digoxin and dihydrodigoxin

Digoxin-like immunoreactive factor (DLIF) and ouabain-like factor (OLF) are the mammalian counterparts to the plant-derived cardiotonic steroids digoxin and ouabain. Compelling evidence indicates that the cardiotonic steroids may have anticancer properties. Recent evidence indicates that low (nanomolar) concentrations of DLIF selectively induce cell death in transformed cells, while sparing normal cells, and is even more potent than the plant-derived compounds. The discovery that these endogenous molecules may play a role in the regulation of cancer cell proliferation provides a potentially new paradigm for the physiologic role of DLIF and OLF. In addition, the possible use of digoxin itself as a therapeutic agent in cancer has been explored, and evidence suggests that its conversion to dihydrodigoxin may be involved in regulating anticancer activity. The mechanism(s) for the pro-apoptotic property of these compounds is not known. In this brief review, we will discuss the proposed mechanism of action of digoxin, ouabain, DLIF, and OLF as anticancer compounds and discuss the effects that metabolic conversion to their dihydro-derivatives may have on this activity. From the perspective of therapeutic drug monitoring, these findings suggest some potential new challenges in the need to measure concentrations of digoxin and dihydrodigoxin as well as their endogenous counterparts DLIF and OLF in serum.

Topics: Adrenal Glands; Animals; Anticarcinogenic Agents; Apoptosis; Cardenolides; Cell Proliferation; Digoxin; Humans; Neoplasms; Ouabain; Saponins

Digoxin-like immunoreactive factor (DLIF) from adrenal glands is an endogenous ligand structurally related to the plant-derived cardiac glycoside digoxin. Cardiac glycosides regulate the activity of the sodium pump and thus play key roles in disease processes involving regulation of ion transport. We now report the discovery of an endogenous dihydro-DLIF analogous to dihydrodigoxin. We used HPLC, ultraviolet spectrophotometry, and cross-reactivity with two antibodies, one specific for digoxin and one for dihydrodigoxin, to support the hypothesis that dihydro-DLIF contains a chemically reduced lactone ring. The spectral absorbance maximum for dihydro-DLIF is at 196 nm, identical to dihydrodigoxin. DLIF and dihydro-DLIF are 975- and 2588-fold less immunoreactive than digoxin and dihydrodigoxin for their respective antibodies. The molar ratio of dihydro-DLIF to DLIF is approximately 5.3 in bovine adrenocortical tissue and approximately 0.38 in human serum. Dihydrodigoxin (reduced lactone ring) added to microsomes isolated from bovine adrenal cortex produced a 4.5-fold increase in digoxin-like immunoreactivity (oxidized lactone ring) after 3 h of incubation. The biotransformation is likely mediated by a cytochrome P-450 NADPH-dependent process. Our findings demonstrate the presence of a dihydro-DLIF in mammals and suggest a metabolic route for synthesis of endogenous DLIF in mammalian tissue.

Topics: Adrenal Cortex; Animals; Cardenolides; Cattle; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Digoxin; Humans; Lactones; NADP; Oxidation-Reduction; Saponins; Spectrophotometry, Ultraviolet

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.

Topics: Animals; Biological Assay; Cats; Digoxigenin; Digoxin; Guinea Pigs; Heart; Humans; Immunoassay; Mice; Ouabain; Sensitivity and Specificity; Sodium-Potassium-Exchanging ATPase

Electrochemical detection of 3,5-dinitrobenzoyl derivatives of digoxin and its metabolites following high-performance liquid chromatography is reported. Partial resolution of derivatized digoxin and dihydrodigoxin was obtained using a Spherisorb ODS II analytical column. Both single- and dual-electrode detection were investigated and a maximum sensitivity equivalent to 0.39 ng of digoxin was found with the dual-electrode method. This system has the necessary sensitivity and selectivity for development into a therapeutic monitoring assay method.

Topics: Chromatography, High Pressure Liquid; Digoxin; Dinitrobenzenes; Electrochemistry; Electrodes; Humans; Monitoring, Physiologic; Spectrophotometry, Ultraviolet

This HPLC/immunoassay procedure measures digoxin in serum with no interference from digoxin metabolites or digoxin-like factors. We used solid-phase (C18 and Diol) extraction, a C18 column with a tetrahydrofuran/water mobile phase, and final quantification by fluorescence polarization immunoassay. Deslanoside and gitoxigenin were used as the internal standard and the retention-time marker, respectively. The average CV for 300-microL samples at digoxin concentrations between 0.9 and 3.9 nmol/L was 9.3%. Minimum column lifetime with daily use was three months. We also compared results, for 49 samples from patients taking digoxin, obtained with the Abbott "TDx FPIA digoxin I" and the present procedure. Discrepancies between the two methods were substantial for 20% of the samples.

