digitoxigenin and digitoxigenin-monodigitoxoside

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of β-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Survival; Digitoxigenin; Humans; Lung Neoplasms; Molecular Docking Simulation; Sodium-Potassium-Exchanging ATPase; Swine; Time Factors

Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.

Topics: A549 Cells; Cardenolides; Cardiac Glycosides; Cell Line, Tumor; Cell Movement; Cell Proliferation; Digitoxigenin; Humans; Lung Neoplasms; Neoplasm Metastasis; Strophanthins

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.

Topics: Biotransformation; Cells, Cultured; Chromatography, High Pressure Liquid; Digitoxigenin; Digoxigenin; Digoxin; Glucuronosyltransferase; Humans; In Vitro Techniques; Liver; Microsomes, Liver

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent Km and Vmax values were 7.37 +/- 0.99 mM and 4.76 +/- 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.

Topics: Acetaminophen; Cetomacrogol; Chromatography, High Pressure Liquid; Digitoxigenin; Enzyme Activation; Glucuronates; Glucuronosyltransferase; Humans; Isoenzymes; Kinetics; Microsomes, Liver; Naphthols; Substrate Specificity

The metabolism of digitoxin is known to occur through several major pathways including oxidation and glucuronidation. Several studies have compared the behavior of the various unconjugated metabolites with respect to reactivity toward Na-K-ATPase and toward the radioimmunoassay (RIA). Other studies with conjugated products synthesized chemically have also been performed. However, the activity of the conjugated products produced in vivo has not been reported and these metabolites are generally assumed to be inactive. In the present studies we have prepared and purified the glucuronide conjugate of digitoxigenin monodigitoxoside formed by rat liver microsomes and determined the activity of this metabolite as measured by inhibition of Na-K-ATPase and by the RIA. The concentration of the conjugated product needed to cause a fifty per cent inhibition of Na-K-ATPase was 5.40 microM compared to 0.68 for digitoxin and 0.08 for digitoxigenin monodigitoxoside. However, the conjugated product had a two-fold greater affinity for the antibody in the RIA procedure than did either digitoxin or digitoxigenin monodigitoxoside. Although these data cannot be extrapolated to the effects of these drugs on cardiac contractility, the results suggest that the contribution of the glucuronide conjugate to the therapeutic or toxic effect of digitoxin may be minimal. This metabolite may, however, lead to inaccurate estimation of blood levels by the RIA.

Topics: Animals; Digitoxigenin; Digitoxin; Glucuronidase; In Vitro Techniques; Radioimmunoassay; Rats; Sodium-Potassium-Exchanging ATPase

Liver microsomes of male Beagle dogs contain a form of UDP-glucuronyltransferase which is capable of conjugating digitoxin and its cleavage products digitoxigenin-bisdigitoxoside and digitoxigenin-monodigitoxoside. The highest reaction rates (Vmax 236 pmoles/mg microsomal protein min) were found for digitoxin and digitoxigenin-monodigitoxoside whereas the lowest Km was obtained for digitoxigenin-bisdigitoxoside (29 microM). Digoxin cannot be glucuronidated and digitoxigenin is glucuronidated only in traces. The result may explain the fast digitoxin elimination in dogs. Mutual induction experiments utilizing cardenolides and model substrates of UDP-glucuronyltransferase result in the conclusion that a specific form of UDP-glucuronyltransferase is responsible for glucuronidating digitoxigenin glycosides.

Topics: Animals; Biphenyl Compounds; Digitoxigenin; Digitoxin; Dogs; Glucuronates; Glucuronosyltransferase; In Vitro Techniques; Kinetics; Male; Microsomes, Liver; Nitrophenols; Pregnanediol; Substrate Specificity; Testosterone

A specific assay for determining the urinary excretion of unchanged digitoxin and its metabolites is described. The procedure includes solvent extraction of urine at pH 8.5, reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) of equivalent fractions. Confidence limits showed good linearity and precision of recovery and high sensitivity, accuracy and specificity. Cross-reactivities were high for digitoxigenin (DGTN) and digitoxigenin bisdigitoxoside (Bis-DGTN), they were low for digitoxigenin monodigitoxoside (Mono-DGTN) or digoxin when [125I]digitoxin RIA was used. The interference of endogenous compounds in urine in the RIA was overcome by using HPLC. Compared with results reported in the literature, the urinary recovery of unchanged digitoxin was lower, being only 8.11 +/- 1.51% of the dose administered. Amounts of 6.52 +/- 1.31% were excreted hydrolysed as Bis-DGTN, Mono-DGTN, DGTN or C12-hydroxylated as digoxin.

Topics: Adult; Biotransformation; Chromatography, High Pressure Liquid; Digitoxigenin; Digitoxin; Digoxin; Humans; Radioimmunoassay

We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

Topics: Animals; Betamethasone; Cells, Cultured; Cytochrome P-450 Enzyme System; Dexamethasone; Digitoxigenin; Enzyme Induction; Female; Glucocorticoids; Glucuronosyltransferase; Gonadal Steroid Hormones; Humans; Hydrocortisone; Microsomes, Liver; Rats; Rats, Inbred Strains; Triamcinolone

The pharmacokinetic behavior of digitoxigenin is affected by the number of glycosides present. This study was conducted to compare the bioavailabilities of the bis- and monodigitoxosides of digitoxigenin in man. Intravenous and oral doses of the two drugs were administered to six normal volunteers. Blood samples were collected up to 28 days after each dose, and assayed for the specific drug administered and for total radioassayable drug. Both drugs were virtually completely absorbed, based on serum concentrations of administered drug plus metabolites. However, the mean bioavailability of unchanged bisdigitoxoside was only 56.3% indicating that substantial metabolism occurred prior to entry into the systemic circulation. Monodigitoxoside was virtually completely metabolized prior to entry into the systemic circulation.

