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Advanced stage cancers acquire anoikis resistance which provides metastatic potential to invade and form tumors at distant sites. Suppression of anoikis resistance by novel molecular therapies would greatly benefit treatment strategies for metastatic cancers. Recently, digitoxin and several of its novel synthetic derivatives, such as α-l-rhamnose monosaccharide derivative (D6-MA), have been synthesized and studied for their profound anticancer activity in various cancer cell lines. In this study, we investigated the anoikis sensitizing effect of D6-MA compared with digitoxin to identify their anti-metastatic mechanism of action. D6-MA sensitized NSCLC H460 cells to detachment-induced apoptosis with significantly greater cytotoxicity (IC50 = 11.9 nM) than digitoxin (IC50 = 90.7 nM) by activating caspase-9. Screening of the Bcl-2 protein family revealed that degradation of anti-apoptotic Mcl-1 protein is a favorable target. Mcl-1 over-expression and knockdown studies in D6-MA and digitoxin exposed cells resulted in rescue and enhancement, respectively, indicating a facilitative role for decreased Mcl-1 expression in NSCLC anoikis. Transfection with mutant Mcl-1S159 attenuated detachment-induced cell death and correlated with a remaining of Mcl-1 level. Furthermore, D6-MA suppressed Mcl-1 expression via ubiquitin proteasomal degradation that is dependent on activation of glycogen synthase kinase (GSK)-3β signaling. In addition, D6-MA also targeted Mcl-1 degradation causing an increased anoikis in A549 lung cancer cells. Anoikis sensitizing effect on normal small airway epithelial cells was not observed indicating the specificity of D6-MA and digitoxin for NSCLC. These results identify a novel cardiac glycoside (CG) sensitizing anoikis mechanism and provide a promising anti-metastatic target for lung cancer therapy.

The purpose of this investigation was to define, under controlled in vitro conditions, the processes contributing to the uptake and accumulation of [3H]digoxin by incubated slices of chicken renal cortex. Progressive uptake was evident in time-course experiments with the slice-to-medium concentration ratio (S/M) reaching 5.25 after 120 min. No metabolism was evident. Increasing the ratio of unlabeled to labeled digoxin resulted in a concentration-dependent decrease in relative uptake of the label, suggesting saturability. Incubation under conditions of metabolic inhibition reduced digoxin S/M by about 50%, indicating that both energy-requiring and passive mechanisms contribute to the overall accumulation process. The structural nature of the uptake process was explored by incubating digoxin in the presence of potential inhibitors of transport. The organic cation quinine and the non-glycosidic steroids digoxigenin and spironolactone were without effect even at greater than 1000-fold excess compared to digoxin. Similarly, the sugar digitoxose had no inhibitory activity on digoxin accumulation by the slices. On the other hand, the glycosides digitoxin, digoxigenin-bis-digitoxoside and digoxigenin-mono-digitoxoside inhibited dogoxin uptake in a concentration-dependent manner. These results indicate a structural preference for an intact glycoside rather than for either the steroidal or sugar portion of the molecule alone. An inhibitory effect of ouabain and a stimulatory effect of reduced medium potassium concentration suggest a possible role for Na+,K(+)-ATPase in the uptake of digoxin by the renal cortex.

Previous investigations on the digitoxin metabolism hardly considered the role of the sulfate ester conjugation. Therefore, this study examined whether digitoxin (dt-3) or one of its cleavage products might be sulfated in vitro . It was proven that digitoxigenin (dt-0) is by far the best substrate for the cytosolic sulfotransferases (ST). Digitoxigenin-monodigitoxoside (dt-1) and digitoxigenin-bisdigitoxoside (dt-2) are sulfated in trace amounts whereas dt-3 is not sulfated at all. The purification of the responsible enzyme was performed by liquid chromatography on Q-Sepharose and hydroxyapatite. During the purification procedure this enzymatic activity corresponded exactly to that towards dehydroepiandrosterone (DHEA). The 134-fold purified and gel electrophoretically homogeneous enzyme protein ( M i 33,000) showed a V max . of 12.5 nmoles dt-0 sulfate/min mg protein and a K M of 37 μmol/L. The purified enzyme conjugated dt-1 and dt-2 in trace amounts only and was inhibited competitively by DHEA. It can be concluded that in the rat a 3β-hydroxy-steroid sulfotransferase is responsible for the sulfation of dt-0. The purified enzyme reacts with dt-1, dt-2 and digoxigenin (dg-0) in traces only, a sulfation of dt-3 is not detectable.

