Quo vadis Cardiac Glycoside Research?

Cardiac glycosides (CGs), toxins well-known for numerous human and cattle poisoning, are natural compounds, the biosynthesis of which occurs in various plants and animals as a self-protective mechanism to prevent grazing and predation. Interestingly, some insect species can take advantage of the CG's toxicity and by absorbing them, they are also protected from predation. The mechanism of action of CG's toxicity is inhibition of Na+/K+-ATPase (the sodium-potassium pump, NKA), which disrupts the ionic homeostasis leading to elevated Ca2+ concentration resulting in cell death. Thus, NKA serves as a molecular target for CGs (although it is not the only one) and even though CGs are toxic for humans and some animals, they can also be used as remedies for various diseases, such as cardiovascular ones, and possibly cancer. Although the anticancer mechanism of CGs has not been fully elucidated, yet, it is thought to be connected with the second role of NKA being a receptor that can induce several cell signaling cascades and even serve as a growth factor and, thus, inhibit cancer cell proliferation at low nontoxic concentrations. These growth inhibitory effects are often observed only in cancer cells, thereby, offering a possibility for CGs to be repositioned for cancer treatment serving not only as chemotherapeutic agents but also as immunogenic cell death triggers. Therefore, here, we report on CG's chemical structures, production optimization, and biological activity with possible use in cancer therapy, as well as, discuss their antiviral potential which was discovered quite recently. Special attention has been devoted to digitoxin, digoxin, and ouabain.

Bibliometric profile of global scientific research on digoxin toxicity (1849-2015).

Digoxin is a cardiac glycoside derived from the common foxglove digitalis purpurea and has been available for several centuries as a medicinal agent. Despite extensive patient experience over many years, there remains some controversy regarding the possibility that digoxin might have a deleterious effect on survival. This study was constructed to assess trends in digoxin toxicity research using well-established qualitative and quantitative bibliometric indicators. The current study is based on publications that have been indexed in Scopus. Articles referring to the subject of digoxin toxicity between 1849 and 2015 were assessed according to the document type, publication language, countries/territories, institutions, journal, impact factors, total number of citations, h-index, average number of citations per publication, and international collaborations. There were 2900 publications that included 2542 (87.7%) original research articles, while 5.3% were reviews and 4.6% letters. The country of origin was the USA in 849 publications, Germany in 241, the UK in 150, and France in 143. The USA and the UK had the highest number of international collaborations. The average number of citations per publications related to digoxin toxicity was 8.1, and the h-index was 59. The USA and Canada had the highest h-indices by country at 46 and 22, respectively. This study presents the first bibliometric analysis on digoxin toxicity publications. The USA was the most important contributors to digoxin toxicity literature with the greatest international collaboration, largest number of articles and highest h-index, followed by Germany and the UK. There has been a trend towards reduced publication numbers related to digoxin toxicity at global level, although it is still an important issue and we present the current research themes related to digoxin toxicity that were identified.

Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides.

AIMS: Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca(2+) handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined. METHODS AND RESULTS: We examined the effects of digitoxin (DGT) on Ca(2+) handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca(2+) waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated. CONCLUSION: These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.

Accidental digitoxin intoxication: an interplay between laboratory and clinical medicine.

INTRODUCTION: Two Italian adults arrived at the Emergency Department referring diarrhea, nausea and vomiting for 4 days; weakness, fatigue and visual hallucinations were also complained of. Patients reported the ingestion of some leaves of a plant, which they supposed to be "donkey ears", a week before. Physical examination showed hypotension and bradycardia and ECG examination disclosed sinus rhythm and repolarization abnormalities (scooping of the ST-T complex) in both patients and a 2:1 AV block in the man. MATERIALS AND METHODS: Digoxin concentration was evaluated twice for each patient (at the admission and after 4 hours) by the automated immunoassay system ADVIA Centaur. Digitoxin concentration was evaluated by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: Despite clinical picture was suggestive of digitalis intoxication, digoxin levels were undetectable. Due to the more severe clinical picture, the male patient was treated with anti-digoxin antibodies (Digifab) achieving a good clinical improvement and remission of the AV block within two hours. Initial diagnosis was confirmed by LC-MS/MS showing high digitoxin concentrations, but digoxin was undetectable. Patients remained stable and 48 hours later were discharged from the hospital. CONCLUSION: Whereas digoxin determination frequently relies on monoclonal antibodies which do not cross-react to digitoxin, polyclonal antibodies constituting Digifab recognize a large spectrum of cardiac glycosides, including digitoxin. This report emphasizes the primary role of the clinical approach to patients in the emergency setting and how an active communication and a continuous sharing of professional experiences between Laboratory and Clinicians ensure an early and correct diagnosis.

Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks.

Cardiac glycosides (e.g., digoxin, digitoxin) constitute a diverse family of plant-derived sodium pump inhibitors that have been in clinical use for the treatment of heart-related diseases (congestive heart failure, atrial arrhythmia) for many years. Recently though, accumulating in vitro and in vivo evidence highlight potential anticancer properties of these compounds. Despite the fact that members of this family have advanced to clinical trial testing in cancer therapeutics, their cytotoxic mechanism is not yet elucidated. In this study, we investigated the cytotoxic properties of cardiac glycosides against a panel of pancreatic cancer cell lines, explored their apoptotic mechanism, and characterized the kinetics of cell death induced by these drugs. Furthermore, we deployed a high-throughput kinome screening approach and identified several kinases of the Na-K-ATPase-mediated signal transduction circuitry (epidermal growth factor receptor, Src, pkC, and mitogen-activated protein kinases) as important mediators downstream of cardiac glycoside cytotoxic action. To further extend our knowledge on their mode of action, we used mass-spectrometry-based quantitative proteomics (stable isotope labeling of amino acids in cell culture) coupled with bioinformatics to capture large-scale protein perturbations induced by a physiological dose of digitoxin in BxPC-3 pancreatic cancer cells and identified members of the interferon family as key regulators of the main protein/protein interactions downstream of digitoxin action. Hence, our findings provide more in-depth information regarding the molecular mechanisms underlying cardiac glycoside-induced cytotoxicity.

Interspecies differences in cancer susceptibility and toxicity.

One of the most complex challenges to the toxicologist represents extrapolation from laboratory animals to humans. In this article, we review interspecies differences in metabolism and toxicity of heterocyclic amines, aflatoxin B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related compounds, endocrine disrupters, polycyclic aromatic hydrocarbons, tamoxifen, and digitoxin. As far as possible, extrapolations to human toxicity and carcinogenicity are performed. Humans may be more susceptible to the carcinogenic effect of heterocyclic amines than monkeys, rats, and mice. Especially, individuals with high CYP1A2 and 3A4 activities and the rapid acetylator phenotype may be expected to have an increased risk. Striking interspecies variation in susceptibility to aflatoxin B1 carcinogenesis is known, with rats representing the most sensitive and mice the most resistant species, refractory to dietary levels three orders of magnitude higher than rats. An efficient conjugation with glutathione, catalyzed by glutathione S-transferase mYc, confers aflatoxin B1 resistance to mice. Extremely large interspecies differences in TCDD-induced toxicity are known. The guinea pig is the most susceptible mammal known, with an LD50 in the range 1-2 micrograms TCDD/kg, whereas the hamster is the most resistant species with an LD50 greater than 3000 micrograms/kg. A number of experts have pointed out to the fact that humans appear to be less sensitive to TCDD than most laboratory animals. Human exposure to background levels of TCDD is not likely to cause an incremental cancer risk. A clear cause--effect relationship has been shown between environmental endocrine-disrupting contaminants and adverse health effects in wildlife, whereas the effects seem to be less critical for humans. Studies on DNA adduct formation and metabolism of the nonsteroidal antiestrogen tamoxifen indicate that rats and mice are orders of magnitude more susceptible than humans.

Involvement of a peripheral mechanism in the emesis induced by cardiac glycosides in Suncus murinus.

The ability of three cardiac glycosides, ouabain, digitonin and digitoxin, to induce emesis and their mechanism(s) of action were investigated in Suncus murinus. The intraperitoneal injection of ouabain but not digitonin nor digitoxin caused vomiting in a dose-dependent manner. However, the administration of ouabain into the cerebroventricle did not cause emesis. Ouabain-induced emesis was partly prevented by surgical abdominal vagotomy. Pretreatment with tropisetron, a selective 5-HT3 (5-hydroxytriptamine) receptor antagonist, did not affect the emetic response evoked by ouabain. These results suggest that ouabain exerts emetic effects via peripheral mechanism(s), but 5-HT3 receptors are not involved in the pathway.

Species differences in the toxicity and cytochrome P450 IIIA-dependent metabolism of digitoxin.

