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The 78 kilodalton glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) molecular chaperone with antiapoptotic properties and a key regulator of the unfolded protein response (UPR). ER-stress induction of GRP78 in cancer cells represents a major pro-survival branch of the UPR. Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease and high level of GRP78 is associated with aggressive disease and poor survival. Recently, we reported that PDAC exhibited high level of ER stress and that GRP78 haploinsufficiency is sufficient to suppress pancreatic tumorigenesis in mice, suggesting the utility of inhibitors of GRP78 expression in combating pancreatic cancer. Screening of clinically relevant compound libraries revealed that cardiac glycosides (CGs) can inhibit ER-stress induction of GRP78 in pancreatic and other types of human cancers. Using the FDA-approved CG compound Lanatoside C (LanC) and human pancreatic cancer cell lines as model systems, we discovered that LanC preferably suppressed ER stress induction of GRP78 and to a lesser extent GRP94. The suppression is at the post-transcriptional level and dependent on the Na+/K+-ATPase ion pump. Overexpression of GRP78 mitigates apoptotic activities of LanC in ER stressed cells. Our study revealed a new function of CGs as inhibitor of stress induction of GRP78, and that this suppression at least in part contributes to the apoptotic activities of CGs in human pancreatic cancer cells in vitro. These findings support further investigation into CGs as potential antineoplastic agents for pancreatic and other cancers which depend on GRP78 for growth and survival.

Neuroinflammation is a key component of virtually all neurodegenerative diseases, preceding neuronal loss and associating directly with cognitive impairment. Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain vasculature, surrounding meninges and the choroid plexus. We designed a high content screening system to target inflammation in human brain-derived cells of the blood–brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers. Screening an FDA-approved drug library we identify digoxin and lanatoside C, members of the cardiac glycoside family, as inflammatory-modulating drugs that work in blood–brain barrier cells. An ex vivo assay of leptomeningeal and choroid plexus explants confirm that these drugs maintain their function in 3D cultures of brain border tissues. These results suggest that cardiac glycosides may be useful in targeting inflammation at border regions of the brain and offer new options for drug discovery approaches for neuroinflammatory driven degeneration., Jansson et al. design a high content screening system to target inflammation in human brain cells of the blood–brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers. They identify cardiac glycosides as powerful regulators of neuroinflammatory pathways in brain barrier tissues such as vasculature, meninges and choroid plexus.

Lanatoside C has a promising anti-tumor activity and is a potential candidate for radiosensitizers. In this study, we have investigated the therapeutic efficacy of the combination of 131I-trastuzumab and lanatoside C for inhibition of human epidermal growth factor receptor 2 (HER2) positive tumor progression in NCI-N87 xenograft model. The combination treatment (131I-trastuzumab and lanatoside C) showed highest cytotoxicity when compared to non-treated control or trastuzumab alone or 131I alone or 131I-trastuzumab alone in vitro. Biodistribution studies using 131I-trastuzumab or combination of 131I-trastuzumab and lanatoside C showed tumor uptake in BALB/c nude mice bearing HER2 positive NCI-N87 tumor xenograft model. The higher tumor uptake was observed in 131I-trastuzumab (19.40 ± 0.04% ID/g) than in the combination of 131I-trastuzumab and lanatoside C (14.02 ± 0.02% ID/g) at 24 h post-injection. Most importantly, an antitumor effect was observed in mice that received the combination of 131I-trastuzumab and lanatoside C (p = 0.009) when compared to control. In addition, mice received lanatoside C alone (p = 0.085) or 131I-trastuzumab alone (p = 0.160) did not significantly inhibit tumor progression compared with control. Taken together, our data suggest that combination of 131I-trastuzumab and lanatoside C might be a potential synergistic treatment for radioimmunotherapy to control the HER2 positive tumor.