We have performed direct and indirect binding studies with [3H]ouabain or [3H]digitoxin on beef or guinea pig cardiac (Na+ + K+)-ATPase to measure the potencies of a broad range of cardiotonic steroids for structure-activity relationship (SAR) studies for comparison with previously determined positive inotropic potencies. The positive inotropic potencies of twelve compounds on contracting guinea pig left atria correlated well with the equilibrium dissociation constants (KD values) from the inhibition of [3H]ouabain binding to guinea pig cardiac (Na+ + K+)-ATPase (r = 0.98 for seven 5 beta-compounds, r = 0.95 for five 5 alpha-compounds). Further we calculated KD values from the inhibition of [3H]ouabain binding data for a total of 33 digitalis derivatives on the digitalis-sensitive beef cardiac (Na+ + K+)-ATPase. For the 27 compounds tested on both beef cardiac (Na+ + K+)-ATPase and guinea pig left atria, the potencies showed a significant correlation (r = 0.92 for 22 5 beta-compounds, r = 0.96 for five 5 alpha-compounds. For seven compounds, KD values were measured on beef cardiac (Na+ + K+)-ATPase using inhibition of binding of [3H]digitoxin. These values correlated well (r = 0.99) with the KD values from the [3H]ouabain studies. These results show that: (1) The significant correlation observed between KD values on guinea pig cardiac (Na+ + K+)-ATPase and positive inotropic potency in guinea pig left atria is further evidence that the pharmacological receptor for inotropy is part of the enzyme, (2) Inhibition of the binding of [3H]ouabain or [3H]digitoxin can be used to determine the relative potencies of unlabelled digitalis derivatives. The similar relative potencies on beef and guinea pig cardiac (Na+ + K+)-ATPase of a broad range of digitalis derivatives indicate that the binding site is similar for both species; and (3) SAR studies indicate that functional groups on these steroids have the same influence on potency on either the positive inotropy or cardiac (Na+ + K+)-ATPase studies.

The specific binding of three cardiac glycosides, 3 H-ouabain, 3 H-digitoxin and 3 H-dihydrodigitoxin, to beef cardiac (Na + + K + )-ATPase was compared. Non-specific binding was defined as that in the presence of 0.1 mM unlabelled compound, or in the absence of ligands. The dissociation constants ( K D -values) calculated from the inhibition of 3 H-ouabain binding were: ouabain, 2.9 × 10 −9 M; digitoxin, 1.1 × 10 −9 M; and dihydrodigitoxin 2.7 × 10 −8 M. The concentrations which inhibited beef cardiac (Na + + K + )-ATPase by 50% were: ouabain, 5.9 × 10 −9 M; digitoxin, 1.6 × 10 −9 M; and dihydrodigitoxin, 2.5 × 10 −8 M. Ouabain and digitoxin showed straight Scatchard plots for one site of high affinity (ouabain, K D = 2.6 × 10 −9 M; digitoxin, K D = 1.7 × 10 −9 M). However, dihydrodigitoxin gave a curved Scatchard plot. Analysis of this binding by the methods of M.J. Weidemann, H. Erdelt and M. Klingenberger ( Eur. J. Biochem . 16 , 313 (1970)) for two binding sites gave the following results: for Mg 2+ ,P i -supported binding, the K D of the high affinity site was 1.6 × 10 −8 M with a capacity similar to that for ouabain of about 30 pmole/mg protein. For binding supported by Na + ,ATP,Mg 2+ , the K D -value of the high affinity site was 5.3 × 10 −8 M of similar capacity. The low affinity binding site ( K D = 4.0 × 10 −6 M for Mg 2+ ,P i ; K D = 5.5 × 10 −6 M for Na + ,ATP,Mg 2+ ) bound about 350 pmole/mg protein. The low affinity site but not the high affinity site was also present in heat-denatured enzyme. Bincling supported by Mg 2+ ,P i showed one low affinity site only for ouabain and dihydrodigitoxin in the presence of 200 mM Na + . The high affinity sites for these three cardiac glycosides were further characterized by measurement of the association and dissociation rate constants. The specific binding of 3 H-ouabain and 3 H-dihydrodigitoxin to human cardiac (Na + + K + )-ATPase was measured. 3 H-Ouabain showed a straight Scatchard plot for one high affinity site only ( K D = 4.5 × 10 −9 M, capacity about 15 pmole/mg protein). 3 H-Dihydrodigitoxin gave two binding sites: a high affinity site ( K D = 1.8 × 10 −8 M) of similar capacity to ouabain, and a low affinity site ( K D = 2.0 × 10 −6 M) of about 10-fold greater capacity. We conclude that there are two binding sites for some cardiac glycosides in cardiac (Na + + K + )-ATPase from the digitalis-sensitive beef and human: a dihydrodigitoxin high affinity site also occupied by ouabain and digitoxin, and a low affinity binding site, for dihydrodigitoxin only, whose biological significance has to be investigated further.

