Digitoxin Concentrations Research Articles

Na+/K+‑ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells.

The alpha subunits (ATP1A1-3) of Na+/K+-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the ATP1A1 and ATP1A3 subunits. In Panc-1 cells, ATP1A3 gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the ATP1A1 gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/β/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the β [E484K], Mink [Y453F] and α/β/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells

BackgroundCardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. MethodsWe treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. ResultsDigitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. ConclusionsOur study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SignificanceDiscovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

Effect of irradiation by gamma rays and the use of benzyl adenine to increase the production of cardiac glycoside compounds from Digitalis lanata in vitro

Seeds of fox glove Digitalis lanata were irradiated with gamma ray with the dose 25, 50 grey in addition to the treatment of the comparison 0 grey, BA was also used at the concentrations of 0, 1, 2, 3, 4 mg. L−1 to determine the effectiveness of the gamma ray and BA and their interaction in stimulating the proportion, speed of seed germination and production of Cardiac glycosides, chlorophyll and carbohydrates in the vegetative branches of the plantlet implanted on the nutritional media. The results showed that the irradiation at the dose of 50 grey was significantly increased the rate of germination percent to 100% and the speed of germination by a rate of 5 seeds / day, compared with control group which gave 30% and 1.1 seeds / day respectively. The same treatment had a significant increase on the rate of production of Digoxin, Digitoxin and Gitoxin glycosides to 45.54, 91.87 and 68.70 μg. g−1 respectively, compared to control group values of 6.40, 20.17 and 13.09 μg.−1 g respectively. The same treatment achieved a significant increase in the total chlorophyll and carbohydrate production rate of 2.10 and 3.80 mg g−1 respectively, compared with the control group values of 1.30 and 2.82 mg. g−1 respectively. Also the benzyl adenine at a concentration of 3 mg.L−1 increased the rate of the same glycoside compounds which to 58.57, 79.15 and 78.14 μg. g−1 respectively, compared to the control values of 25.97, 56.02 and 40.90 μg. g−1 respectively. Also, the concentration of total chlorophyll and carbohydrates was 2.74 and 4.39 mg. g−1 respectively, compared to the control group values of 1.70 and 3.31 mg. g−1 respectively. As for the effect of the interference of irradiation treatment at 3 mg. g−1 concentration with adenine on Digoxin and Gitoxin the concentrations were 79.11 and 118.20 μg. g−1, respectively, and the chlorophyll concentration was 3.65 mg. g−1. The irradiation treatment at 4 mg. L−1 achieved the highest concentration of Digitoxin (118.23 μg. g−1) and the highest concentration of carbohydrates of (5.13 mg. g−1).

Digitoxin Inhibits Epithelial-to-Mesenchymal-Transition in Hereditary Castration Resistant Prostate Cancer.

Castration Resistant Prostate Cancer (CRPC) is thought to be driven by a collaborative mechanism between TNFα/NFκB and TGFβ signaling, leading to inflammation, Epithelial-to-Mesenchymal-Transition (EMT), and metastasis. Initially, TGFβ is a tumor suppressor, but in advanced metastatic disease it switches to being a tumor promoter. TGFBR2 may play a critical role in this collaboration, as its expression is driven by NFκB and it is the primary receptor for TGFβ. We have previously reported that the cardenolide drug digitoxin blocks TNFα/NFκB-driven proinflammatory signaling. We therefore hypothesized that digitoxin might break the collaborative process between NFκB and TGFβ by also inhibiting expression of TGFBR2. We therefore tested whether TGFβ-driven EMT and resulting metastases would be suppressed. Here we show, in vitro, that digitoxin inhibits NFκB-driven TGFBR2 expression, as well as Vimentin, while elevating E-cadherin expression. Digitoxin also significantly reduces HSPB1 mRNA and the HSPB1/RBFOX2 mRNA ratio in PC3 cells. In vivo, in a syngeneic, immune competent rat model of metastatic CRPC, we show that digitoxin also suppresses Tgfbr2 expression, as well as expression of other genes classically driven by NFκB, and of multiple EMT genes associated with metastasis. Concurrently, digitoxin suppresses tumor growth and metastasis in these animals, and prolongs survival. Gross tumor recurrence following tumor resection also appears prevented in ca 30% of cases. While the existence of a collaboration between NFκB and TGFβ to drive EMT and metastasis has previously been appreciated, we show here, for the first time, that chronic, low concentrations of digitoxin are able to block CRPC tumor progression, EMT and the ensuing metastatic disease.

Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors.

Cardiac glycosides are Na+ /K+ -ATPases inhibitors used to treat congestive heart failure and cardiac arrhythmias. Epidemiological studies indicate that patients on digitalis therapy are more protected from cancer. Evidence of a selective cytotoxicity against cancer cells has suggested their potential use as anticancer drugs. The effect on angiogenesis of clinically used cardiac glycosides has not been extensively explored. We studied the effect of digoxin, digitoxin and ouabain on early events of the angiogenic process in HUVECs. We determined HUVEC viability, proliferation, migration and differentiation into capillary tube-like structures. We also tested drug activity using an in vivo angiogenesis model. Activation of protein tyrosine kinase 2 (FAK) and signalling proteins associated with the Na+ /K+ -ATPase signalosome was determined by Western blotting. Digitoxin and ouabain (1-100nM) inhibited HUVEC migration, concentration-dependently, without affecting cell viability, while digoxin induced apoptosis at the same concentrations. Digitoxin antagonized growth factor-induced migration and tubularization at concentrations (1-25nM) within its plasma therapeutic range. The anti-angiogenic effect of digitoxin was confirmed also by in vivo studies. Digitoxin induced Src, Akt and ERK1/2 phosphorylation but did not affect FAK autophosphorylation at Tyr397 . However, it significantly inhibited growth factor-induced FAK phosphorylation at Tyr576/577 . Therapeutic concentrations of digitoxin inhibited angiogenesis and FAK activation by several pro-angiogenic stimuli. These novel findings suggest a potential repositioning of digitoxin as a broad-spectrum anti-angiogenic drug for diseases where pathological angiogenesis is involved.

Drug safety associated with the change of digitalis drug in Norway

BACKGROUND In 2011, following a period with delivery problems, the only registered digitoxin drug in Norway was replaced with digoxin. As a result, approximately 21000 patients had to replace digitoxin with digoxin. There are important pharmacokinetic differences between digitoxin and digoxin (the general term for both drugs is digitalis), which must be taken into account when changing therapy. The aim of this study was to investigate compliance of drug security, during the transition from digitoxin to digoxin in Norway.MATERIAL AND METHOD Enquiries addressed to the Norwegian Poison Information Centre and reports of fatal adverse effects to the Regional Drug Information Centres (RELIS) regarding intake of digitalis were analysed. Serum concentrations of digitoxin and digoxin analyzed at Oslo University Hospital were reviewed. All data sources were reviewed for the years 2010-2014 and patients > 20 years were included.RESULTS The total number of enquiries addressed to the Norwegian Poison Information Centre, fatal adverse drug reactions reported to RELIS, and patient samples in the toxic range analyzed at Oslo University Hospital increased from 2012, timewise related to the transition to digoxin.INTERPRETATION Despite extensive information from the Norwegian Medicines Agency, a small, transient increase was observed in the number of overdoses and reported deaths from digitalis related to change in therapy. The cause of the overdose was in many cases unknown. This study revealed several cases of incorrect dosage, simultaneous use of digitoxin and digoxin, and washout time that was insufficient or lacking before initiation of digoxin.

Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients

We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.

A medieval case of digitalis poisoning: the sudden death of Cangrande della Scala, lord of verona (1291–1329)