Purpose This study investigated the effect of cedilanid on retinal neovascularization in a mouse model of oxygen-induced retinopathy. Methods Seven-day-old C57BL/6 mice were exposed to 75% ± 1% oxygen for 5 days and were then returned to room air to induce retinal neovascularization. Cedilanid (0.025–0.2 μg) was intravitreally injected into the left eye of each mouse on postnatal day 12 (P12) and P15. PBS was intravitreally injected into the right eye as a control. Retinal neovascularization was evaluated with isolectin GS-IB4 staining of the retinal blood vessels. The function of reestablishment blood vessels was evaluated with angiography with the injection of fluorescein isothiocyanate (FITC)-dextran followed by isolectin GS-IB4 staining. Real time (RT)–PCR and western blot were used to examine the mRNA and protein expression of hypoxia inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF), respectively. Results Retinal neovascular areas and obliterative areas were statistically significantly smaller in the eyes injected with cedilanid (0.05 μg, 0.1 μg, and 0.2 μg) compared with the control eyes. The inhibitory effect of cedilanid was observed in a dose-dependent manner. In addition, the retinal neovascular areas and the obliterative areas in the eyes injected with 0.2 μg cedilanid on P12 were statistically significantly smaller than those in the eyes injected with the same dose of cedilanid on P15. Cedilanid promoted the circulative function of reestablished blood vessels in the obliterative areas. Cedilanid inhibited the expression of HIF-1α and VEGF in mice treated with hyperoxia. Conclusions Cedilanid inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy. Early treatment with cedilanid produces better inhibition of retinal neovascularization. Cedilanid may be a potential treatment of neovascular diseases.

Cardenolides isolated from genus Digitalis are natural compounds used in treating cardiac insufficiencies. Recent investigations have also suggested the use of cardenolides in oncology. Due to the medicinal importance of the genus Digitalis, in vitro regeneration techniques would be useful for propagating the best genotypes selected from natural sources. To achieve this, Digitalis ferruginea subsp. schischkinii (Ivanina) K. Werner was collected from different locations along the Black Sea region of Turkey. In the cardenolide analysis, samples from Giresun and Trabzon showed a greater diversity than those from other locations, and thus, the Trabzon population was selected for plant tissue culture studies. In vitro regeneration was performed using cotyledonary leaves and hypocotyl segments on Murashige and Skoog (MS) medium containing various concentrations (0.5, 1.0, or 2.0 mg L-1) of 6-benzyladenine (BA), kinetin (KIN), or thidiazuron (TDZ) alone or in combination with indole-3-acetic acid (IAA) at 0.5 or 1.0 mg L-1. Among the media used for shoot regeneration, the highest frequency of shoot organogenesis was obtained from MS medium containing 2.0 mg L-1 TDZ and 0.5 mg L-1 IAA, producing 11.0 shoots per explant with 100% regeneration frequency from hypocotyl explants after 4 wk. For successful rooting, IAA was the most effective at 1.0 or 2.0 mg L-1, producing 16.3 or 17.3 roots per shoot, respectively, after 4 wk. Regenerants from several medium types were grown in a greenhouse for 6 mo, and all displayed similar cardenolide contents, i.e., the lanatoside C level was around 0.3% (w/w) while the levels of lanatosides A and B, digoxin, and digitoxin were less than 0.08% (w/w), similar to plants grown from seeds collected from Trabzon district.

Cardiac glycosides are clinically used for cardiac arrhythmias. In this study, we investigated the mechanism responsible for anti-cancer and radiosensitizing effects of lanatoside C in colorectal cancer cells. Lanatoside C-treated cells showed classic patterns of autophagy, which may have been caused by lanatoside C-induced mitochondrial aggregation or degeneration. This mitochondrial dysfunction was due to disruption of K+ homeostasis, possibly through inhibition of Na+/K+-ATPase activity. In addition, lanatoside C sensitized HCT116 cells (but not HT-29 cells) to radiation in vitro. γ-H2AX, a representative marker of DNA damage, were sustained longer after combination of irradiation with lanatoside C, suggesting lanatoside C impaired DNA damage repair processes. Recruitment of 53BP1 to damaged DNA, a critical initiation step for DNA damage repair signaling, was significantly suppressed in lanatoside C-treated HCT116 cells. This may have been due to defects in the RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A that increases 53BP1 recruitment to DNA damage sites. Although lanatoside C alone reduced tumor growth in the mouse xenograft tumor model, combination of lanatoside C and radiation inhibited tumor growth more than single treatments. Thus, lanatoside C could be a potential molecule for anti-cancer drugs and radiosensitizing agents.