Treatment of rats with pregnenolone-16 alpha-carbonitrile (PCN) markedly induces rat liver microsomal cytochrome P-450p and UDP-GT-dt1, a glucuronosyltransferase active towards the digitoxin metabolite, digitoxigenin monodigitoxoside. The present study characterizes the regulation of these two enzymes in rats treated with different xenobiotics. Like PCN, treatment of rats with dexamethasone, spironolactone, troleandomycin or erythromycin estolate markedly induced both UDP-GT-dt1 and cytochrome P-450p (measured as erythromycin demethylase and testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activities). However, compared to PCN and dexamethasone, both troleandomycin and erythromycin estolate preferentially induced cytochrome P-450p, whereas spironolactone preferentially induced UDP-GT-dt1. Treatment of rats with the polychlorinated biphenyl mixture, Aroclor 1254, increased both cytochrome P-450p and UDP-GT-dt1 activity to about 40% of that in liver microsomes from rats induced with PCN or dexamethasone. Treatment of rats with phenobarbital or chlordane caused a relatively small increase in cytochrome P-450p and UDP-GT-dt1 activity. Neither enzyme was induced by treatment of rats with 3-methylcholanthrene, rifampin or digitoxin. The induction of cytochrome P-450p and UDP-GT-dt1 by PCN followed similar dose-response curves. Although cytochrome P-450p and UDP-GT-dt1 are differentially affected by the age and the sex of rats, the enzymes responded similarly, but not identically, to xenobiotic treatment. This suggests that cytochrome P-450p and UDP-GT-dt1 are co-inducible but not coordinately regulated.

Liver musomes 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 ( V max 236 pmoles/mg musomal protein min) were found for digitoxin and digitoxigenin-monodigitoxoside whereas the lowest K m was obtained for digitoxigenin-bisdigitoxoside (29 μM). 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.

Isolated left guinea pig auricles and cardiac Na,K-ATPase from calf heart were incubated with highly unsaturated phosphatidylcholine (PC) for 2 hr. Thereafter the actions of ouabain on Na, K-ATPase, and of ouabain, digoxin, digitoxin, isoproterenol, acetylcholine, pentobarbital and different extracellular Ca2+ concentrations on contractile force of the auricles were investigated. PC itself altered neither the ATPase activity nor the intracellular ionic homeostasis, nor contractile force of the auricles. Similarly, the staircase phenomenon, the contractile response to different extracellular Ca2+ concentrations, to pentobarbital and ouabain, and the extent of ouabain-induced inhibition of the ATPase were not influenced. In addition the toxicity of ouabain was not altered. However, the rate of development of the ouabain-induced ATPase inhibition, as well as of the positive inotropism and toxic effects of ouabain, digoxin and digitoxin was markedly reduced. The maximum inotropic effect of digoxin and digitoxin, and the toxicity of digitoxin were significantly enhanced. Remarkably, the binding of [3H]digitoxin was significantly diminished in PC-pre-incubated auricles, binding of [3H]ouabain, however, remained unchanged. The dose-response curves to isoproterenol and acetylcholine, the latter also in the presence of physostigmine, were markedly parallel-shifted to the right. The modifying effect of highly unsaturated PC on the action of the drugs studied is suggested to be due to an altered physico-chemical state (fluidity) of the outer surface of the cellular membrane.

22Na fluxes (J) and [3H]ouabain binding were studied in vitro in the distal portion of the rabbit colon, mounted as a membrane, separating two perspex chambers. JMSNa (flux of 22Na from mucosal to serosal chamber) was 2.1 and JSMNa was 1.0 μmoles cm2 × hr· JSMNa was completely abolished by 10−3 M ouabain, placed in the serosal side. JSMNa was unaffected by ouabain. [3H] Ouabain binding to the serosal surface of the colon was inhibited to the same maximal effect by ‘cold’ ouabain (I50 = 5 × 10−7 M), by K+ placed in the serosal chamber (I50 = 3.5 mM) and by replacement of Na+ with choline on the serosal side. Increased tissue concentration of Na+ did not affect [3H]ouabain binding, suggesting that extracellular rather than intracellular sodium plays a major role in cardiac glycoside binding. Various cardiac glycosides (digoxin, digitoxin. ouabain) inhibited [3H]ouabain binding to the serosal side of the colon but other steroids (estriol, testosterone, desoxycorticosterone) had not effect. Efflux of [3H]ouabain, bound to the serosal side of the colon, was differentiated into dissociation of nonspecifically bound [3H]ouabain (95 per cent in 60 min) and dissociation of specifically bound [3H]ouabain (20 per cent in 60 min). Dissociation of specifically bound [3H]ouabain was accelerated when Na was replaced by choline (50 per cent in 60 min). The dissociation rate of nonspecifically bound ouabain was unaffected by replacement of Na+· [3H]Ouabain binding to mucosal surface was unaffected by ‘cold’ ouabain, by increased K+ in the medium or by replacement of Na+. It is concluded that there is no specific binding of [3H]ouabain to the mucosal surface. Omission of Ca, Mg or phosphate from the medium did not affect [3H]Ouabain binding to either mucosal or serosal surfaces of rabbit colon.

