Digitoxins (4-1000 nM, 24-48 h) have anti-tumor effects in MHCC97H, A549, HCT116, and HeLa cells [3]. Digitoxins (4-100 nM, 24-48 h) interfere with the cell cycle of HeLa cells [3]. Digitoxins (20-500 nM, 48 h) activate HeLa cells in South Africa [3]. Digitoxins (0-80 nM, 72 h) reduce the death rate of PC12 cells [1].

In mice without skin, digitalisin (1-2 mg/kg, intraperitoneal injection, once day for 19 days) exhibits anti-cancer properties [3]. Digitoxin (0.3–3 μg/kg, injected intraperitoneally once daily for four days in a row)

Cell Viability Assay[3]
Cell Types: MHCC97H, A549, HCT116 and HeLa Cell
Tested Concentrations: 4-1000 nM
Incubation Duration: 24 hrs (hours), 48 hrs (hours)
Experimental Results: Reduces the viability of these cancer cells in a dose and time dependent manner, IC50 value range ranged from 0.075 to 0.395 µM after 24 hrs (hours) of digoxigenin treatment and from 0.028 to 0.077 µM after 48 hrs (hours) of digoxigenin treatment.

---

Cell cycle analysis [3]
Cell Types: HeLa Cell
Tested Concentrations: 4 nM, 20 nM, 100 nM
Incubation Duration: 24 hrs (hours), 36 hrs (hours), 48 hrs (hours)
Experimental Results: The G2/M phase cell population increased from 16.27 to 18.36, 23.46 and the concentration was At 20 nM, it was 31.51% at 12, 24 and 36 hrs (hours). At concentrations of 4, 20 and 100 nM, the average cell population in G2/M phase increased from 16.27% to 28.07% within 24 hrs (hours). Dramatically diminished protein expression levels of total CDK1 and phosphorylated CDK1.

---

Apoptosis analysis [3]
Cell Types: HeLa Cell
Tested Concentrations: 20 nM, 100 nM, 500 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: Bax expressio

Animal/Disease Models: Nude mice carrying HeLa tumor xenografts [3]
Doses: 1 mg/kg, 2 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: diminished tumor volume from 330.71±45.61 mm to 214.56.93±73.25 mm. Strongly increases protein levels of cleaved caspase-3. The number of Ki-67 positive cells diminished.

---

Animal/Disease Models: cotton rat [4]
Doses: 0.3 μg/kg, 1 μg/kg, 3 μg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results:Blocked cytokine storm. Cytokine expression is affected to varying degrees. Immune cell density remains intact in virus-infected lungs.

Absorption, Distribution and Excretion
Postmortem digitoxin levels in the choroid-retina and vitreous humor of patients who had undergone digitoxin therapy (therapeutic group) and in one case of suicidal digitoxin poisoning were measured and compared with levels in femoral vein blood, myocardium, kidney and liver. The results were interpreted in light of the medical history of each patient. The digitoxin level in the choroid-retina of the single case of suicidal poisoning was far higher than the choroid-retinal levels in the therapeutic group. In the latter, variation in choroid-retinal levels was comparable to that in the other tissues. In cases where the choroid-retina of the right and left eyes were examined, digitoxin levels in both eyes were essentially equal. There was no indication of significant changes in choroid-retinal levels due to postmortem diffusion of digitoxin into the vitreous body. Based on these results, determination of digitoxin levels in the choroid-retina could contribute to improving postmortem diagnosis of lethal digitoxin poisoning.
In cats ... 100% of digitoxin is absorbed in 80-100 min following duodenal admin ... digitalis glycosides are transported by blood ... bound to ... albumin, and in part free. ... Tissue distribution is not primarily to heart ... /highest concentration/ ... in excretory organs (liver, bile, intestinal tract, kidney) ... .
It is not predictably absorbed from gut of dogs... .
...Prolonged biological half-life of digitoxin and its metabolites appears to depend on recirculation of free drug molecules after biliary excretion as glucuronide and sulfate conjugate.
For more Absorption, Distribution and Excretion (Complete) data for Digitoxin (14 total), please visit the HSDB record page.