In rats, cytochrome P450 (P450) IIIA enzymes are an important determinant of digitoxin toxicity. Induction of these liver microsomal enzymes decreases the toxicity of digitoxin by increasing its oxidative cleavage to digitoxigenin bis- and monodigitoxoside (dt2 and dt1). The present study shows that the susceptibility of different mammalian species to digitoxin toxicity is inversely related to liver microsomal P450 IIIA activity (measured as testosterone 6 beta-hydroxylase activity). Based on this correlation, we correctly predicted that hamsters, which have the highest P450 IIIA activity, are extremely resistant to digitoxin toxicity. To further examine the relationship between digitoxin toxicity and P450 IIIA activity, the pathways of digitoxin metabolism catalyzed by liver microsomes from nine mammalian species were examined by high performance liquid chromatography. The overall rate of digitoxin metabolism varied approximately 90-fold and followed the rank order: hamster greater than rat greater than guinea pig greater than dog greater than mouse approximately monkey greater than rabbit approximately cat greater than human. The qualitative differences in digitoxin metabolism were as striking as the quantitative differences. Formation of 16- and/or 17-hydroxydigitoxin was the major pathway of digitoxin oxidation catalyzed by liver microsomes from hamster, guinea pig, rabbit, cat, dog, and cynomolgus monkey. Guinea pig and, to a lesser extent, hamster liver microsomes also converted digitoxin to an unknown metabolite, the formation of which was catalyzed by P450. None of the species examined catalyzed the 12-hydroxylation of digitoxin to digoxin at a high rate. Similarly, none of the species examined catalyzed a high rate of conversion of digitoxin to dt2, with the notable exception of the rat. However, dt2 formation was the major pathway of digitoxin metabolism catalyzed by human liver microsomes, although humans were much less active (approximately 2%) than rats in this regard. The rate of dt2 formation varied approximately 41-fold among 22 samples of human liver microsomes, which was highly correlated (r = 0.841) with the rate of testosterone 6 beta-hydroxylation. Antibody against rat P450 IIIA1 inhibited the high rate of dt2 formation by rat liver microsomes and the low rate catalyzed by mouse, guinea pig, dog, monkey, and human liver microsomes. In contrast, anti-P450 IIIA1 did not inhibit the 12-, 16-, or 17-hydroxylation of digitoxin (or the formation of the unknown metabolite), despite the fact that anti-P450 IIIA1 strongly inhibited (greater than 70%) the 6 beta-hydroxylation of testosterone by liver microsomes from each of the species examined (except rabbit liver microsomes, which were inhibited only approximately 30%).(ABSTRACT TRUNCATED AT 400 WORDS)

Digoxin-specific Fab fragments in the treatment of oleander toxicity in a canine model.

STUDY OBJECTIVE: To examine the efficacy of digoxin-specific Fab fragments (dsFab) in the treatment of experimentally induced Nerium oleander cardiac glycoside toxicity in a dog model. DESIGN: A nonblined, placebo-controlled experiment. SUBJECTS: Ten adult greyhound dogs of either sex divided into treatment and control groups of five dogs each. INTERVENTIONS: A tincture of oleander was prepared and administered intravenously to each animal. After the onset of cardiotoxicity, the treatment group received 60 mg/kg dsFab IV. MEASUREMENTS AND MAIN RESULTS: All dogs exhibited dysrhythmias meeting our criteria for cardiac glycoside cardiotoxicity within 27 minutes of beginning the infusion. Three of five control dogs had lethal dysrhythmias during the three-hour observation period. The remaining two control dogs exhibited dysrhythmias throughout the three-hour experiment. All five of the dsFab-treated dogs survived and converted to normal sinus rhythm within eight minutes of dsFab infusion. Three treatment animals reverted back to nonlethal and hemodynamically stable dysrhythmias after a mean of 107 minutes. CONCLUSION: Large doses of dsFab are efficacious in the treatment of dysrhythmias in this canine model of N oleander cardiac glycoside poisoning.

The positive inotropic drugs DPI 201-106, BDF 9148, and veratridine increase ouabain toxicity and [3H]ouabain binding in guinea pig heart.

The interaction of three positive inotropic compounds, which are modulators of sodium channels, with the cardiac glycoside ouabain was investigated in isolated guinea pig atria. In the presence of DPI 201-106 (3 x 10(-7) M), of its new acetidine derivative BDF 9148 (10(-7) M), or of veratridine (10(-6) M), the threshold ouabain concentration to induce toxicity was lowered by a factor of 2. This effect can be explained by the observation that specific equilibrium [3H]ouabain binding in intact atria was elevated by these compounds in the appropriate concentrations. The binding results were analyzed by means of a previously established model of "positive cooperative ouabain binding to intact myocardium," which describes the relationship between cellular sodium homeostasis and ouabain binding. The extent to which the compounds increased ouabain binding was in good quantitative agreement with the observed shift in the threshold concentration of ouabain toxicity. The increase of specific [3H]ouabain binding is likely initiated by a gain in cytosolic sodium. It takes place at the cellular level only, since a direct enhancement of [3H]ouabain binding to isolated cardiac membranes was not found. In conclusion, at least under some conditions, new inotropic drugs acting as sodium channel modulators can increase the risk of digitalis toxicity.

