem and 13.3 +/- 6 ng/mL postmortem). Postmortem redistribution of digoxin was assessed by injection of 125I-labelled digoxin with or without pretreatment with the unlabelled drug. The results indicate that after death passive redistribution of digoxin may take place. When the SDC are within the therapeutic or low toxic range, digoxin may reenter the blood. High antemortem serum concentrations of digoxin may prevent such passive redistribution. Therefore, antemortem digoxin intoxication cannot be reliably inferred on the basis of high postmortem levels of the drug. Digoxin intoxication can be ruled out when postmortem SDC remain within the therapeutic range. The above changes cast doubt on some of the forensic and cardiologic literature, which has in the past been based on incorrect assumptions concerning postmortem behavior of digoxin.

The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection of the concentration of digoxin spiked into in a plasma sample. The assay is based on a shift in the equilibrium of a DNA strand displacement competition reaction, and can be performed in 30 min for concentrations above 10 nM. The equilibrium shift occurs upon binding of anti-digoxigenin antibody. As a model, the assay provides a potential alternative to current small-molecule detection methods used for therapeutic drug monitoring.

Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1-10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes.

Numerous in vitro studies have suggested that digoxin suppresses inflammation and alters lipid metabolism. However, the effect of dioxin on atherosclerosis is poorly understood. The present study was conducted to determine whether digoxin affects the development of atherosclerosis in a murine model of atherosclerotic disease.

The role of digoxin (cardiac glycoside) in controlling the heart rate (HR) for the treatment of atrial fibrillation (AF) patients has not been explored in depth.

The effect of intravenous (IV) furosemide (2.5 mg/kg) on serum digoxin concentration, urine flow rate, and myocardial contractility and rhythm was studied in normal dogs and dogs treated with digoxin. Furosemide IV to animals which had not been receiving digoxin resulted in increased urine flow rate from 0.11 +/- 0.03 to 0.43 +/- 0.1 ml/min/kg, but had no effect on myocardial contractility, blood pressure, or cardiac rhythm. Furosemide IV to animals with steady-state therapeutic serum concentrations of digoxin (2.0 +/- 0.58 ng/ml) increased urine flow similarly but did not significantly alter serum digoxin concentration, blood pressure, myocardial contractility, or cardiac rhythm. Thus, contrary to some previous reports, furosemide does not interact with digoxin to produce an elevation in serum digoxin concentration or enhancement of the glycoside's myocardial action.

Sterile inflammation after tissue damage is a ubiquitous response, yet it has the highest amplitude in the liver. This has major clinical consequences, for alcoholic and non-alcoholic steatohepatitis (ASH and NASH) account for the majority of liver disease in industrialized countries and both lack therapy. Requirements for sustained sterile inflammation include increased oxidative stress and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independently of PKM2 kinase activity results in chromatin remodeling and downregulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin that can effectively protect the liver from ASH and NASH.

Digoxin has long been used for rate control in atrial fibrillation (AF); its safety remains controversial.We performed a literature search using MEDLINE (source PubMed, January 1, 1966, to July 31, 2015) and EMBASE (January 1, 1980, to July 31, 2015) with no restrictions. Studies that reported relative risk (RR) estimates with 95% confidence intervals (CIs) for the associations of interest were included. Pooled effect estimates were obtained by using random-effects meta-analysis.Twenty-two studies involving 586,594 patients were identified. Patients taking digoxin, as compared with those who took no digoxin, experienced an increased risk of death from any cause (RR: 1.29[95% CI 1.16-1.43]), even after reported adjustment for propensity scores (RR: 1.28[95% CI 1.18-1.39]). The risk of death was increased with patients with or without heart failure (RR: 1.12[95% CI 1.02-1.23] and RR: 1.26[95% CI 1.15-1.29], respectively), and patients taking or not taking beta blockers (RR: 1.17 [95% CI 1.06-1.30] and RR: 1.28 [95% CI 1.08-1.51], respectively). Digoxin use was also associated with increased risk of cardiovascular death (RR: 1.32 [95% CI 1.07-1.64]), arrhythmic death (RR: 1.38 [95% CI 1.07-1.79]), and stroke (RR: 1.20 [95% CI 1.004-1.44]). Digoxin treatment is associated with an absolute risk increase of 19 (95% CI 13-26) additional deaths from any cause per 1000 person-years.Digoxin use is associated with a significant increased risk for death from any cause in patients with AF. This finding suggests a need for reconsideration of present treatment recommendations on use of digoxin in AF.