---

Metabolism / Metabolites
Hepatic.
Eliminated by hepatic degradation ...to inactive genins ...Stepwise hydrolysis of 3 molecules of digitoxose converts glycoside to aglycone digitoxigenin, which is ...converted to inactive epidigitoxigenin. Because enterohepatic recirculation occurs, approx 25% of metabolic end products appear in stool.
Studies with various tissues of guinea pig showed that liver, kidney, and adrenal tissues converted digitoxin to digoxin.
Digitoxin in bile of rats was excreted largely in form of glucuronide of digitoxigenin monodigitoxoside.
Cardiac glycosides undergo varying degrees of hepatic metabolism, enterohepatic circulation, and renal filtration and reabsorption depending on their polarity and lipid solubility. ... Less polar glycosides such as digitoxin are metabolized extensively before they are excreted. Metabolism includes stepwise cleavage of the sugar molecules, hydroxylation, epimerization, and formation of glucuronide and sulfate conjugates. /Cardiac glycosides/
For more Metabolism/Metabolites (Complete) data for Digitoxin (6 total), please visit the HSDB record page.
Hepatic.

---

Biological Half-Life
The digitoxin half-life in elderly patients in the eight and ninth decade was more prolonged (mean +/- SD: 25 +/- 9 days) than in younger people (6.7 +/- 1.7). These elderly patients accumulated digitoxin even on a dose of 0.05 mg/ day. The symptoms of digitoxin intoxication disappeared on discontinuation of medication. When digitoxin is used in the treatment for heart failure in the very elderly patients, one should be aware of the possibility of digitoxin intoxication, even on a low dose.
After administration of 0.6 mg digitoxin mean serum digitoxin half-life of 4.3 days and 8.1 days respectively observed in cholecystectomized heart patients and control subjects.
The elimination half-life of digitoxin is usually 5-7 days, but may range from 4-14 days. The elimination half-life of digitoxin is generally unchanged in patients with renal failure. In patients with biliary fistulas, plasma half-life is decreased by about 50%. Variability among patients in the degree of enterohepatic recycling of digitoxin may account for part of the variability in plasma half-life in some patients. The elimination half-life of digitoxin is prolonged in hypothyroid patients and decreased in hyperthyroid patients.

[1]. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels . Proceedings of the National Academy of Sciences, 2008, 105(7): 2610-2615.

[2]. Anti-HSV activity of digitoxin and its possible mechanisms . Antiviral research, 2008, 79(1): 62-70.

[3]. Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo . International Journal of Oncology, 2020, 57(2): 562-573.

[4]. Pollard B S, Blanco J C, Pollard J R. Classical drug digitoxin inhibits influenza cytokine storm, with implications for COVID-19 therapy . in vivo, 2020, 34(6): 3723-3730.

[5]. Haux J. Digitoxin is a potential anticancer agent for several types of cancer . Medical hypotheses, 1999, 53(6): 543-548.

Digitoxin appears as odorless white or pale buff microcrystalline powder. Used as a cardiotonic drug. (EPA, 1998)
Digitoxin is a cardenolide glycoside in which the 3beta-hydroxy group of digitoxigenin carries a 2,6-dideoxy-beta-D-ribo-hexopyranosyl-(1->4)-2,6-dideoxy-beta-D-ribo-hexopyranosyl-(1->4)-2,6-dideoxy-beta-D-ribo-hexopyranosyl trisaccharide chain. It has a role as an EC 3.6.3.9 (Na(+)/K(+)-transporting ATPase) inhibitor. It is functionally related to a digitoxigenin. It is a conjugate acid of a digitoxin(1-).
A cardiac glycoside sometimes used in place of digoxin. It has a longer half-life than digoxin; toxic effects, which are similar to those of digoxin, are longer lasting. (From Martindale, The Extra Pharmacopoeia, 30th ed, p665)
Digitoxin has been reported in Digitalis viridiflora, Digitalis grandiflora, and other organisms with data available.
Digitoxin is a lipid soluble cardiac glycoside that inhibits the plasma membrane sodium potassium ATPase, leading to increased intracellular sodium and calcium levels and decreased intracellular potassium levels. In studies increased intracellular calcium precedes cell death and decreased intracellular potassium increase caspase activation and DNA fragmentation, causing apoptosis and inhibition of cancer cell growth. (NCI)
Digitoxin is only found in individuals that have used or taken this drug. It is a cardiac glycoside sometimes used in place of digoxin. It has a longer half-life than digoxin; toxic effects, which are similar to those of digoxin, are longer lasting. (From Martindale, The Extra Pharmacopoeia, 30th ed, p665)Digitoxin inhibits the Na-K-ATPase membrane pump, resulting in an increase in intracellular sodium and calcium concentrations. Increased intracellular concentrations of calcium may promote activation of contractile proteins (e.g., actin, myosin). Digitoxin also acts on the electrical activity of the heart, increasing the slope of phase 4 depolarization, shortening the action potential duration, and decreasing the maximal diastolic potential.
A cardiac glycoside sometimes used in place of DIGOXIN. It has a longer half-life than digoxin; toxic effects, which are similar to those of digoxin, are longer lasting. (From Martindale, The Extra Pharmacopoeia, 30th ed, p665)
See also: Acetyldigitoxin (is active moiety of); Digitalis (annotation moved to).