Effect of the position of the cyano-group of cyanopregnenolones on their drug metabolic inducing activity.

The effect of the position of the cyano-group of several cyanopregnenolones on the body's resistance to drugs and on drug metabolism was investigated. Female rats were pretreated with 2 alpha-, 6-, 16 alpha-, 17 alpha-cyano- or 16 alpha-cyanomethyl-pregnenolone or with pregnenolone, and the (in vivo) resistance to zoxazolamine, digitoxin and indomethacin, as well as the in vitro drug metabolism (post mitochondrial fraction) of zoxazolamine and ethylmorphine were determined. It was found that the 16-derivative was the most active in this respect, the 2- and 17-cyanopregnenolones were less active but significantly potent compared to controls, while the 6-cyano, the 16-cyanomethyl derivatives and pregnenolone were essentially inactive. These differences were explained in terms of an effective or poor fit of the steroids to their receptor. The poor performance of pregnenolone-16 alpha-acetonitrile was attributed to electronic effects. A hypothesis of some structural features of the receptor site for its interaction with the cyanopregnenolone inducers was presented.

Pathogenesis of digitoxin-induced duodenal ulcers in pregnant rats. Roles of gastric acid and duodenal alkaline secretion.

Late-stage pregnant rats (day 17) had higher rates of gastric acid secretion (45-55 mu eq/15 min) as compared to nonpregnant and middle-stage pregnant (day 10) rats (20-25 mu eq/15 min). In contrast, basal rates of duodenal alkaline secretion were significantly lower (2-3 mu eq/15 min) in pregnant rats (day 10 and 17) than those in nonpregnant rats (approximately 5 mu eq/15 min), although the duodenal mucosa responded to acid with a significant rise in HCO3- output in these three groups of rats. In pregnant rats (day 17), a single injection of digitoxin, a Na+ K+-ATPase inhibitor (10 mg/kg, subcutaneously), had no effect on basal acid and alkaline secretions, but significantly blocked the acid-induced HCO3- secretion for more than 18 hr from 6 hr after administration. This drug, when given once daily for four days (10 mg/kg, subcutaneously), produced well-defined ulcers in the proximal duodenum with few lesions in the stomach of female rats, and the severity and incidence were significantly higher in late-stage pregnant rats than in the other two groups of rats. Following repeated administration of digitoxin (10 mg/kg) to late-stage pregnant rats (days 17-20), acid secretion significantly declined after two days of treatment, while the acid-induced HCO3- secretion was significantly attenuated after one day of treatment and remained inhibited during the whole period. These results suggest that an impairment of the mechanisms related to acid-induced HCO3- secretion may be associated with the induction of duodenal ulcers caused by digitoxin in female rats, and the high incidence of these ulcers in late-stage pregnant rats may be due to acid hypersecretion.

Digitoxin metabolism by liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase and its role in the protection of rats from digitoxin toxicity by pregnenolone-16 alpha-carbonitrile.

The aim of the present study was to investigate whether the mechanism by which pregnenolone-16 alpha-carbonitrile (PCN) protects rats from digitoxin toxicity was dependent on the induction of liver microsomal cytochrome P-450p and/or the UDP-glucuronosyltransferase active toward digitoxigenin monodigitoxoside (UDP-GT-dt1). Evidence is presented that suggests troleandomycin is a selective inhibitor of cytochrome P-450p in vivo, based on the pattern of inhibition observed when zoxazolamine paralysis time and hexobarbital sleeping time were measured in rats treated with different cytochrome P-450 inducers. A single dose of troleandomycin completely reversed the ability of PCN to protect rats from digitoxin toxicity, establishing the importance of cytochrome P-450p induction in the protective effect of PCN. The postpubertal decline in constitutive cytochrome P-450p levels in female but not male rats was paralleled by a female-specific, age-dependent decline in the rate of digitoxin sugar cleavage (i.e., digitoxosyl oxidation of digitoxin to 15'-dehydrodigitoxin and digitoxosyl cleavage to digitoxigenin bisdigitoxoside). This resulted in a marked sex difference in the rate of digitoxin sugar cleavage catalyzed by liver microsomes from mature rats (male/female approximately 6). However, no sex difference in digitoxin toxicity was observed in either immature or mature rats. In contrast to cytochrome P-450p, liver microsomal UDP-GT-dt1 activity increased dramatically with age in both male and female rats (mature/immature approximately 10). However, no age differences in digitoxin toxicity were observed in rats of either sex. The results indicate that cytochrome P-450p and UDP-GT-dt1 can be independently regulated in rat liver and that large changes in the constitutive levels of these microsomal enzymes have no effect on digitoxin toxicity. This suggests that the induction of cytochrome P-450p and UDP-GT-dt1 does not fully account for the mechanism by which PCN protects rats from digitoxin toxicity.