Recent studies have revealed that cardiac glycosides, such as digitalis and digoxin, have anticancer activity and may serve as lead compounds for the development of cancer treatments. The poor prognosis of hepatocellular carcinoma (HCC) patients reflects the development of resistance to current chemotherapeutic agents, highlighting the need for discovering new small-molecule therapeutics. Here, we found that lanatoside C, an anti-arrhythmic agent extracted from Digitalis lanata, inhibited the growth of HCC cells and dramatically decreased tumor volume as well as delayed tumor growth without obvious body weight loss. Moreover, lanatoside C triggered mitochondrial membrane potential (MMP) loss, activation of caspases and translocation of apoptosis-inducing factor (AIF) into the nucleus, which suggests that lanatoside C induced apoptosis through both caspase-dependent and -independent pathways. Furthermore, we discovered that lanatoside C activated protein kinase delta (PKCδ) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKCδ reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKCδ activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKCδ activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKCδ activation.

Acylation of lanatoside C in the presence of organocatalyst 5 gave the C(4'''')-O-acylate in up to 90% regioselectivity (catalyst-controlled regioselectivity). Various functionalized acyl groups can be introduced at the C(4'''')-OH by a mixed anhydride method in the presence of 5 or the related organocatalyst. On the other hand, DMAP-catalyzed acylation of lanatoside C gave the C(3'''')-O-acylate in up to 97% regioselectivity (substrate-controlled regioselectivity). Thus, diverse regioselective introduction of acyl groups among eight free hydroxy groups of lanatoside C was achieved.

Lanatoside C’s impact on atherosclerosis is poorly understood. The present study was conducted to determine whether lanatoside C affects the development of atherosclerosis in apolipoprotein E-deficient (ApoE–/–) mice. ApoE–/– mice were administered either phosphate-buffered saline (PBS) containing 0.1% DMSO (the vehicle control group) or lanatoside C at low (1 mg/kg per day) or high (2 mg/kg per day) doses and fed a Western diet for 12 weeks. Lanatoside C dose-dependently aggravated the development of atherosclerosis in the ApoE–/– mice compared with the vehicle control group. In an effort to determine the mechanism by which lanatoside C increased atherosclerosis, we found that lanatoside C significantly promoted the uptake of oxidised low-density lipoprotein (oxLDL) and increased foam-cell formation by upregulation of scavenger receptor class A (SR-A) and the class B scavenger receptor (CD36) in macrophages. Meanwhile, the effects of lanatoside C were abolished using small interfering RNA (siRNA) inhibition of peroxisome proliferator-activated receptors β/δ (PPARβ/δ). Overall, our data demonstrate that lanatoside C aggravates the development of atherosclerosis by inducing PPARβ/δ expression, which mediates upregulation of SR-A and CD36 and promotes oxLDL uptake and foam-cell formation.

Background Hepatocellular carcinoma is the second deadliest cancer with limited treatment options. Loss of PTEN causes the P13K/Akt pathway to be hyperactive which contributes to cell survival and resistance to therapeutics in various cancers, including the liver cancer. Hence molecules targeting this pathway present good therapeutic strategies for liver cancer. Hypothesis It was previously reported that Cardiac glycosides possessed antitumor activity by inducing apoptosis of multiple cancer cells through oxidative stress. However, whether Cardiac glycoside Lanatoside C can induce oxidative stress in liver cancer cells and induce cell death both in vitro and in vivo remains unknown. Methods Cell viability was measured by SRB assay. Cell death analysis was investigated by propidium iodide staining with flow cytometry and PARP cleavage. DCFH-DA staining and cytometry were used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Antitumor activity was investigated on mice xenografts in vivo. Results In this study, we found that Cardiac glycosides, particularly Lanatoside C from Digitalis ferruginea could significantly inhibit PTEN protein adequate Huh7 and PTEN deficient Mahlavu human liver cancer cell proliferation by the induction of apoptosis and G2/M arrest in the cells. Lanatoside C was further shown to induce oxidative stress and alter ERK and Akt pathways. Consequently, JNK1 activation resulted in extrinsic apoptotic pathway stimulation in both cells while JNK2 activation involved in the inhibition of cell survival only in PTEN deficient cells. Furthermore, nude mice xenografts followed by MRI showed that Lanatoside C caused a significant decrease in the tumor size. In this study apoptosis induction by Lanatoside C was characterized through ROS altered ERK and Akt pathways in both PTEN adequate epithelial and deficient mesenchymal liver cancer cells. Conclusion The results indicated that Lanatoside C could be contemplated in liver cancer therapeutics, particularly in PTEN deficient tumors. This is due to Lanatoside C's stress inducing action on ERK and Akt pathways through differential activation of JNK1 and JNK2 by GSK3β. © 2015 Elsevier GmbH. © 2016 Elsevier GmbH. All rights reserved.

Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.