Topics: Chromatography, High Pressure Liquid; Digoxin; Fluorescence Polarization; Humans; Time Factors

Topics: Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Digoxin; Spectrophotometry, Ultraviolet

An efficient high-performance liquid chromatographic (HPLC) separation for digoxin and its metabolites has been developed. Quantitation of digoxin at plasma levels was possible after the column effluent was passed through a fluorogenic post-column reactor. A study of the optimum post-column conditions was undertaken using a combination of ascorbic acid, hydrogen peroxide and hydrochloric acid, which was known to induce fluorescence in the digoxin molecule. Digoxin and its metabolites were separated on a 15 cm X 4.6 mm I.D., 3-microns reversed-phase (C18) HPLC column using methanol--ethanol--isopropanol--water (52:3:1:45) as the mobile phase at a flow-rate of 0.3 ml/min. A solution of 1.1 X 10(-3) M hydrogen peroxide in a 0.1% ascorbic acid solution and concentrated hydrochloric acid were added into the post-column reactor through a peristaltic pump at a combined flow with a flow-rate of 0.23 ml/min. The mixture was passed into the 20-m reaction coil maintained at 79 +/- 1 degrees C. The resulting digoxin fluorophore was monitored with a fluorescence detector. Detector responses were linear from 1.5 to 10 ng injected on-column. The overall performance demonstrated that this system has the sensitivity, linearity and stability desired in a digoxin plasma level determination. The total chromatographic time including the postcolumn derivatization step was about 40 min.

Topics: Chromatography, High Pressure Liquid; Digoxin; Humans; Spectrometry, Fluorescence; Temperature

The anaerobic bacterium Eubacterium lentum, a common constituent of the intestinal microflora, inactivates digoxin by reducing the unsaturated lactone ring. Reduction of the cardiac glycoside by growing cultures of E. lentum ATCC 25559 proceeded in a stereospecific manner, with the 20R-dihydrodigoxin constituting more than 99% of the product formed. This is in contrast to the 3:1 ratio of 20R and 20S epimers formed in the chemical catalytic hydrogenation. Formation of the reduced glycosides proceeded quantitatively when an overall concentration of 10 micrograms/ml was added to the cultures. E. lentum did not hydrolyze the digitoxose sugars from C-3 of the parent glycoside. However, the synthetically prepared sugar-hydrolyzed metabolites (digoxigenin, digoxigenin monodigitoxoside, and digoxigenin bisdigitoxoside) were reduced to the corresponding dihydro metabolites. Repetition of the experiments with a feces sample from a volunteer who was known to be a converter of digoxin to dihydrodigoxin gave results identical to those obtained with pure E. lentum cultures.

Topics: Chromatography, High Pressure Liquid; Digoxin; Eubacterium; Feces; Humans

Inhibition of Na,K-ATPase by cardiac glycosides is at least partially antagonized by K+. The kinetics of the antagonism, however, appear complicated because K+ is capable of reducing both association and dissociation rate constants for the glycoside-enzyme interaction. In order to better understand the effect of K+, inhibition of partially purified Na,K-ATPase obtained from rat brain, guinea-pig heart and rat heart by ouabain, digoxin, digoxigenin, dihydrodigoxin and cassaine were compared in the presence of 1, 3 or 10 mM K+. Higher concentrations of K+ caused a parallel shift to the right in the concentration-inhibition curves for these compounds. For ouabain or digoxin, the extent of the shift was minimal with rat brain enzyme, intermediate with guinea-pig heart enzyme and more substantial with rat heart enzyme. For digoxigenin, dihydrodigoxin or cassaine, the extent of the shift was substantial in all enzyme preparations. These results could not be explained from either the affinity of the enzyme for the compound or its lipid solubility alone. The concentrations of these compounds required to cause a 50 percent inhibition of enzyme activity were markedly different with rat brain enzyme, but relatively similar with rat heart enzyme. The effects of K+, which depend on the source of the enzyme and chemical structures of the compounds, have to be considered in studies on comparative effects of various compounds on Na,K-ATPase, [3H]ouabain binding, sodium pumping and the force of myocardial contraction.