After oral administration of 25 μg/kg 3 H-labelled digitoxin (sp. act. 26.2 mCi/mg) to female rats, the total radioactivity in blood and in urine was eliminated with a half-life time of 2 and 1.7 days, respectively. The fecal elimination half-life time had a. biphasic course. The chloroform-soluble and chloroform-insoluble metabolites excreted in urine and feces were determined in order to explain the much shorter half-life time of 0.4 days in feces during the early phase of elimination. In the feces, 45 per cent of the dose excreted within 5 days consisted of chloroform-soluble substances. In this fraction, the main excretion product was digoxigenin-bis-digitoxoside (20 per cent), whereas the percentages of the other glycosides, after the last collection period, amounted to significantly less: 9% digitoxin, 9% digoxin. 5% digitoxigenin-bis-digitoxoside, and 2% digitoxigenin-mono-digitoxoside. The Chromatographic analysis of the chloroform-insoluble fraction, which accounted for 15 per cent of the dose. revealed a conjugation of glucuronic and sulfuric acid with digoxin, and digoxin, 5% digitoxigenin-bis-digitoxosidc. and 2% digitoxigenin-mono-digitoxoside. The contrast, sulfuric acid alone was the main conjugation partner of 3-epi-digitoxigenin. In urine, 4.6 per cent of the administered radioactivity was represented by digoxin, 2 per cent by digitoxin, 1 per cent by digoxigenin-bis-digitoxoside, and 1.4 per cent by polar metabolites. Only traces of digitoxigenin-bis-digitoxoside cind digoxigenin-mono-digitoxoside were detected. The much shorter half-life time of the eliminated radioactivity in feces seems to be due to the higher portion of poorly reabsorbed conjugation products and digoxigeninbis-digitoxoside.

Guinea-pig hearts were perfused with Krebs-Henseleit (K-H) medium 30 min for equilibration, then 4, 16, 32 or 64 min with Deslanatoside C- 3 H 10 −7 M in K-H medium and then again with K-H medium alone to wash extracellular spaces at a constant flow of 3 ml/min at 28 C. Deslanatoside C incorporated into the ventricles and atria increased from 4 to 64 min, the ventricles invariably showing a higher degree of incorporation. The concentration of Deslanatoside C also increased from 4 to 64 min in microsome. mitochondrial and nuclear fractions, the microsome traction invariably showing the highest specific concentration. Glycoside in the supernatant fraction was not tightly bound to any macromolecular structure. A β-adrencrgie blocking agent SD 1601 10 −7 M, and Creatinol O -phosphate 10 −5 M both increased myocardial uptake of Deslanatoside C- 3 H and its incorporation into the pellet fractions. Daunomycin 10 −5 M increased the myocardial uptake of the labelled glycoside and its concentration in both supernatant and pellet fractions. Neither phenobarbital sodium 10 −6 M nor Ouabain 10 −7 M affected uptake or the subcellular distribution of Deslanatoside C. Ouabain 10 −6 M decreased both uptake by the heart and the subcellular concentrations of Deslanatoside C.

The effects of digitonin and digitoxin on the phospholipid metabolism of mammalian tissue culture cells have been studied. Digitonin stimulates the incorporation of inorganic orthophosphate- 32 P into the total phospholipid. The phospholipid was then separated on silicic acid-impregnated glass paper chromatography, and the specific activity of each phosphatide was calculated. It was found that digitonin caused a linear increase in the specific activity of phosphatidyl inositol and diphosphatidyl glycerol at the time periods studied. The increase in the specific activities of phosphatidyl choline and phosphatidyl ethanolamine was delayed but to the same extent (more than twofold) after 120-min incubation. The specific activities of phosphatidyl serine and sphingomyelin were low and not significantly affected. The increase was similar in specific activity values of phospholipids of digitonintreated HeLa cells as that of the controls in the nuclear, mitochondrial, and microsomal fractions. Digitonin failed to stimulate the incorporation of palmitic-l- 14 C acid into the phospholipids of HeLa cells. A nonsteroid surface-active agent (Triton X-100) did not stimulate the incorporation of 32 P, into the phospholipids of HeLa cells, but other steroids, desoxycholate, and estradiol did stimulate such incorporation of 32 P i . Digitoxin had little effect on the phospholipid labeling of HeLa cells. However, digitoxin stimulated the incorporation of 32 P, into phosphatidyl inositol and sphingomyelin fraction of human heart culture cells. The specific interaction of various steroids with the target-cell membranes and its relation to phospholipid metabolism is discussed.

The methodology of isolating and purifying large quantities of two highly polar metabolites of digotoxin-7αT by giving KC1 concurrently to rabbits is described. The effect of concomitant KC1 administration and the effect of large doses of digitoxin on the metabolism of the glycoside were evaluated. The cardiotoxicity of the isolated metabolites in cats has been determined. The data show that KC1 does not qualitatively alter the metabolism of digitoxin in rabbits, although the toxicity of the glycoside at the dose employed is abolished. Evidence is also presented which indicates that the large doses of glycoside did not qualitatively alter the metabolism of the drug. When the two isolated metabolites of digitoxin were bioassayed in cats, it was found that they were at least three times more toxic than the parent compound. Thus it was concluded that digitoxin metabolism in the rabbit was unique since other species generally convert the digitoxin to less toxic compounds.