Topics: Administration, Oral; Adult; Aged; Biological Availability; Digitoxigenin; Female; Humans; Injections, Intravenous; Male; Middle Aged; Radioimmunoassay

A series of digitoxigenin glycosides was studied: five with beta-D-sugars varying stepwise in sugar structure from beta-D-digitoxose to beta-D-galactose, including one beta-D/alpha-D pair. I50 values for these glycosides and digitoxigenin were determined with hog kidney Na+, K+-ATPase. These data suggest a major and unexpected role for 4'-OH conformation in the sugar. All the glycosides with an equatorial 4'-OH were more active than the two with the 4'-OH axial [digitoxigenin beta-D-galactoside (6) I50 = 6.45 X 10(-8) M; digitoxigenin 2'-deoxy-alpha-D-ribo-hexopyranoside (alpha-3a) I50 = 9.33 X 10(-8) M; digitoxigenin I50 = 1.17 X 10(-7) M]. Stereochemistry of the 3'-OH had much less of an activity role than that of the 4'-OH, in contrast to existing models of "sugar-site" binding.

Topics: Animals; Carbohydrate Conformation; Cardiac Glycosides; Crystallography; Digitoxigenin; Kidney; Sodium-Potassium-Exchanging ATPase; Swine

The kinetics of digitoxin and two of its metabolites, the bis- and monodigitoxosides of digitoxigenin, were determined in six normal subjects. Mean t 1/2s and total body clearances were 134.4, 15.4, and 0.59 hr and 2.66, 27.3, and 1071 ml/min. Mean renal clearance of the monodigitoxoside was more rapid (7.24 ml/min) than those of digitoxin (0.81 ml/min) or the bisdigitoxoside (0.94 ml/min). The volumes of distribution were of the same order, 0.45 l/kg for digitoxin, 0.57 l/kg for the bisdigitoxoside, and 0.83 l/kg for the monodigitoxoside. The short t 1/2 of monodigitoxoside would make it unsuitable for clinical use, but the bisdigitoxoside of digitoxigenin has a t 1/2 of an intermediate length and may have significant therapeutic advantages.

Topics: Adult; Aged; Digitoxigenin; Digitoxin; Drug Evaluation; Female; Half-Life; Humans; Injections, Intravenous; Kinetics; Male; Middle Aged; Radioimmunoassay

The kinetics of digitoxin and two of its major metabolites, the bis- and monodigitoxosides of digitoxigenin, were determined in six subjects with renal insufficiency and compared to those in six age- and sex-matched normal control subjects. No significant differences between the two groups were found in elimination t 1/2, total body clearance, or volume of distribution. Average renal clearances of all three drugs were reduced in subjects with renal failure, but the differences were significant only in the case of digitoxin. The bis-digitoxoside of digitoxigenin has kinetic properties that offer clinical advantages.

Topics: Acute Kidney Injury; Adult; Aged; Creatinine; Digitoxigenin; Digitoxin; Drug Evaluation; Female; Half-Life; Humans; Injections, Intravenous; Kinetics; Male; Middle Aged; Radioimmunoassay; Random Allocation

A specific assay is described for measuring the concentration of digitoxin and the bis- and monoglycosides of digitoxigenin in serum. The procedure includes: (1) addition of a tracer amount of tritium labeled parent compound to the serum in order to measure percentage recovery; (2) solvent extraction to separate polar and non-polar metabolites; (3) reversed-phase thin-layer chromatography of the non-polar fraction to separate digoxigenins from digitoxigenins; (4) thin-layer chromatography to isolate digitoxin, and the bis- and monoglycosides of digitoxigenin; and (5) use of an 125I-radioimmunoassay to determine the concentration of the glycosides. Each of these three glycosides was administered intravenously to a normal subject, and the concentration of parent compound was measured in the serum at various times.

Topics: Adult; Digitoxigenin; Digitoxin; Humans; Male; Radioimmunoassay; Stereoisomerism; Time Factors

The aim of the present study was to investigate the specificity of the UDP-glucuronosyltransferase (EC 2.4.1.17) involved in the conjugation of digitoxigenin monodigitoxoside. By in vitro assays with detergent activated liver microsomes it was found that (1) digitoxigenin monodigitoxoside is by far the best substrate of all cardenolides and cardenolide digitoxosides tested. (2) In the presence of saturating UDP-glucuronate concentrations an apparent Km of 5.8 microM was obtained from linear Lineweaver-Burk plots together with a Vmax of about 150 pmoles/mg microsomal protein/min (3) Neither phenobarbital nor polycyclic aromatic hydrocarbons caused a considerable induction of the enzyme without change of the apparent Km, but spironolactone did. (4) The conjugation of the substrate (4 microM) could only be inhibited by the 3'-epi-digitoxoside of digitoxigenin. (5) 25-50 microM substrate inhibited only the conjugation of the 3'-epimer and that of digoxigenin monodigitoxoside. It is suggested that there is a form of glucuronyltransferase which specifically conjugates digitoxigenin monodigitoxoside.

Topics: Animals; Chemical Phenomena; Chemistry; Digitoxigenin; Enzyme Induction; Glucuronosyltransferase; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Substrate Specificity