OBJECTIVE To investigate whether the impaired reflex response to cardiopulmonary baroreceptor unloading in hypertensive patients with left ventricular hypertrophy can be promptly improved by a pharmacological challenge. For this purpose we studied the effects of acute digitalis administration on cardiopulmonary baroreflex, evaluated by forearm noradrenaline spillover. METHODS Eleven hypertensives with left ventricular hypertrophy and 10 age- and sex-matched normotensives underwent the application of -5 and -10 mmHg lower-body negative pressure (LBNP) before and after the administration of digitalis. Forearm noradrenaline spillover, measured using a tracer technique, was used to estimate the reflex sympathetic response. RESULTS Under control conditions LBNP evoked a similar fall in right atrial pressure in the two study groups. In the normotensives there was a significant increase in forearm noradrenaline spillover. In the hypertensives no significant changes in forearm noradrenaline spillover were found. Intravenous administration of 0.02 mg/kg lanatoside C was associated with an increase in systolic blood pressure and a reduction in forearm noradrenaline spillover in both groups. In the normotensives the percentage change in forearm noradrenaline spillover induced by LBNP increased significantly in response to digitalis administration. However, digitalis restored the response of forearm noradrenaline spillover to LBNP in the hypertensives, so that no significant difference in this response was detected between the two study groups. Digitalis did not modify the effects of LBNP on cardiac pressures in either group. CONCLUSIONS The present results demonstrate that administration of lanatoside C restores the response of forearm noradrenaline spillover to cardiopulmonary baroreceptor unloading in hypertensive patients with left ventricular hypertrophy. This indicates that the impairment of cardiopulmonary baroreflexes in these patients can be reversed by acute pharmacological treatment. Therefore, impairment of this reflex response seems to be related to functional rather than to structural abnormalities of the hypertrophied ventricle.

One case of the erroneous administration of deslanoside and high level of drug in antemortem plasma and postmortem specimens has been reported owing to the unusual surrounding circum-stances. Deslanoside in antemortem plasma was determined by FPIA and the analysis was done by HPLC in the postmortem tissue samples. The analytical results and methods used in the examinations are discussed in the following paper.

The antibodies of two commercially available digoxin-radioimmunoassay kits showed complete equimolar cross-reactivity with deslanoside and were used to determine serum levels of the latter glycoside. The serum concentrations of deslanoside were measured after a single intravenous dose of 1.2 mg in four patients recuperating after acute myocardial infarction. A phase of log-linear elimination (pseudoequilibrium) was reached after 4--8 h, and the biological half-life was 38-77 h (median 51 h). In a second study, the serum concentration was determined daily in 15 patients given multiple intravenous doses of the drug for atrial fibrillation and/or congestive heart failure. Symptoms or signs of digitalis toxicity occurred in six patients given a loading dose of deslanoside followed by daily maintenance doses of 0.4--0.6 mg. One of the patients tolerated a steady-state serum concentration of 3.9 micrograms/L (4.1 nmol/L) without toxicity symptoms. The findings in the remaining 14 patients suggest that the upper limit of the "therapeutic" concentration range is approximately 2.5 micrograms/L (2.7 nmol/L). A significant positive correlation (p less than 0.001) was found between the serum concentration of the drug and serum creatinine. Reduction of the maintenance dose of deslanoside is recommended in patients with impaired renal function.

Controversy exists over the rapidity of onset of the inotropic effect of various digitalis glycosides. Shortening of the systolic time intervals (STI) provides a quantitative measure of the inotropic effect of digitalis glycosides in human subjects. Total electromechanical systole corrected for heart rate (QS 2 I) is the most sensitive of the STI since it combines the shortening effect of digitalis glycosides on both the pre-ejection period and ejection time. Normal volunteer subjects were studied serially following i.v. injection of 1.6 mg cedilanid-D (C) (n = 18), 1.0 mg ouabain (O) (n = 12), 1.6 mg digoxin (D) (n = 16), and 1.6 mg digitoxin (DT) (n = 9). The shortening of QS 2 I was corrected for the molecular weight of the digitalis glycoside. The onset of shortening of the QS 2 I/ mole proved to be exponential for each digitalis glycoside. This allowed estimation of the maximum shortening of QS 2 I/mole (A) which would occur assuming zero excretion, from which the time constant (t c ) of the curves could be determined. There was no significant difference in A among the digitalis glycosides. The t c , were 5.8 min (O), 7.2 min (C), 23 min (D), and 56 min (DT). These t c were significantly different except for O and C. Thus both C and O have a rapid onset of activity which is significantly shorter than either D or DT. The t c for C in patients with congestive heart failure is the same as normals. This study provides a heretofore unavailable, accurate measure of the differences among commonly used glycosides.