Topics: Abietanes; Alkaloids; Animals; Brain; Cardiac Glycosides; Digoxigenin; Digoxin; Dose-Response Relationship, Drug; Guinea Pigs; In Vitro Techniques; Male; Myocardium; Ouabain; Potassium; Rats; Rats, Inbred Strains; Sodium-Potassium-Exchanging ATPase; Solubility; Structure-Activity Relationship

The authors investigated the cross-reactivity of the major known digoxin metabolites--digoxigenin, digoxigenin monodigitoxoside, digoxigenin bisdigitoxoside, and dihydrodigoxin--and of digitoxin in three 125I-radioimmunoassays and one enzyme immunoassay for digoxin. Digitoxin and dihydrodigoxin exhibit low cross-reactivity and nonparallel dilution responses for these assays. The cross-reactivities of the other three substances are significant for all assays studied with digoxigenin and monodigitoxoside having nonparallel and enhanced tracer displacement compared with digoxin itself. The authors demonstrate that because of nonparallel tracer displacement estimates of cross-reactivity calculated by the 50% displacement method fail to adequately predict the error induced in digoxin assays by digitoxin. They conclude that digoxin metabolites in serum are measured to various extents as the parent digoxin compound by all of the immunoassays they studied. In view of the varying biologic activity of digoxin metabolites and the large patient to patient variations in digoxin metabolism, the cross-reactivities the authors observe may help to explain the discrepancies in correlation of clinical response to measured serum digoxin values reported in other studies.

Topics: Cross Reactions; Digitoxin; Digoxigenin; Digoxin; Humans; Immunoenzyme Techniques; Radioimmunoassay; Reagent Kits, Diagnostic; Reference Standards

A capsule preparation containing small, enteric-coated granules of digoxin was developed to prevent acid hydrolysis of the drug in the stomach and to diminish the variation in plasma glycoside concentration during the intervals between doses. The absorption and metabolism of tritiated digoxin after a single oral loading dose of this formulation (Formulation C) were compared to those after ingestion of a digoxin solution (Formulation S) by 8 healthy men. Drug concentrations were measured by radioimmunoassay (RIA) and liquid chromatography (LC). The percentage of the digoxin dose excreted in the urine during 72 h, as measured by RIA, was significantly lower after the capsule (20.5 +/- 2.0% vs 36.2 +/- 3.0% after S, mean +/- SEM) but total urinary radioactivity after the two treatments was similar (C 35.3 +/- 5.2 and S 41.2 +/- 2.6%; p greater than 0.05). The discrepancy was mainly due to significantly greater excretion of dihydrodigoxin after the capsule (m 12.8%, range 0-28.6% of the dose) than after the digoxin solution (m 5.4%, range 0-14.5%). Dihydrodigoxin was not measured by the RIA. The recovery of hydrolysis metabolites (LC) was greater during the first 24 h after S (2.3 +/- 0.6% vs 0.9 +/- 0.3% after C; p less than 0.05). The peak plasma concentration of digoxin (RIA) was significantly reduced and delayed after intake of C (2.5 +/- 0.4 nmol/l at 3.8 +/- 0.3 h vs. 8.3 +/- 0.8 nmol/l at 0.9 +/- 0.1 h after S), and so was the shortening of electromechanical systole at 1.5 h, 2.5 h, and 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Biotransformation; Capsules; Digoxigenin; Digoxin; Humans; Hydrogen-Ion Concentration; Hydrolysis; Male; Time Factors

A high-performance liquid chromatographic method is described for the determination of digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside, digoxin, and dihydrodigoxin as the 3,5-dinitrobenzoyl esters. The method is applied to a 10 ml urine sample by adding digitoxigenin as internal standard, extracting with methylene chloride, derivatizing with 3,5-dinitrobenzoyl chloride in pyridine, chromatographing with a normal-phase system and detecting at 254 nm. Derivatized digoxigenin, digoxigenin mono- and bis-digitoxoside, and digoxin each yielded one symmetrical peak with the limit of sensitivity of the method being approximately 100 ng/ml. Analysis of a commercially obtained sample of dihydrodigoxin resulted in two well-separated, symmetrical peaks that represent the two epimers of derivatized dihydrodigoxin. Data indicate rapid and complete esterification of all primary and secondary alcohol moieties in the various molecules and the derivatives are shown to be stable in chloroform for at least four days. The procedure appears to be suitable for metabolic investigations and as a prototype for future analytical developments.