The contractile strength of isolated rabbit heart is dependent on the Ca 2+ concentration in the perfusion medium. At suboptimal Ca 2+ concentration, the addition of exogenous phospholipids extracted from beef heart improves myocardial contractility. Evidence is presented that phospholipids significantly potentiate the inotropic effect of digitoxin. The possible mechanism by which phospholipids facilitate Ca 2+ transport is discussed.

A method of obtaining specifically labeled digitoxin-7αT from Digitalis purpurea plants previously fed pregnenolone-7αT is described. The metabolism of digitoxin-7αT by normal rabbits and by rabbits with surgically induced heart failure was investigated. Digitoxin-7αT was injected into both groups of rabbits, and the urinary excretion of radioactivity was evaluated. By means of countercurrent distribution and thin-layer chromatography, a difference in metabolism of digitoxin-7αT between the two groups was observed. The data show that rabbits with heart failure required much longer periods of time to excrete an equal amount of radioactivity as compared with the normal rabbits. Further, although the radiochromatographic patterns of the two groups were qualitatively identical, the profiles showed that in the group with heart failure the conversion of digitoxin-7αT to its relatively more polar metabolites was substantially hindered.

The Na + + K + + Mg 2+ —dependent adenosine triphosphate phosphohydrolase system (transport ATPase) from the heart muscle of dog, cat, man (fetus), pigeon, guinea-pig, frog, rat and toad was studied. The transport ATPases of the eight species are equally strongly inhibited by calcium ions, they show, however, great variations of the susceptibility to inhibition by the cardiotonic drugs ouabain, digitoxin, cassaine and prednisolone-3,20-bis-guanylhydrazone. The species variations in the inhibitory activity of these cardiotonics are not due to accidental variables or to preparative artefacts, but represent characteristics of the transport ATPases of the various animal species. The cardiotonics compared one with another, differ in the relative strength of the inhibitory effect on the enzymatic systems of various species. The complementarity between the surface of the steroid or terpenoid component of the cardiotonics on the one hand and the surface in the neighbourhood of the K + —centre of the transport ATPase on the other hand is presumed to be the decisive factor for the inhibitory activity of the compounds, because it appears to determine essentially the affinity between the cardiotonic and the enzyme system. Thus, the species variations in the susceptibility of the transport ATPase to inhibition might be caused by species differences in the topography of the neighbourhood of the K + —centre at the outer surface of the cell membrane. The observations on the enzyme level are examined whether they offer an explanation for the species differences of the susceptibility to the therapeutic or toxic actions of ouabain, digitoxin, cassaine or prednisolone-bis-guanylhydrazone. The species dependency of their effects on the enzyme system, the contractility and the intact animal is compared. The results favour the conclusion that the species variations in the susceptibility of the heart muscle and the intact animal to cardiotonics are largely based on corresponding variations in the susceptibility of the different transport ATPases to inhibition by these compounds.

The influence of the four cardiac glycosides, ouabain, digitoxin, digoxin and gitoxin on the uptake of 5-HT by blood platelets has been investigated. They all inhibited the uptake in a dose-dependent manner. Ouabain which has been studied separately inhibited the 5-HT uptake by a non-competitive mechanism. The inhibition was less if K +-ions were omitted from the incubation medium and enhanced by an increase in K+-ions. The uptake of 5-HT by the blood platelets has been already diminished by the withdrawal of K+ from the medium. The results have been discussed in terms of the carrier theory of the transport of norepinephrine proposed by Bogdanski and Brodie.

The antagonistic action of digitoxin and prostaglandin E 1 on the norepinephrine-stimulated lipolysis has been studied qualitatively and quantitatively. The actions of the two drugs are somewhat similar: (a) their antagonism appears, from the log dose-effect curves, to be of a mixed type; (b) Ca 2+ concentration in the medium clearly influences their antagonistic effect. A relation between the action of these drugs on lipolysis and active ion transport or oxidative phosphorylation cannot be entirely excluded. Calcium movements appear more directly involved. The importance of Ca 2+ in the process studied is pointed out.