In order to determine the excretion pattern of urinary catecholamines and ensure the role of the catecholamines in the chronic congestive heart failure, 24-hour urinary catecholamines were determined by THI-method on 100 patients with chronic congestive heart failure caused by various underlying diseases. 1) The level of urinary catecholamines, especially that of norepinephrine, in both hydrolyzed and non-hydrolyzed form, were found to be increased progressively corresponding to the aggravation of severity of heart failure. This result suggests the extreme acceleration of sympathetic nervous system in congestive heart failure. 2) The observation that the elevation of venous pressure corresponded to an increase in urinary norepinephrine may be a reflection of the presence of neurohumoral regulation of venous pressure. 3) Correlation between the urinary norepinephrine value and decrease rate of vital capacity were observed. 4) There was fairly close correlation between the urinary norepinephrine value and renal plasma flow. This result suggests the contribution of sympatho-adrenal stimulation to the reduced renal hemodynamics in the heart failure. Fifteen patients were laid on time-course study. 5) The urinary norepinephrine level decreased gradually corresponding to the decrease in severity of heart failure. 6) The temporary rise in the urinary norepinephrine level was generally observed in the initial phase of digitalisation. It may be reasonable to assume that the augmented sympathetic nervous system may play a certain role, preferably supportive, to gain the effect of digitalis glycoside on the failing myocardium. 7) Five cases, including 3 with refractory heart failure, were treated with diuretics in addition to the digitalisation. The results obtained may indicate that the "enforced diuresis" by the diuretics, unlike "natural" or "physiologic diuresis" by digitalisation, may produce the augmented sympathetic nervous system until the occurrence of adaptation response to the conditions brought about by the "enforced diuresis" and that repeated performance of the "enforced diuresis" finally led the heart failure to refractory, and the patient to death when the reinforcement of the augmented sympathetic nervous system is deprived by its exhaustion. 8) Moreover, the indication that intensification of the heart failure was observed in the 2 cases laid on the administration of antiadrenergic drugs, may imply the adrenergic support to myocardial function regulating the heart failure. In conclusion, these results may be interpreted to mean with certainty that the sympathetic nervous system plays an important compensatory role in the circulatory regulation of patient to congestive heart failure.

The effect of bretylium on ventricular tachycardia induced by digitalis was studied in Dialurethane-anesthetized cats with continuous monitoring of the electrocardiogram and arterial blood pressure. Ventricular tachycardia was produced by repeated injections of deslanoside (25 µg/kg) at 15 min intervals. Only one of eight animals showed conversion of ventricular tachycardia to sinus rhythm with bretylium administration (2-100 mg/kg i.v.). In each animal, bretylium increased the ventricular rate. The effect of bretylium (30 mg/kg i.v.) on duration of ventricular tachycardia induced by deslanoside (0.75 µg/kg/min) was also studied. Duration was shortened from 65 ± 8.0 min to 7 ± 1.0 min, but the reduction was due to the intervention of either ventricular fibrillation or severe hypotension caused by bretylium. Pretreatment with propranolol (0.5 mg/kg i.v.) prevented these deleterious effects and unmasked an antiarrhythmic effect of bretylium since duration of ventricular tachycardia was significantly shorter (40 ± 6.0 min) than the controls (65 ± 8.0 min). In addition, an antiarrhythmic effect of bretylium was noted when it was administered as a dose of 30 mg/kg ip to animals 24 and 4 hours prior to deslanoside intoxication; 231 µg/kg deslanoside were now required for inducing ventricular tachycardia as compared to 175 µg/kg deslanoside in controls. Thus, bretylium given alone during a digitalis-induced ventricular tachycardia increases both the rate of ventricular tachycardia and the likelihood of ventricular fibrillation. These deterious effects appear to be related to a norepinephrine-releasing action and may be avoided by either pretreatment with propranolol or administration of bretylium several hours prior to digitalis intoxication.