---

Drug Indication
For the treatment and management of congestive cardiac insufficiency, arrhythmias and heart failure.

---

Mechanism of Action
Digitoxin inhibits the Na-K-ATPase membrane pump, resulting in an increase in intracellular sodium and calcium concentrations. Increased intracellular concentrations of calcium may promote activation of contractile proteins (e.g., actin, myosin). Digitoxin also acts on the electrical activity of the heart, increasing the slope of phase 4 depolarization, shortening the action potential duration, and decreasing the maximal diastolic potential.
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.
Pro-inflammatory processes initiated in the endothelium represent a crucial step in the pathogenesis of inflammatory cardiovascular disease, such as atherosclerosis. Recent observations pointed to potential anti-inflammatory properties of the cardiac glycoside digitoxin. Therefore, the present study investigated potential anti-inflammatory and vasoprotective properties of digitoxin as well as the underlying signaling pathways affected in endothelial cells (EC). Digitoxin, employing therapeutical concentrations used in patients (3-30 nM), potently inhibited the IL-1beta-induced expression of MCP-1 and VCAM-1 in EC and the capacity of corresponding cell culture supernatants on monocyte migration as well as monocyte adhesion to endothelial monolayers, respectively. Furthermore, digitoxin prevented the IL-1beta-induced activation of p44/42-MAPK and NF-kappaB without affecting activation of JNK and p38-MAPK. Inhibition of NF-kappaB signaling but not p44/42-MAPK mimicked the observed inhibitory effects of digitoxin on MCP-1 expression and monocyte migration. Moreover, digitoxin inhibited NF-kappaB signaling at the level of TAK-1/IKK. Additionally, digitoxin prevented TNF-alpha-induced apoptosis in EC accompanied by activation of Akt. Blockade of PI-3-kinase, activator of Akt, prevented the anti-apoptotic properties of digitoxin and impaired its inhibitory action on NF-kappaB signaling and MCP-1 expression. Finally, digitoxin activated endothelial NO-synthase, which was blocked by inhibition of PI-3-kinase, Ca(2+)/Calmodulin-dependent-proteinkinase-II and chelation of intracellular calcium. Digitoxin elicits anti-inflammatory and vasoprotective properties by blocking NF-kappaB and activating PI-3-kinase/Akt signaling as well as Ca(2+)/Calmodulin-dependent-proteinkinase-II in EC. These observations indicate a potential therapeutical application of digitoxin in the treatment of cardiovascular diseases, such as atherosclerosis.
Cardiac glycosides have been used in the treatment of arrhythmias for more than 200 years. Two-pore-domain (K2P) potassium channels regulate cardiac action potential repolarization. Recently, K2P3.1 [tandem of P domains in a weak inward rectifying K+ channel (TWIK)-related acid-sensitive K+ channel (TASK)-1] has been implicated in atrial fibrillation pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. We hypothesized that blockade of cardiac K2P channels contributes to the mechanism of action of digitoxin and digoxin. All functional human K2P channels were screened for interactions with cardiac glycosides. Human K2P channel subunits were expressed in Xenopus laevis oocytes, and voltage clamp electrophysiology was used to record K+ currents. Digitoxin significantly inhibited K2P3.1 and K2P16.1 channels. By contrast, digoxin displayed isolated inhibitory effects on K2P3.1. K2P3.1 outward currents were reduced by 80% (digitoxin, 1 Hz) and 78% (digoxin, 1 Hz). Digitoxin inhibited K2P3.1 currents with an IC50 value of 7.4 uM. Outward rectification properties of the channel were not affected. Mutagenesis studies revealed that amino acid residues located at the cytoplasmic site of the K2P3.1 channel pore form parts of a molecular binding site for cardiac glycosides. In conclusion, cardiac glycosides target human K2P channels. The antiarrhythmic significance of repolarizing atrial K2P3.1 current block by digoxin and digitoxin requires validation in translational and clinical studies.
Cardiac glycosides inhibit the activity of sodium-potassium-activated adenosine triphosphatase (Na+-K+-ATPase), an enzyme required for active transport of sodium across myocardial cell membranes. Inhibition of this enzyme in cardiac cells results in an increase in the contractile state of the heart and it was believed that benefits of cardiac glycosides in heart failure were mainly associated with inotropic action. However, it has been suggested that benefits of cardiac glycosides may be in part related to enzyme inhibition in noncardiac tissues. Inhibition of Na+-K+-ATPase in vagal afferents acts to sensitize cardiac baroreceptors which may in turn decrease sympathetic outflow from the CNS. In addition, by inhibiting Na+-K+-ATPase in the kidney, cardiac glycosides decrease the renal tubular reabsorption of sodium; the resulting increase in the delivery of sodium to the distal tubules leads to the suppression of renin secretion from the kidneys. These observations led to the hypothesis that cardiac glycosides act in heart failure principally by attenuating the activation of the neurohormonal system, rather than by a positive inotropic action. Toxic doses of cardiac glycosides cause efflux of potassium from the myocardium and concurrent influx of sodium. Toxicity results in part from loss of intracellular potassium associated with inhibition of Na+-K+-ATPase. With therapeutic doses, augmentation of calcium influx to the contractile proteins with resultant enhancement of excitation-contraction coupling is involved in the positive inotropic action of cardiac glycosides; the role of Na+-K+-ATPase in this effect is controversial. /Cardiac glycosides/
Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na+,K+-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na+,K+-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na+,K+-ATPase a1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na+,K+-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.