Topics: Chromatography, High Pressure Liquid; Digoxin; Humans; Nitrobenzoates; Stereoisomerism

Digitalis sensitivity of the heart is increased in patients with ischemic heart disease. Whether this elevation of digitalis sensitivity occurs as the result of ischemia-induced changes in the cardiac tissue and whether changes in the sarcolemmal Na,K-adenosine triphosphatase (ATPase) or reserve capacity of the sodium pump are responsible for the increased digitalis sensitivity were examined using isolated heart preparations obtained from guinea pigs. Ligation of the left anterior descending coronary artery (LAD) in Langendorff preparations 40 min before perfusion with a toxic concentration (either 1.8 or 2.5 microM) of digoxin decreased the time to the onset of arrhythmias. LAD-ligation by itself did not cause arrhythmias. The time to the onset of arrhythmias during digoxin perfusion was slightly longer in preparations obtained from reserpine-treated animals; however, the reserpine pretreatment failed to alter the effect of LAD ligation on digitalis sensitivity, indicating that the release of catecholamines is not involved in the sensitization. The effects of ischemia on Na,K-ATPase and sodium pump activities, glycoside binding to the enzyme and reserve capacity of the sodium pump were examined in globally ischemic Langendorff preparations. The preparations were perfused with a Krebs-Henseleit bicarbonate buffer solution (pH 7.4) saturated with a 95% O2-5% CO2 gas mixture at a control flow rate of 2.5 ml/g of tissue per min or at 5 or 0% of the control flow rate. After 6 hr of zero perfusion, Na,K-ATPase activity and the number of specific ouabain binding sites were reduced in ventricular muscle homogenates. However, the remaining Na,K-ATPase was not altered in its sensitivity to dihydrodigoxin-induced inhibition or affinity of binding sites for ouabain, sodium or potassium. Similar results were observed after reperfusion following 2 or 5 hr of zero perfusion. A 5% perfusion for 2 or 6 hr, or zero perfusion for 2 hr failed to affect Na,K-ATPase activity in muscle homogenates. Sodium pump activity in ventricular slices, estimated from the ouabain-sensitive 86Rb+ uptake, was unchanged after 5% perfusion or zero perfusion for 2 hr, but was significantly reduced after a 20-min reperfusion following 2 hr of zero perfusion. Reserve capacity of the sodium pump, as estimated from the differences in 42K+ uptake by right ventricular strips under 1.5 and 7 Hz stimulation, was unaffected by 2 hr of 5% perfusion. These results indicate that coronary artery occlusion

Topics: Animals; Coronary Disease; Digitalis Glycosides; Digoxin; Female; Guinea Pigs; Heart; Male; Myocardium; Ouabain; Perfusion; Potassium; Rubidium; Sodium-Potassium-Exchanging ATPase

The cardioinactive digoxin metabolite, dihydrodigoxin, has been conjugated to bovine serum albumin and to bovine pancreatic ribonuclease by the periodate oxidation method. Rabbits immunized with the dihydrodigoxin-bovine serum albumin conjugate formed antibodies which bound a radioiodinated dihydrodigoxin-ribonuclease conjugate. This binding was inhibited by dihydrodigoxin. After affinity chromatography on a digoxin-ribonuclease-Sephacryl immunoadsorbent to remove antibodies which cross-reacted with digoxin, dihydrodigoxin was 300 times more effective than digoxin in inhibiting the binding of tracer by antibody. Digoxin-absorbed antidihydrodigoxin antibodies were coupled to Sephacryl and were used to develop a solid-phase radioimmunoassay capable of detecting 250 to 500 pg of dihydrodigoxin in 1 ml of human serum or urine. This radioimmunoassay has been used to define the pharmacokinetics of the metabolite in four normal human volunteers who ingested 125 to 500 micrograms of dihydrodigoxin by mouth. Dihydrodigoxin was quickly absorbed, with maximal serum concentrations achieved within 45 to 105 min, followed by a rapid fall in serum immunoreactivity over 2 to 4 hr and then by a slower, more gradual decline. The terminal half-life (beta) in serum varied from 4.24 to 11.9 hr (mean +/- S.E. = 8.1 +/- 1.3 hr). Most of the administered dose was excreted in the urine, with cumulative urinary recovery varying inversely with the dose. Urinary half-lives averaged 13.8 +/- 2.1 hr, and renal clearance rates were similar to those of creatinine. Dihydrodigoxin is rapidly absorbed and excreted in man and appears to be eliminated from the body at a faster rate than digoxin.

Topics: Adult; Animals; Antigens; Cattle; Digoxin; Humans; Kinetics; Male; Middle Aged; Rabbits; Radioimmunoassay; Ribonucleases; Time Factors

After oral intake of enteric-coated granules containing [3H]-digoxin extensive metabolism was observed. Maximum 66% of the 24 h urinary excretion was identified as [3H]-dihydrodigoxin, using high performance liquid chromatography for the analysis. It is suggested that metabolism of digoxin may depend on the absorption site.