Background: Malnutrition among hospitalized patients increases length of stay (LOS) and carries extra hospitalization costs. Objective: To review the impact of malnutrition on hospital LOS and costs in Europe. Methods: PubMed and Google Scholar search. All articles from January 2004 until November 2014 were identified. Reference lists of relevant articles were also manually searched. Results: Ten studies on LOS and nine studies on costs were reviewed. The methods used to assess malnutrition and to calculate costs differed considerably between studies. Malnutrition led to an increased LOS ranging from 2.4 to 7.2 days. Among hospitalized patients, malnutrition led to an additional individual cost ranging between 1640 V and 5829 V. At the national level, the costs of malnutrition ranged between 32.8 million V and 1.2 billion V. Expressed as percentage of national health expenditures, the values ranged between 2.1% and 10%. Conclusions: In Europe, malnutrition leads to an increase in LOS and in hospital costs, both at the individual and the national level. Standardization of methods and results reported is needed to adequately compare results between countries. © 2015 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

Binding of indomethacin to HSA was studied in vitro by equilibrium dialysis. Our results show that binding to HSA is 97 per cent at therapeutic levels. Binding is characterized by several saturable numbers of binding sites (n = 8) with a moderate association constant ( K = 1.2. 10 4 M −1 ) and by another nonsaturable phenomenon ( nK = 4200 M −1 ). Furthermore, acetylsalicylic acid (ASA), salicylic acid (SA), 1 anilinonaphtalene-8-sulfonic acid (ANS), chlorophenoxyisobutyrate (CPIB), digitoxin, tryptophan and warfarin do not alter indomethacin binding. On the other hand, free fatty acids (FFA) decrease indomethacin binding in human serum albumin (HSA). Moreover indomethacin markedly decreases warfarin binding.

Stable mutants highly resistant to the cardiac glycoside ouabain (OuaR) and the erythrophleum alkaloid cassaine (CasR) are obtained at similar frequencies in different experiments in mutagen-treated cultures of HeLa cells. All of the OuaR and CasR mutants examined (twelve of each kind from two independent experiments) exhibited identical cross resistance patterns to ouabain and cassaine and to a number of other cardiac glycosides and aglycones such as oleandrin, strophanthidin, digitoxigenin and digitoxin, which were examined. The apparent identity of OuaR and CasR mutants as suggested by these studies indicates that resistance to these two groups of compounds results from the same primary lesion, which from earlier studies on the OuaR mutants is known to involve the plasma membrane Na+, K+-ATPase. Results of genetic studies support the notion that these two groups of compounds, i.e. cardiac glycosides and erythrophleum alkaloids, possess common structural determinants which are responsible for their biological activities, and mutants are selected in both cases against these common determinants. The OuaR and CasR mutants, however, did not show any cross resistance to other types of inhibitors of Na+,K+-ATPase function such as ethacrynic acid, sanguinarine, quindonium bromide, suramin and oligomycin, indicating that the site of action of these inhibitors is different from that of cardiac glycosides and erythrophleum alkaloids.

We have performed direct and indirect binding studies with [3H]ouabain or [3H]digitoxin on beef or guinea pig cardiac (Na+ + K+)-ATPase to measure the potencies of a broad range of cardiotonic steroids for structure-activity relationship (SAR) studies for comparison with previously determined positive inotropic potencies. The positive inotropic potencies of twelve compounds on contracting guinea pig left atria correlated well with the equilibrium dissociation constants (KD values) from the inhibition of [3H]ouabain binding to guinea pig cardiac (Na+ + K+)-ATPase (r = 0.98 for seven 5 beta-compounds, r = 0.95 for five 5 alpha-compounds). Further we calculated KD values from the inhibition of [3H]ouabain binding data for a total of 33 digitalis derivatives on the digitalis-sensitive beef cardiac (Na+ + K+)-ATPase. For the 27 compounds tested on both beef cardiac (Na+ + K+)-ATPase and guinea pig left atria, the potencies showed a significant correlation (r = 0.92 for 22 5 beta-compounds, r = 0.96 for five 5 alpha-compounds. For seven compounds, KD values were measured on beef cardiac (Na+ + K+)-ATPase using inhibition of binding of [3H]digitoxin. These values correlated well (r = 0.99) with the KD values from the [3H]ouabain studies. These results show that: (1) The significant correlation observed between KD values on guinea pig cardiac (Na+ + K+)-ATPase and positive inotropic potency in guinea pig left atria is further evidence that the pharmacological receptor for inotropy is part of the enzyme, (2) Inhibition of the binding of [3H]ouabain or [3H]digitoxin can be used to determine the relative potencies of unlabelled digitalis derivatives. The similar relative potencies on beef and guinea pig cardiac (Na+ + K+)-ATPase of a broad range of digitalis derivatives indicate that the binding site is similar for both species; and (3) SAR studies indicate that functional groups on these steroids have the same influence on potency on either the positive inotropy or cardiac (Na+ + K+)-ATPase studies.

HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not. The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured. In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured. The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr −1 , similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain. In the sucrose gradient experiments it was found that [ 3 H]ouabain, digoxin and digotoxin all initially co-distribute with the plasma membrane marker, 5′-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37°, to co-distribute with the lysosomal marker, β-hexosaminidase. At 2° the ouabain remains co-distributed with the plasma membrane marker. The rate of transfer is estimated to be some 90% hr −1 , much faster than previously thought. Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate. These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell. The main barrier for ouabain occurs at a stage later than this. The consequences of this model on other aspects of pump activity is discussed.