C41H64O13

764.95

764.434

71-63-6

Gitoxin;4562-36-1

441207

White to off-white solid powder

1.3±0.1 g/cm3

902.3±65.0 °C at 760 mmHg

240ºC (dec.)(lit.)

269.5±27.8 °C

0.0±0.6 mmHg at 25°C

1.594

2.44

5

13

7

54

1410

20

C[C@@H]1[C@H]([C@H](C[C@@H](O1)O[C@@H]2[C@H](O[C@H](C[C@@H]2O)O[C@@H]3[C@H](O[C@H](C[C@@H]3O)O[C@H]4CC[C@]5([C@@H](C4)CC[C@@H]6[C@@H]5CC[C@]7([C@@]6(CC[C@@H]7C8=CC(=O)OC8)O)C)C)C)C)O)O

WDJUZGPOPHTGOT-XUDUSOBPSA-N

InChI=1S/C41H64O13/c1-20-36(46)29(42)16-34(49-20)53-38-22(3)51-35(18-31(38)44)54-37-21(2)50-33(17-30(37)43)52-25-8-11-39(4)24(15-25)6-7-28-27(39)9-12-40(5)26(10-13-41(28,40)47)23-14-32(45)48-19-23/h14,20-22,24-31,33-38,42-44,46-47H,6-13,15-19H2,1-5H3/t20-,21-,22-,24-,25+,26-,27+,28-,29+,30+,31+,33+,34+,35+,36-,37-,38-,39+,40-,41+/m1/s1

3-[(3S,5R,8R,9S,10S,13R,14S,17R)-3-[(2R,4S,5S,6R)-5-[(2S,4S,5S,6R)-5-[(2S,4S,5S,6R)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy-14-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]phenanthren-17-yl]-2H-furan-5-one

NSC 7529 NSC-7529 NSC7529 Carditalin EPA Pesticide Chemical Code 097002 Digitoxin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~130.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)