The natural mummy of Cangrande della Scala was exhumed from its tomb in the church of Santa Maria Antiqua in Verona and was submitted to a multidisciplinary study, that included archaeological, palaeopathological, palynological, toxicological and historical investigation. The body of Cangrande, still wearing his precious clothes, was in a good state of preservation. Palynological analyses demonstrated the presence of pollen grains of Digitalis sp./foxglove in the rectum content, along with Matricaria chamomilla/chamomille and Morus nigra/black mulberry. Toxicological analyses showed toxic concentrations of digoxin and digitoxin, two Digitalis glycosides, in the liver and faeces samples. Both palynological and toxicological data suggest an intoxication through the oral administration of an infusion or decoction of leaves and flowers of Digitalis.Cangrande died on July 22 1329, four day after his triumphal entrance in the city of Treviso. The sudden death was preceded by vomit and diarrhoea with fever that, according to written documents, he had contracted a few days before by “drinking from a polluted spring”. The gastrointestinal symptoms manifested by Cangrande in his last hours of life are compatible with the early phase of Digitalis intoxication and the hypothesis of poisoning is mentioned by some local historical sources. The palaeopathological analyses confirm a Digitalis poisoning. The most likely hypothesis on the causes of death is that of a deliberate administration of a lethal amount of Digitalis. Although several cases of poisoning through the use of organic substances are known from historical sources, no other direct evidences are documented in the palaeopathological literature.

The Role of Digitalis Pharmacokinetics in Converting Atrial Fibrillation and Flutter to Regular Sinus Rhythm

This report examined the role of digitalis pharmacokinetics in helping to guide therapy with digitalis glycosides with regard to converting atrial fibrillation (AF) or flutter to regular sinus rhythm (RSR). Pharmacokinetic models of digitoxin and digoxin, containing a peripheral non-serum effect compartment, were used to analyze outcomes in a non-systematic literature review of five clinical studies, using the computed concentrations of digitoxin and digoxin in the effect compartment of these models in an analysis of their outcomes. Four cases treated by the author were similarly examined. Three literature studies showed results no different from placebo. Dosage regimens achieved ≤11 ng/g in the model's peripheral compartment. However, two other studies achieved significant conversion to RSR. Their peripheral concentrations were 9-14 ng/g. In the four patients treated by the author, three converted using classical clinical titration with incremental doses, plus therapeutic drug monitoring and pharmacokinetic guidance from the models for maintenance dosage. They converted at peripheral concentrations of 9-18 ng/g, similar to the two studies above. No toxicity was seen. Successful maintenance was achieved, using the models and their pharmacokinetic guidance, by giving somewhat larger than average recommended dosage regimens in order to maintain peripheral concentrations present at conversion. The fourth patient did not convert, but only reached peripheral concentrations of 6-7 ng/g, similar to the studies in which conversion was no better than placebo. Pharmacokinetic analysis and guidance play a highly significant role in converting AF to RSR. To the author's knowledge, this has not been specifically described before. In my experience, conversion of AF or flutter to RSR does not occur until peripheral concentrations of 9-18 ng/g are reached. Results in the four cases correlated well with the literature findings. More work is needed to further evaluate these provocative findings.

Real-time analysis of the effects of toxic, therapeutic and sub-therapeutic concentrations of digitoxin on lung cancer cells

Digitoxin belongs to a naturally occurring class of cardiac glycosides (CG); digitoxin is clinically approved for heart failure and known for its anti-cancer effects against non-small lung cancer cells (NSCLC). However, concerns associated with its narrow therapeutic index and its concentration-dependent mechanism of action are rising. Thus, before digitoxin implementation in designing and developing safer and more effective CG-based anti-cancer therapies, its pharmacological and safety profiles need to be fully elucidated. In this research we used a combinatorial approach to evaluate the anti-cancer mechanisms of digitoxin in real-time. Our approach employed a non-invasive electric cell impedance sensing technique as a proxy to monitor NSCLC behavior post-exposure to toxic, therapeutic and sub-therapeutic concentrations of the drug. By developing structure–function combinatorial relations we showed that digitoxin targets cancer cells in a time and dose-dependant manner by activating pro-apoptotic and anti-proliferative signaling cascades that results in strengthening cellular adhesion and sequestration of key regulatory proliferation protein from the nucleus.