In the present studies, glucuronidation of [ 3 H]digoxigenin monodigitoxoside and [ 3 H]digitoxigenin monodigitoxoside has been investigated in vitro using rat liver microsomes. During a 40-min incubation period, the conjugation of digitoxigenin monodigitoxoside was five times greater than of digoxigenin monodigitoxoside at a substrate concentration of 5 μm and three times greater at a 20 μM substrate concentration. No difference in conjugation rates was observed between male and female rats, with either substrate. Pretreatment with spironolactone (100 mg/kg/day for 3 days) stimulated the conjugation of both substrates in females, but no significant changes were observed in males with either substrate. Pretreatment (75 mg/kg/day) with pregnenolone-16α-carbonitrile (PCN) caused enhanced glucuronidation of both substrates in both sexes. The conjugation rates in the PCN-pretreated rats were approximately twice those of the spironolactone-pretreated rats. The possible role of this increased conjugation in the protective effect of spironolactone and PCN against digitalis toxicity is discussed.

The cross-resistance patterns of two different types of mutants of HeLa cells selected for resistance to the digoxin analog SC4453 (SCR mutants) in which the Na+/K+-ATPase is affected [A. Chopra and R. S. Gupta, J. biol. Chem. 261, 2034 (1986)], and towards numerous cardiac glycosides (CGs) and genins, were examined. One type of SCR mutant (designated as group C) was highly resistant to all CGs and genins investigated. In contrast, the other type of SCR mutant (group D) showed a high degree of cross-resistance towards selected CG derivatives (viz. digoxin, SC4453, digoxigenin, lanatoside C, alpha- and beta-methyldigoxin, dihydrodigoxin, alpha- and beta-acetyldigoxin, alpha,beta-diacetyldigoxin), all of which contained a free 12 beta-OH group in the steroid structure. Slight cross-resistance of the group D mutants was also observed for other compounds (viz. ouabain, ouabagenin, dihydroouabain) that contain a free 11 alpha-OH group in the molecule. However, these mutants exhibited no cross-resistance to other CG derivatives, which either lacked the above groups (viz. digitoxin, digitoxigenin, dihydrodigitoxin, digitoxigenin mono- and bisdigitoside, nerifolin, gitoxigenin, gitoxin, 16-acetylgitoxin, lanatosides A and B, cymarin, convallatoxin, oleandrin, strophanthidin, actodigin and bufalin) or in which the 12 beta-OH group was acetylated (viz. as in the case of 12-acetyldigoxin). Since the 12 beta-OH group is not required for CG-like activity, to account for these observations it is suggested that the genetic lesion in the group D mutant leads to the creation of a new binding site in the digitalis receptor, which specifically interacts with the 12 beta-OH group (the site presumably also interacts weakly with the 11 alpha-OH group) and either prevents or distorts the binding of the compounds to the drug binding site on the receptor. Further investigations with the different classes of CG-resistant mutants at the molecular level should prove very useful in identifying the drug receptor site and in understanding how these drugs interact with it.

Myocardial and liver uptake and subcellular distribution in the two organs of [ 3 H]Deslanatoside C, [ 3 H]Ouabain and [ 3 H]Digitoxin (100 μg/kg, i.v.) were investigated in conscious guinea-pigs. Myocardial uptake, the specific concentrations in its pellet fractions and the ventricle/plasma concentration ratio observed with Deslanatoside C and Ouabain were higher than those observed with Digitoxin, this being the inverse behaviour of that observed in vitro . This suggests that myocardial uptake depends on plasma protein in unbound glycoside, which is 100 per cent with Ouabain, about 70–80 per cent with Deslanatoside C and only about 10 per cent with Digitoxin. The liver uptake of Digitoxin was much higher than that of Ouabain and Deslanatoside C. Plasma protein binding thus seems to not interfere with the liver mechanism uptake. Subcellular distribution of the three glycosides in the liver did not differ. In conclusion, for a given glycoside a substantial difference can be observed between uptake in perfused and in in situ hearts as well as between heart and liver uptake mechanisms, heart uptake provoking contractility and liver uptake biliary excretion.

In rats, microsomal mono-oxygenases are involved in the cleavage and hydroxylation of digitoxin (dt-3).† Previous to hydrolytic cleavage the terminal digitoxosyl has to be oxidized to the corresponding dehydro-digitoxosyl. The microsomal formation rate of 15'-dehydro-dt-3 could be enhanced 3 to 7 fold by pretreatment with pregnenolone-16α-carbonitril, spironolactone, cancrenoate and cyproterone. Phenobarbital and polychlorinated biphenyls had no effect, whereas polyaromatic hydrocarbons caused a significant decrease of both C12-β-hydroxylation and sugar oxidation. The inducing effects of the steroids were less pronounced in the formation of 9'-dehydro-dt-2 and 3'-dehydro-dt-1. For the latter, phenobarbital evoked an inducing effect by factor 1.7. The sugar chain oxidation could be inhibited by metyrapone, spironolactone, cancrenoate and digitoxigenin when added in vitro . a-Naphthoflavone was a weak inhibitor only. The results indicate that the terminal digitoxosyl oxidation is due to a specific isocytochrome P450 (or pattern of isocytochromes) inducible by some synthetic steroids.