Plasma iron excess can lead to iron accumulation in heart, kidney and liver. Heart failure is a clinical widespread syndrome. In thalassemia, iron overload cardiomyopathy is caused by iron accumulation in the heart that leads to cardiac damage and heart failure. Digoxin increases the intracellular sodium concentration by inhibition of Na+/K+-ATPase that affects Na+/Ca2+ exchanger (NCX), which raises intracellular calcium and thus attenuates heart failure. The mechanism of iron uptake into cardiomyocytes is not exactly understood.

Digoxin is a cardiac glycoside that has been reported to inhibit growth of multiple tumor cell types in vitro. The present study was assessing the cytogenetic effects of this drug on Chinese Hamster Ovary (CHO) cells. All experiments were performed in triplicate. The IC50 was 22.5 ± 0.8 µg/ml. To investigate the clastogenic effects of drug, chromosomal aberration in metaphase cells were analyzed. Chromatid breaks and polyploidy were the main types of aberration. Mitomycin-C and sodium arsenite were used as positive controls. CHO cells were exposed to different concentration of drug (5, 10, 15, 20 µg/ml) in 24 hours. All of the study aberrations and frequency of aberrant cells significantly increased as a function of digoxin concentration (for chromatid breaks: r = 0.881, df = 13, P < 0.001; for polyploidy: r = 0.777, df = 13, P = 0.001; for cells with aberrations: r = 0.926, df = 13, P < 0.001). The mitotic index negatively correlated with the concentration of digoxin (r = -0.978, df = 13, P < 0.001). All concentrations that cause chromosomal aberrations are in the cytotoxic range of the drug. The peak serum digoxin concentration (5 - 20 ng/ml) was very lower than concentrations we used in the present experiments. Further studies on valuation of chromatid breaks, micronuclei, and sister chromatid exchange in lymphocytes of patients who received digoxin, were recommended.

The poor outcome of treatments for fungal infections is a consequence of the increasing incidence of resistance to antifungal agents, mainly due to the overexpression of efflux pumps. To surpass this mechanism of resistance, a substance able to inhibit these pumps could be administered in association with antifungals. Saccharomyces cerevisiae possesses an efflux pump (Pdr5p) homologue to those found in pathogenic yeast. Digoxin is a natural product that inhibits Na+, K+-ATPase. The aim of this study was to evaluate whether digoxin and its derivatives (i.e., DGB, digoxin benzylidene) can inhibit Pdr5p, reversing the resistance to fluconazole in yeasts. An S. cerevisiae mutant strain that overexpresses Pdr5p was used in the assays. The effects of the compounds on yeast growth, efflux activity, and Pdr5p ATPase activity were measured. All derivatives enhanced the antifungal activity of fluconazole against S. cerevisiae, in comparison to fluconazole alone, with FICI values ranging from 0.031 to 0.500. DGB 1 and DGB 3 presented combined effects with fluconazole against a Candida albicans strain, with fractional inhibitory concentration index (FICI) values of 0.625 and 0.281, respectively The compounds also inhibited the efflux of rhodamine 6G and Pdr5p ATPase activity, with IC50 values ranging from 0.41 μM to 3.72 μM. The results suggest that digoxin derivatives impair Pdr5p activity. Considering the homology between Pdr5p and efflux pumps from pathogenic fungi, these compounds are potential candidates to be used in association with fluconazole to treat resistant fungal infections.

In previous studies we have demonstrated a natriuretic factor of small molecular weight (less than 1,000 Daltons) in the serum and urine of salt loaded subjects. This factor isolated from salt loaded animals inhibits the Na-K-ATPase enzyme system. In addition, the natriuretic material isolated from plasma of salt-loaded dogs was shown to bind to specific digoxin antibodies. It was therefore suggested that a digitalis-like endogenous natriuretic factor (endoxin) is released in response to saline loading. In the present study we therefore investigated the presence of such an endogenous natriuretic digitalis-like activity in the urine of healthy volunteers during high salt intake. Using Sephadex G-25 for chromatographic separation of urine a material elutes as a single peak in the natriuretic post-salt fraction IV which is specifically bound to digoxin antiserum complex. Mean peak activity amounted to 1.55 +/- 0.48 ng/ml digoxin equivalents. We further purified the natriuretic material by immunoprecipitation with the digoxin antiserum complex. This purification procedure resulted in a more than 10-fold increase in specific natriuretic activity from 2.7 +/- 0.4 to 30.4 +/- 5.8 muEq Na+ x min-1 x mg-1. Thus the digitalis-like natriuretic activity previously observed in the plasma of saline loaded dogs is also present in the urine of healthy subjects during high dietary salt intake. Immunoprecipitation may offer a meaningful tool for further isolation and identification of the natriuretic hormone(s).