Evaluation of a straightforward and rapid method for the therapeutic drug monitoring of digitoxin by LC-MS/MS

ObjectivesTherapeutic drug monitoring of digitoxin is strongly recommended but metabolites of digitoxin and digitoxin-like immunoreactive substances may interfere with widely used immunoassays. Recently evaluated assays on LC-MS/MS have the drawback of long turnaround time. We sought to evaluate a specific method on LC-MS/MS optimizing sample preparation thereby significantly reducing turnaround time. Design and methodsLinearity, functional sensitivity, and precision of the method were established. External quality control samples were used for the evaluation of accuracy of the LS-MS/MS method. In addition, digitoxin concentrations in 221 samples were measured by LC-MS/MS and immunoassay. ResultsLinearity was validated between 0.15 and 80ng/mL. Limit of quantification was established at 0.14ng/mL. Between-day imprecision lay between 1.4 and 4.9% and meets the conditions required for routine analysis. Comparison to results obtained by immunoassay revealed a mean difference of −1.2ng/mL. ConclusionsBy optimizing preparation steps turnaround time was shorter for LC-MS/MS than for immunoassay. This did not result in increased susceptibility to matrix effects. Analytical performance was sufficient for routine analysis. Therefore, the method is suitable for routine therapeutic drug monitoring of digitoxin.

Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells

Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potency than digitoxin. In comparison, non-tumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.

Dosering av digitoksin i klinisk praksis

In 2007, new Norwegian guidelines suggested that serum concentrations of digitoxin should be in the interval 8 - 15 nmol/l, which is about 50 % lower than previous recommendations. We studied trends in dosing and serum concentrations of digitoxin in the period 2000 - 08, based on 13 054 serum samples sent to our laboratory for analysis in that period. The median serum concentration of digitoxin was stable until the end of 2006 (at about 25 nmol/l); then it gradually decreased from 2007 until the end of 2008 (to 19 nmol/l). The mean daily dose decreased from 66 microg to 56 microg in the study period. At a given dose, patients above 85 years of age had serum concentrations that were almost twice as high as those for patients younger than 55 years. This age effect was particularly pronounced from 65 years. Serum concentrations of digitoxin have gradually decreased after 2007, but are often higher than the new reference range. To obtain serum concentrations within the new reference interval, doses at least as low as those currently recommended should be used, particularly in the oldest patients.

Anti-HSV activity of digitoxin and its possible mechanisms

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC 50) of 0.05 μM. The 50% cytotoxicity concentration (CC 50) of digitoxin is 10.66 μM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.

The association of ABCB1 polymorphisms and elevated serum digitoxin concentrations in geriatric patients

Digitoxin is a known substrate of the efflux pump P-glycoprotein (gene name: ABCB1). P-glycoprotein expression was shown to be modulated by single nucleotide polymorphisms in the ABCB1 gene, but it remains unclear whether these polymorphisms influence digitoxin blood levels. Our objective was to examine the association of ABCB1 C3435T genotype and elevated serum digitoxin concentrations (SDC) in a cohort of 77 geriatric patients consecutively admitted to a geriatric department over a 12-month period. The impact of ABCB1 3435 CC, CT, and TT genotypes on SDC and SDC normalized for daily digitoxin dosage and body weight was assessed by multivariate regression analysis. Among participants, 18 (23%) had the CC, 36 (47%) the CT, and 23 (30%) the TT genotype. Adjusting for relevant covariates, no significant association of ABCB1 C3435T genotype and SDC or normalized SDC was detected. Mean SDC was 22.4 ng/ml (95% CI 18.9-25.9) for the TT, 21.8 ng/ml (95% CI 18.1-25.5) for the CT, and 25.7 ng/ml (95% CI 20.6-30.8) for the CC genotype. The means for normalized SDC were 5.2 kg.l(-1) (95% CI 4.3-6.1) for the TT, 6.1 kg.l(-1) (95% CI 4.7-7.5) for the CT, and 6.2 kg.l(-1) (95% CI 4.6-7.7) for the CC genotype. In this sample of frail geriatric patients, the impact of ABCB1 C3435T genotype on serum digitoxin concentration was not of major relevance. Regular monitoring of digitoxin blood levels and surveillance of appropriate drug use remain the best ways to prevent digitoxin intoxications in the elderly.