The amount of cardiac glycosides tightly bound isolated jejunal brush borders of rat and guinea pig decreases in the following order: peruvosid, digitoxin, proscillaridin, digoxin and ouabain. In rat and guinea pig the p-nitrophenylphosphatase(p-NPPase) and the Na, K-ATPase of the brush borders are about five times more sensitive to digitoxin than to ouabain. In brush borders of both species the sensitivity of the Na, K-ATPase to cardiac glycosides is more than 100 times higher than that of the p-NPPase. In the guinea pig the Na, K-ATPase of the brush borders is 70 times more sensitive to cardiac glycosides than in rat.

On the basis of the xanthydrol reaction and a highly sensitive thin-film chromatographic detection method, content and composition of glycoside metabolites were determined in the heart muscle and in the blood of rats and guinea-pigs after single and repeated i.p. injections of digitoxin. In the guinea-pig's heart we found a maximum of 1 μg/g, in the rat's heart 7–8 μg glycoside/g heart muscle. A pre-treatment by reserpine did not influence the uptake of digitoxin. Digitoxin metabolites, inclusive of genins, were found in the heart muscle most frequently 14–24 hr after application of digitoxin, C 12 -hydroxylation products 6 hr at the earliest after injection. The proportion of metabolites was always relatively small in comparison with unchanged digitoxin. After subacute digitoxin injection the proportion of digitoxin in the heart muscle was much bigger than after only one injection. If the content of digitoxin and its metabolites was compared in the blood and in the heart muscle, a correlation with the digitoxigenin derivatives would be seen. The digoxigenin derivatives, almost without exception, were more frequently detected in the blood rather than in the heart muscle.

A method is described, by means of which the glycosides digitoxin, digitoxigenin- bis -digitoxoside, digitoxigenin-mono-digitoxoside, digoxin, digoxigenin- bis -digitoxoside, digoxigenin-mono-digitoxoside as well as the genins digoxigenin and digitoxigenin are isolated from animal tissue (the blood and heart of rats) after extraction by chloroform and cleaning through Sephadex G 200 on the basis of thin-film chromatography ; these compounds can be prepared separately. Compared with conventional methods, this procedure is about one decimal power more sensitive and permits the isolation as well as the test reaction of each 25 ng of the mentioned compounds in the heart or the blood of an animal.

Digitoxigenin is metabolized in vitro by rat liver homogenates and subcellular fractions. No metabolism by cardiac tissue homogenates was observed, with only limited conversion by kidney tissue homogenates. The primary product is 3-epidigitoxigenin, with smaller amounts of 3-dehydrodigitoxigenin and more polar metabolites being detected. The microsomal hydroxylation system can be induced by treatment of the animals with phenobarbital.

The influence of spironolactone, phenobarbital and β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) pretreatment on the tritium levels in blood, liver and heart of rats given [ 3 H]digitoxin was investigated at various time periods. Blood tritium levels of spironolactone-pretreated rats were significantly lower than control animals 1 hr (38 per cent of control), 2 hr (26 per cent of control), 4 hr (38 per cent of control) and 8 hr (6 per cent of control) after i.p. injection of [ 3 H]digitoxin. Tritium levels in the blood of phenobarbital-pretreated animals were not different from control animals at any of the time periods. SKF 525-A-pretreated animals had blood levels of tritium less than control animals at 1 hr (64 per cent of control) and greater than control animals at 8 hr (174 per cent of control), but were not different from control animals at either 2 or 4 hr. Radioactivity in the heart of spironolactone-pretreated animals was less than that in control animals at all but the 1-hr time interval, while radioactivity in phenobarbital-pretreated animals was not different from that of control animals at any time period and in SKF 525-A-pretreated animals was different only at the 8-hr period (172 per cent of control). Tritium levels in the liver of rats pretreated with spironolactone were greater than control animals at 1 hr (140 per cent of control) and less at 4 hr (66 per cent of control), but were not different at 2 or 8 hr. Phenobarbital pretreatment altered liver levels of tritium only at 1 hr (141 per cent of control), while SKF 525-A did not affect liver levels of radioactivity at any time period. These results indicate that spironolactone pretreatment markedly increases the metabolism and/or excretion of digitoxin, while phenobarbital pretreatment apparently does not affect these processes. The apparent inhibitory effects of SKF 525-A on digitoxin metabolism are seen only at 8 hr after the administration of digitoxin.

An essentially quantitative extraction method for the recovery of microgram quantities of 3 H-digoxin from tissue has been used to study the conversion of digoxin to its genin by liver tissue in vitro . Contrary to a previous report of rapid genin conversion of digitoxin in vitro , only trace amount of genin was formed with digoxin as the parent glycoside. Similar results were obtained with both slices and homogenates under aerobic and anaerobic conditions.