Digoxin extracted from the foxglove plant is a widely prescribed natural product for treating heart failure. It is listed as an essential medicine by the World Health Organization. However, how the foxglove plant synthesizes digoxin is mostly unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450scc), which catalyzes the first and rate-limiting step. Here we identify the long-speculated foxglove P450scc through differential transcriptomic analysis. This enzyme converts cholesterol and campesterol to pregnenolone, suggesting that digoxin biosynthesis starts from both sterols, unlike previously reported. Phylogenetic analysis indicates that this enzyme arises from a duplicated cytochrome P450 CYP87A gene and is distinct from the well-characterized mammalian P450scc. Protein structural analysis reveals two amino acids in the active site critical for the foxglove P450scc's sterol cleavage ability. Identifying the foxglove P450scc is a crucial step toward completely elucidating digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Digoxin is a high-alert medication because of its narrow therapeutic range and high drug-to-drug interactions (DDIs). Approximately 50% of digoxin toxicity cases are preventable, which motivated us to improve the treatment outcomes of digoxin. The objective of this study is to apply machine learning techniques to predict the appropriateness of initial digoxin dosage. A total of 307 inpatients who had their conditions treated with digoxin between 2004 and 2013 at a medical center in Taiwan were collected in the study. Ten independent variables, including demographic information, laboratory data, and whether the patients had CHF were also noted. A patient with serum digoxin concentration being controlled at 0.5-0.9 ng/mL after his/her initial digoxin dosage was defined as having an appropriate use of digoxin; otherwise, a patient was defined as having an inappropriate use of digoxin. Weka 3.7.3, an open source machine learning software, was adopted to develop prediction models. Six machine learning techniques were considered, including decision tree (C4.5), k-nearest neighbors (kNN), classification and regression tree (CART), randomForest (RF), multilayer perceptron (MLP), and logistic regression (LGR). In the non-DDI group, the area under ROC curve (AUC) of RF (0.912) was excellent, followed by that of MLP (0.813), CART (0.791), and C4.5 (0.784); the remaining classifiers performed poorly. For the DDI group, the AUC of RF (0.892) was the best, followed by CART (0.795), MLP (0.777), and C4.5 (0.774); the other classifiers' performances were less than ideal. The decision tree-based approaches and MLP exhibited markedly superior accuracy performance, regardless of DDI status. Although digoxin is a high-alert medication, its initial dose can be accurately determined by using data mining techniques such as decision tree-based and MLP approaches. Developing a dosage decision support system may serve as a supplementary tool for clinicians and also increase drug safety in clinical practice.

Digoxin is used in patients with chronic heart failure (CHF) who remain symptomatic despite optimal medical treatment. Impaired renal function is commonly associated with CHF. We investigated the relation between digoxin use and change in renal function over time in patients with CHF.

Previous in vitro studies have reported the inhibitory effect of green tea on p-glycoprotein (p-gp) encoded by ABCB1. This study aimed to investigate the effect of green tea on the pharmacokinetics of digoxin, a typical probe drug of p-gp.

The aim of the study is to examine the possible therapeutic effects of a known cardiac glycoside, digoxin, on a rat model of MTX-induced hepatotoxicity.

Cardiac glycosides, such as digoxin, inhibit Na+/K+-ATPases and cause secondary activation of Na+/Ca2+ exchangers. Preclinical investigations have suggested that digoxin may have anticancer properties. In order to clarify the functional mechanisms of digoxin in cancer, we performed an integrative analysis of clinical and bioinformatics databases. The US Food and Drug Administration Adverse Event Reporting System and the Japan Medical Data Center claims database were used as clinical databases to evaluate reporting odds ratios and adjusted sequence ratios, respectively. The BaseSpace Correlation Engine and Connectivity Map bioinformatics databases were used to investigate molecular pathways related to digoxin anticancer mechanisms. Clinical database analyses suggested an inverse association between digoxin and four cancers: gastric, colon, prostate and haematological malignancy. The bioinformatics database analysis suggested digoxin may exert an anticancer effect via peroxisome proliferator-activated receptor α and apoptotic caspase cascade pathways. Our integrative analysis revealed the possibility of digoxin as a drug repositioning candidate for cancers.