Targeting Na+/K+-ATPase in non-small cell lung cancer (NSCLC)

18144 Background: Non-small cell lung cancer patients that present with grade IIIB or stage IV disease have a median survival of 5–7 months if left untreated. With modern chemotherapy overall survival may be 11–12 months, but still no patients are cured. We have investigated the impact of modulation of the a-1 subunit of Na+/K+-ATPase in NSCLC. Methods: Cancer tissue from 59 patients with NSCLC (30 adenocarcinomas and 29 squamous cell cancers) and 25 normal lung samples as well as four human NSCLC cell lines (A549, Cal-12T, NCI-H727, A427) were assessed with regard to expression of the a-1 subunit of Na+/K+-ATPase (sodium pump) by use of immunohistochemistry. In addition, A549 cells were transfected with specific a-1 siRNA for study of a-1 subunit expression and of cell proliferation and migration. Protein expression was analyzed by Western blotting. Cell proliferation was assessed by MTT and cell migration by video microscopy. Cell lines were exposed to varying concentrations of ouabain, digoxin, digitoxin and UNBS1450, a novel cardenolide targeting the a-1 subunit of Na+/K+-ATPase for study of proliferation, migration, and inhibition of the target. Results: Expression of the a-1 subunit of Na+/K+- ATPase was elevated in almost half of the tissue samples from patients with NSCLC compared to normal controls. The a-1 subunit was also overexpressed in A549, Cal-12T and NCI-H727 cells. Transfection of A549 cells with siRNA resulted in markedly decreased expression of the a- 1 subunit and also to reduced migration and proliferation of such cells. UNBS1450 at 10 and 100 nM for 72 hours reduced A549 cell migration and proliferation similar to that observed with anti- a-1 siRNA. Digoxin had no activity at these concentrations. Conclusions: Inhibition of the a-1 subunit of Na+/K+-ATPase is associated with significant decrease of cell migration and proliferation and has potential as a therapeutic strategy in NSCLC. No significant financial relationships to disclose.

Role of P-Glycoprotein Inhibition for Drug Interactions

We determined in vitro the potency of macrolides as P-glycoprotein inhibitors and tested in hospitalised patients whether coadministration of P-glycoprotein inhibitors leads to increased serum concentrations of the P-glycoprotein substrates digoxin and digitoxin. In vitro, the effect of macrolides on polarised P-glycoprotein-mediated digoxin transport was investigated in Caco-2 cells. In a pharmacoepidemiological study, we analysed the serum digoxin and digitoxin concentrations with and without coadministration of P-glycoprotein inhibitors in hospitalised patients. All macrolides inhibited P-glycoprotein-mediated digoxin transport, with concentrations producing 50% inhibition (IC(50)) values of 1.8, 4.1, 15.4, 21.8 and 22.7 micromol/L for telithromycin, clarithromycin, roxithromycin, azithromycin and erythromycin, respectively. Coadministration of P-glycoprotein inhibitors was associated with increased serum concentrations of digoxin (1.3 +/- 0.6 vs 0.9 +/- 0.5 ng/mL, p < 0.01). Moreover, patients receiving macrolides had higher serum concentrations of cardiac glycosides (p < 0.05). Macrolides are potent inhibitors of P-glycoprotein. Drug interactions between P-glycoprotein inhibitors and substrates are likely to occur during hospitalisation.

Determination of Digitoxin in Human Serum Using Stable Isotope Diluted Liquid Chromatography/Electrospray Ionization‐Tandem Mass Spectrometry

A method for the determination of digitoxin in human serum using a liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS) technique is reported. Digitoxin and the internal standard, [21,21,22‐2H3]digitoxin, were extracted from 200 µL of human serum using a solid phase extraction cartridge and analyzed by LC/ESI‐MS/MS in the selected reaction monitoring mode. The intra‐ and inter‐assay reproducibility and accuracy were satisfactory within the quantification range of 5–100 ng/mL, which was sensitive enough to measure the digitoxin in real samples. The concentrations of digitoxin in serum samples obtained from digitalized patients (n=19) were in the range of 5.3–24.1 ng/mL, and these correlated well with those obtained by radioimmunoassay using a very specific antiserum.

Digitalis-Induced Signaling by Na+/K+-ATPase in Human Breast Cancer Cells

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.