Hemodynamic responses to administration of metaproterenol and deslanoside were studied in 5 and 3 patients with aortic regurgitation, respectively. Metaproterenol produced an increase in forward stroke volume and aortic flow and a reduction in aortic regurgitant flow. In pure aortic regurgitation, these effects were related to a decreased left ventricular work. Deslanoside uniformly increased stroke volume, forward and regurgitant flows. Left ventricular work also increased after deslanoside administration. Calculated regurgitant valve orifice was changeable in association with alteration of inotropy and heart rate following administration of these drugs. The change of regurgitant flow was positively correlated with the change of regurgitant orifice area (r=0.988) and diastolic time (r=0.696). There was no close correlation between the regurgitant flow and systemic vascular resistance or diastolic pressure gradient from the aorta to left ventricle. It was concluded that when the hemodynamics in aortic insufficiency were altered by drug administration, the size of regurgitant orifice area and the duration of diastole were the important determinant of aortic regurgitation.

1 x 10 -6 M Lanatoside C has been demonstrated to produce a sustained increase in oxygen consumption in human myocardial tissue slices as previously demonstrated by Burdette. This acceleration of qO 2 is independent of the substrates employed with the exception of succinate. This qO 2 acceleration has been demonstrated to be calcium dependent in human heart tissue slices similar to the known interrelationship of calcium and digitalis on myocardial contractility. The observation that the greatest increase in qO 2 occurred with glucose and without exogenous substrate suggests that the increase in qO 2 induced by the glycoside is dependent upon a stimulatory effect on glycolysis.

Summary 1 A titration assay with two end points is described for comparison of the emetic and lethal potencies of digitalis-like drugs. 2 A drug was infused at constant rate to a conscious, unrestrained cat, through an indwelling venous cannula. At the moment of vomiting the cat was rapidly anaesthetized and infusion continued at the same rate until the moment of cardiac arrest. 3 With very slow and very fast infusions, the emetic and lethal doses tended to rise. In the range between these extremes (which varied from drug to drug) they were independent of time. 4 The observations could be accounted for by analogue computation, assuming that the drugs entered an initial pool and were distributed at finite rates to receptors in the CNS (vomiting centre) and heart. 5 Half times of metabolic loss derived from this computation for digitoxin, digoxin and ouabain (17, 9·9 and 1·8 h, respectively) were in the same ratio as the threefold longer half times reported for these drugs in man. 6 When measured with infusion rates in the time independent range, the ratio of lethal to emetic doses did not vary between the drugs studied. All caused vomiting at 40% of the lethal dose. 7 From a review of the literature, the emetic and cardiotoxic actions of digitalis-like drugs appear inseparable and probably share a common biochemical mechanism. 8 It is concluded that foreseeable improvements in digitalis-like drugs are small and would depend on the elimination of any local emetic effect on gut receptors which they may have.

The administration of deslanoside to normal subjects was associated with an abbreviation in the duration of all phases of mechanical systole in the absence of a change in cardiac output. The decrease in the ejection time index following deslanoside is an expression of the [see figure in the PDF file] diminution in the duration of ventricular ejection relative to heart rate and reflects the enhanced mean rate of left ventricular ejection following the drug. These observations lend additional evidence for the positive inotropic effect of digitalis in the normal heart. Serial determinations of the left ventricular ejection time index following deslanoside afforded observations on the temporal course of the effect of the drug. The decrease in the left ventricular ejection time index proved to be dose dependent, lending evidence for a direct relationship between the positive inotropic effect and the decreased duration of left ventricular ejection.

A total of fourty-eight cases of various cardiovascular diseases were throughly examined clinico-pathologically, classifying according to the types and degrees of CHF into four groups, namely A-, B-, C-, and D-groups, of which, however, D-group alone was essentially free of apparent CHF and thus to be considered as the control to the others. Thus from these studies there may be revealed to some extent the characteristic correlations between the clinical features and the pathological findings, especially concerned with the therapeutic responsibilities thereof. In conclusion it may be emphasized in this paper that the active or progressive myocardial degeneration not infrequently leading to some protracted shock-like states without evident backward congestion as typically demonstrated in the present A-group may be mainly responsible for the so-called "refractory " CHF in the strict sense, though there the mechanical factors such as valvular deformities per se seem rather mild, and in general vice versa as verified somewhat in the other groups.

The purpose of this investigation was to study the effect of Cedilanid on the mechanical and electrical activity of extracted and nonextracted heart muscle strips of dogs. The glycoside did not influence the work, the speed of contraction, or the length-resting tension relationship of extracted heart muscle. It produced changes in membrane action potentials, but none in membrane resting potentials. The problem of whether digitalis is bound to the membrane or the cell protein was investigated by temporary destruction of the cell membrane by water. The effects of cardiac glycosides on contractile proteins and on membrane activity are probably inseparable; they act through changes in ionic gradient between muscle cells and the surrounding medium.