Topics: Biotransformation; Capsules; Digoxin; Humans; Intestinal Absorption; Male; Solutions; Tablets, Enteric-Coated

Topics: Cross Reactions; Digoxin; Humans; Radioimmunoassay; Reagent Kits, Diagnostic

The urinary excretion of the relatively cardioinactive reduced metabolites of digoxin, dihydrodigoxin and related compounds was measured by radioimmunoassay in 131 normal subjects during studies of the bioavailability of digoxin preparations. Digoxin reduction products (DRP) constitute more than 5 percent of the excretion of digoxin and its metabolites in one-third of the volunteers after the administration of single or multiple doses of digoxin. There was little or no output of DRP during the first 8 hours after a single dose, with maximal excretion usually occurring on the second day. Most subjects who excreted more than 5 percent DRP on one occasion did so with each subsequent exposure to digoxin. Six volunteers, however, in whom substantial amounts of DRP had previously been found, failed to excrete detectable quantities after subsequent doses. In two, this change occurred shortly after they took erythromycin. Urinary DRP were less after the intravenous administration compared to the oral administration of digoxin. After oral doses, DRP excretion tended to vary inversely with the bioavailability of the preparation. The findings are consistent with the hypothesis that DRP are formed as the result of the activity of a variable component of the intestinal flora. Prospective studies will be necessary to prove this hypothesis.

Topics: Adult; Aged; Biological Availability; Digoxin; Female; Humans; Male; Middle Aged; Oxidation-Reduction; Radioimmunoassay

A liquid chromatographic method for the determination of digoxin, digoxigenin, its mono- and bisdigitoxoside and dihydrodigoxin in urine is described. Doses of 100 muCi of [12 alpha-3H]digoxin and 0.5 mg (640 nmol) of digoxin were administered orally to eight healthy volunteers. The compounds were extracted from urine with methylene chloride containing 3% of heptafluorobutanol. After separation, fractions corresponding to digoxin and the metabolites were measured by liquid scintillation counting. Conjugates of the glycoside metabolites were determined indirectly after pre-treatment of the samples with beta-glucuronidase-arylsulphatase. The detection limit was 0.1 nmol/l. Metabolites amounting to 0.5% of digoxin were assayed with a relative standard deviation of 5%. The advantages of the method are a high recovery in the extraction step, short separation times and the possibility of separate assay of dihydrodigoxin.

Topics: Chromatography, High Pressure Liquid; Digoxin; Humans; Time Factors

Topics: Animals; Binding Sites; Digitalis Glycosides; Digoxin; Doxorubicin; Female; Guinea Pigs; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Ouabain; Sodium-Potassium-Exchanging ATPase

We evaluated four commercially available 125I-digoxin radioimmunoassay kits with regard to their ability to cross react with the digoxin metabolite dehydrodigoxin. We prepared dihydrodigoxin serum samples in digoxin-free serum over the concentration range 0.4 to 5.0 microgram/liter and assayed them with each kit according to the manufacturer's instructions. The metabolite was able to displace the 125I-labeled digoxin derivative from the antibody supplied with all four kits. However, the extent of the cross reactivity depended on the kit, ranging from essentially zero to a high degree of interference. Dihydrodigoxin is the only metabolite of digoxin to have been quantitiated in human serum, and may comprise up to 30% of total glycosides. Over the clinical and therapeutic range of serum digoxin concentrations, enough dihydrodigoxin can be produced to interfere in the determination of serum digoxin concentrations by this method. We suggest that laboratories evaluate their specific kit with regard to cross reactivity to this metabolite.

Topics: Antibodies; Cross Reactions; Digoxin; Iodine Radioisotopes; Radioimmunoassay; Reagent Kits, Diagnostic

Topics: Chromatography, DEAE-Cellulose; Chromatography, Paper; Chromatography, Thin Layer; Digoxin; Gels; Silicon Dioxide; Solvents; Spectrometry, Fluorescence

Topics: Chromatography, Ion Exchange; Digoxin; Humans; Tritium

Topics: Animals; Cardiac Glycosides; Digitalis Glycosides; Digitoxin; Digoxin; Dogs; Infusions, Parenteral; Ouabain; Pharmacology; Research; Strophanthidin; Strophanthins

Topics: Animals; Biotransformation; Digoxin; Guinea Pigs; Muscles; Ouabain; Pharmacology; Research; Toxicology

Topics: Animals; Digitalis; Digitoxin; Digoxin; Dogs; Heart; Ouabain; Strophanthins