[Pharmacologic study of a semisynthetic digitalis derivative lacking the hydroxyl group on position 14 of the steroid nucleus]. / Estudio farmacológico de un digitálico semisintético desprovisto del grupo oxhidrilo en la posición 14 del núcleo esteroideo.

All natural cardiac glycosides (CG) have a hydroxyl (OH) group attached to carbon 14 (C 14) of the steroid nucleus which has been considered important for their pharmacological action. To investigate the relation between chemical structure and biological activity of CG, we studied the cardiac effects of a semisynthetic derivative if gluco-digitoxigenin that lacks the C 14 hydroxyl group; the compound was named Dig-3 and corresponds to the number of a series of semisynthetic glycosides being studied. On the failing heart of the Starling's heart-lung preparation Dig-3 reverts experimental cardiac failure. Electrophysiological experiments in anesthetized dogs (morphine chloralose) have shown that Dig-3 shortens the functional refractory period of the ordinary atrial myocardium (OAM), and lengthens that of the specialized atrial tissue (SAT). The basal excitability is reduced in both tissues, however OAM is more susceptible to Dig-3 action. The conduction velocity of impulses in SAT diminishes 50% with one tenth of the lethal dose (LD) of Dig-3 whereas in the OAM, an equivalent decrease is achieved with 60% of LD. A-V dissociation induced by the infusion of toxic doses of Dig-3 reverted to sinus rhythm in about 6 min after the administration was stopped. We conclude that the presence of the OH group attached to C 14 of digitalis molecule does not constitute a structural requirement for the preservation of its inotropic and electrophysiological actions on the heart, although its potency is greatly diminished.

[Digitoxin poisoning: reversing ventricular fibrillation with Fab fragments of anti-digoxin antibody]. / Intoxication par la digitoxine: réversibilité d'une fibrillation ventriculaire par fragments Fab d'anticorps antidigoxine.

Purified Fab fragments of ovine anti-digoxin antibodies (Wellcome Foundation) were used to treat a patient who attempted suicide by absorbing 10 mg of digitoxin (serum concentration 265 micrograms/l). The poor prognosis, as assessed clinically and from serum potassium levels (7.5 mEq/l), seemed to warrant such a treatment. The weak (6.85%) cross-reactivity elicited in vitro between the anti-digoxin antibodies and digitoxin was compensated by increasing the doses, but improvement was observed with 3.6 g, i.e. about half the effective dosage initially considered. The criteria of effectiveness were clinical, electrocardiographic (reversal of the ventricular fibrillation), biochemical (simultaneous and opposite changes in extra- and intracellular potassium levels, suggesting that ATPase inhibition by digitalis is a reversible process) and toxicological: there was an increase in digitoxin serum levels suggesting displacement of the drug from tissue sites to plasma and other extracellular compartments where the Fab fragments are distributed, and Fab-bound digitoxin appeared fairly rapidly in the urine, which suggested shunting of the normal hepatic metabolic pathway.

Effect of age, clonidine or propranolol on behavioral toxicity induced with digitoxin in mice.

In three experiments, behavioral toxicity induced by 5 different dose levels of digitoxin was investigated in mice that were treated with either clonidine or propranolol or were either young (70-140 days), middle aged (240-260) or old (450-600). Observed on four drug-induced behavior categories, it was found that both clonidine and propranolol attenuated the toxic effects of relatively low dose levels of digitoxin, while at higher digitoxin dose levels only propranolol was able to reduce significantly the ED50 and the LD50. The results were discussed in terms of adrenergic mechanisms mediating digitoxin toxicity. Aged mice were found to be the least and middle aged the most resistant to the toxic effects of digitoxin.