A method is described for determining the duration of action of cardiac glycosides and aglycones in the guinea-pig. It is based on their property of potentiating the cardiac response to adenosine. The method is particularly suitable for those drugs with a short duration of action, whereas previous methods are more suitable for those drugs with longer durations of action. The duration of action of one-fifth of the lethal dose has been found for: digoxigenin, lanatoside C, ouabain, digitoxigenin-3-one, digitoxin, 3-acetyldigitoxigenin, digoxin, digitoxigenin, lanatoside A; these drugs are arranged in order of increasing duration of action. The possible relationship between the elimination of these drugs and their duration of action can provide an estimate of their rates of elimination.

We sought to determine whether digitalis augments cardiopulmonary baroreflex control of forearm vascular resistance in normal young men. Cardiopulmonary baroreceptor input was reduced with lower body negative pressure (LBNP) at 10 and 20 mm Hg, which decreased central venous pressure but did not alter blood pressure or heart rate. Decreases in forearm blood flow and increases in forearm vascular resistance with LBNP were greater after administration of lanatoside C (Cedilanid) than before, and the slope of the regression line relating changes in central venous pressure and those in forearm vascular resistance was steeper after lanatoside C. Vasoconstrictor responses to the cold pressor test did not differ before and after lanatoside C, which suggested that augmented responses to LBNP after the drug were not caused by a generalized change in reflex control. These results suggest that lanatoside C augments the tonic inhibitory influence of cardiopulmonary baroreceptors in normal men.

The chronotropic effects of digoxin and deslanoside were studied in canine atria cross-perfused with heparinized arterial blood from donor dogs. Intravenous injections of either drug (100 micrograms/Kg) into the donor dog produced bradycardia followed by ventricular tachyarrhythmia, with or without hypertension, in the donor dog. A significant increase in the developed tension was observed in the isolated atria, with or without slight sinus acceleration. These effects continued over 150 min after the injection. Digoxin (200 micrograms/Kg, i.v.) caused an immediate bradycardia followed by ventricular tachycardia. In addition, ventricular fibrillation occurred in 3 out of 5 donor dogs within 20 min of the drug administration. In isolated atria, there was a marked increase in the developed tension, usually with a little sinus tachycardia. Deslanoside (200 micrograms/Kg, i.v.) caused almost the same response patterns as digoxin. However, this dose of deslanoside caused ventricular fibrillation in all 6 experiments. Drug concentrations in the donor's arterial blood decreased rapidly for 15-20 min and then decreased slowly in all experiments. It is concluded that digoxin and deslanoside have no significant direct accelerating action on the SA node in doses which produced marked increases in the developed tension; only extremely high doses cause a direct, slight sinus acceleration.

EVIDENCE indicates that digitalis drugs administered intravenously increase central sympathetic outflow and that this in turn results in cardiac arrhythmias1–6. The central nervous system transmitter(s) that mediates this effect is not known. According to Saito et al., noradrenaline, the primary transmitter studied so far, is not involved in the case of guinea pigs7. 5-Hydroxytryptamine (5-HT) however, may be an important chemical mediator regulating central sympathetic outflow8,9, and it is involved in the respiratory arrest induced in rats by intravenous administration of digitoxigenin10. We now report evidence that suggests that brain 5-HT is involved in digitalis-induced cardiac arrhythmias.

Activation of Na+, K+-ATPase has previously been suggested to be the triggering signal in mitogen-induced cell activation. Using a digitalis glycoside known to be a potent polyclonal B-cell activator, this hypothesis could be tested since digitalis activates ATPase at different concentrations in various species, depending on the degree of sensitivity to the toxic effects of glycosides. Lanatoside C was found to stimulate lymphocytes from glycoside resistant species such as rat, mouse and hamster. The possible involvement of Na+, K+-ATPase was made less likely by the similarity in dose--response profile in these cells although they have been reported to display varying degrees of glycoside resistance. Furthermore, using lymphocytes from digitalis-sensitive species such as man, guinea-pig or rabbit, no mitogenicity could be recorded, strongly suggesting a lack of correlation between glycoside-induced effects on Na+, K+-ATPase and cell activation.

We studied the direct effects of digitalis on sinoatrial conduction time (SACT), atrial rate (AR) and developed tension (DT) in isolated atrial muscle, using an isolated dog atrial preparation perfused with heparinized blood from the carotid artery of an anesthetized donor dog. After digoxin or deslanoside (100 micrograms or 200 micrograms/Kg) was given intravenously to the donor dog, SACT, AR and DT of the isolated atrium were continuously measured. Following administration of 100 micrograms/Kg of digoxin, an immediate sinus bradycardia followed by various kinds of ventricular arrhythmias was observed in the donor dog, and the DT of the isolated atrium was augmented without any effects on the SACT and AR in all 3 experiments. With 200 micrograms/Kg of digoxin or deslanoside, ventricular fibrillation was induced in all 5 donor dogs within 75 min after digitalis administration. In these cases, the DT of the isolated atrium was immediately increased to over 200% of control DT, but SACT and AR were not affected until 20 min later, at which time increases in SACT and AR were finally induced. From these results, it is concluded that a large amount of digitalis has no distinct direct effect on SA nodal pacemaker activity and SA conductivity. Moreover, extremely large doses of digitalis which cause ventricular fibrillation might also induce a prolongation of SACT in addition to sinus tachycardia.

A study, which arose out of a general-practitioner teaching programme in clinical pharmacology, was designed to assess the use of digitalis in 391 patients in general practice. Clinical, biochemical, and pharmacological data were used to assess whether digitalis treatment should be discontinued (89 patients; 22.8%); the dose kept unchanged (47; 12%); or the dose increased (47; 12%) or decreased (24; 6.1%). Serum concentrations of cardiac glycoside were below a defined therapeutic range in 159 patients (40.7%), above this range in 37 (9.7%), and within the range in 195 (49.3%). Studies of this kind may help to promote a more critical attitude to prescribing widely used drugs such as digitalis.

Clinical open trials of beta-methyldigoxin were carried out in 15 institutions in order to examine the effect, usefulness and ease of its oral administration. In the case of oral digitalization with 0.2 mg, 3 times daily, an effect was obtained in all of 13 cases of congestive heart failure accompanied by atrial fibrillation or flutter. The average time and dose required for digitalization were about 50 hours and 1.27 mg respectively. In 9 of the 13 cases, the effect was achieved within 48 hours. The average maintenance does of beta-methyldigoxin in 102 cases of congestive heart failure with atrial fibrillation was 0.177 mg per day. About 75% of the cases were maintained with 0.15 to 0.2 mg. This range of dose of beta-methyldigoxin was much smaller than that of digoxin in our series. This can be ascribed to a higher absorption rate of beta-methyldigoxin from the digestive tract. Studies on the cases in which patients previously treated with other glycosides were switched over to beta-methyldigoxin revealed that 1 mg of beta-methyldigoxin is equivalent to 1.8 mg of digoxin or to 0.59 mg of digitoxin. The usefulness and ease of beta-methyldigoxin in maintenance was evaluated as being somewhat superior to other cardiac glycosides, according to the global judgement of the physicians. The observed side effects were similar to those of other glycosides in frequency and character.

Intra-atrial, intranodal and His-Purkinje conduction times were determined in perfused rabbit hearts. In one series, the concentration of lanatoside C was increased by 0.4 mg/liter every 30 minutes, in the presence of normal (4.5) or high (7.5 mM) K + in the perfusion fluid. At high (K + ) the glycoside produced a greater increase in intra-atrial and His-Purkinje conduction times but caused a smaller increase of intranodal conduction time than at normal (K + ). Second-degree A-V block always occurred intranodally, but the incidence was lower with high (K + ). In another series, (K + ) was increased by 3 mM every 30 minutes, in the absence or presence (0.4, 0.8 or 1.2 mg/liter) of lanatoside C. No difference was seen between hearts treated with lanatoside C and those untreated at the level of (K + ) producing failure of conduction. At all levels of glycoside, conduction delay caused by high (K + ) was greatest within the atria, less in the His-Purkinje system and insignificant within the A-V node. Intra-atrial and His Purkinje block were observed but never an intranodal block. It is concluded that (1) cardiac glycoside and high (K + ) primarily affect different regions of the A-V conducting system, and (2) aggravation of glycoside-induced A-V block by high (K + ) may result from additional intra-atrial and His-Purkinje block rather than from further depression of intranodal conduction. Electrophysiological mechanisms underlying these interactions were also discussed.