Mechanism of cardioprotective effect of adenocine and non-glycoside cardiotonic drugs during experimental chronic cardiac insufficiency.

The therapeutic action of adenocine during cardiac insufficiency (heart failure) caused by ischemic (stenosis) or reperfusion (removal of ligature) injury to the myocardium prevents depletion of ATP, the major energy source for myocytes in the right and left ventricles, and a drop in NAD/NADH ratio. The development of energy shortage during heart failure cannot be eliminated by ß-acetyldigoxin, levosimendan, or milrinone: the content of ATP in the right and left ventricular myocardium remained below the normal level by 28 and 29%, 37 and 33%, 32 and 28%, respectively; the NAD/NADH ratio of the energy supply system in cardiomyocytes did not return to normal. Adenocine increased the content of NAD to the normal level in both the right and left ventricles, while it remained below the normal level after administration of ß-acetyldigoxin (by 24 and 19.5%, respectively), levosimendan (by 27 and 29%), and milrinone (by 26 and 24%). In contrast to ß-acetyldigoxin, levosimendan, and milrinone, adenocine inhibited activity of poly(ADP-ribose) polymerase in both ventricles. It is concluded that adenocine directly inhibits the key enzyme triggering apoptosis; we also hypothesized that this drug activates the regulatory and signal mechanisms arresting apoptotic alterations in the myocardium during heart failure.

Comparative therapeutic efficacy of adenocin and non-glycoside cardiotonic drugs in chronic heart failure at rest and under conditions of heart overload.

Experiments were performed on the model of chronic heart failure. Functional capacity of myocardial structures under conditions of maximum pressure overload was within the upper limit of normal after treatment with Adenocin. The myocardial functional reserve and potential capacity index were shown to increase to normal under these conditions. Dobutamine, levosimendan, and milrinone increased functional capacity under conditions of maximum pressure overload. Treatment with adenocin restored diastolic function of the heart under conditions of maximum pressure overload. The end-diastolic pressure increased, but remained 1.7 times below the level observed in heart failure. After treatment with dobutamine and milrinone, the end-diastolic pressure (8th episode of ligation) did not differ from the level observed in heart failure, while after administration of levosimendan this parameter decreased by 31%. Contraction-relaxation coupling was completely restored under the influence of Adenocin in all episodes of ligation both before and after removal of the ligature. Nearly all animals with heart failure were resistant to 8 episodes of ligation after treatment with Adenocin (89 vs. 96% under normal conditions). Under these conditions, the survival rate of animals after administration of levosimendan, milrinone, and dobutamine was 65, 60, and 61%, respectively, (the mortality rate of animals with heart failure was 75%). Adenocin, a cardiotonic drug with cardioprotective properties, in contrast to other cardiotonic drugs, has a modulatory effect on the system of cell energy supply, restores myocardial reserves, and improves myocardial function under conditions of overload.

Submolecular mechanisms underlying in vitro and in vivo effect of cardiac glycosides on contractile activity of myocardial myofibrils during heart failure.

The development of severe heart failure associated with toxicoallergic myocarditis is accompanied by profound structural and conformational changes in the outer domain of actin (major protein in a thin filament of cardiomyocyte sarcomere). These changes were revealed in subdomains 1 (Cys374 and Cys10) and 2 (Lys61 and Tyr69). Structural and conformational changes in the monomer and protomer of the actin thread during heart failure were energetically forbidden. Variations in the distance between amino acid residues exceeded 0.26 nm. They were partly or completely reversible in vivo under the influence of cardiotropic drug refracterin with high antihypoxic activity, as well as in vitro after treatment with digitalis preparations optimizing the concentration of ATP.

Efficiency of refracterin in patients with chronic cardiac insufficiency caused by coronary heart disease.

Composite preparation refracterin administered in a dose of 300 mg/day for 3 days in addition to routine therapy significantly improved the results of treatment of severe cardiac insufficiency of ischemic genesis compared to placebo. Improvement of clinical status of patients is determined by positive dynamics of systolic and diastolic functions of the left ventricle.

Efficiency and mechanisms of the antioxidant effect of standard therapy and refracterin in the treatment of chronic heart failure in elderly patients with postinfarction cardiosclerosis.

Refracterin therapy of patients with chronic heart failure caused by coronary heart disease and postinfarction cardiosclerosis markedly promoted improvement in the pulmonary and systemic circulation in comparison with patients receiving traditional therapy. The mean functional class of chronic cardiac failure decreased by 43% under the effect of refracterin vs. 27% decrease in the group receiving traditional therapy. After 1-month refracterin course the end-systolic and end-diastolic sizes of the left ventricle decreased by 12 and 7%, respectively, ejection fraction increased by 7.2% in comparison with the initial level, total oxidant activity and MDA content in the plasma decreased significantly, while total antioxidant activity, catalase and SOD activities, cytochrome C, NADH, and NADPH levels increased. The prooxidant-antioxidant system was shifted towards antioxidants, which attests to activation of the defense and adaptive mechanisms after administration of refracterin, which is especially important in elderly patients with initially decreased reserve potentialities of the antioxidant defense system.

Amiodarone-associated "torsade de pointes". Relevance of concomitant cardiovascular medication in a patient with atrial fibrillation and structural heart disease.

A 69 year old female with history of coronary heart disease, myocardial infarction and paroxysmal atrial fibrillation suffered from occipital apoplexy. Under treatment with amiodarone 600 mg daily and concomitant medication with beta-acetyldigoxine (0.1 mg daily) and bisoprolole (1.25 mg daily), significant QT-prolongation (max. 700 ms; QTc: 614 ms) could be documented. Out of normofrequent sinus rhythm but as well out of bradycardia, the patient developed repetitive short-lasting "torsade de pointes" tachycardias (320 bpm) which terminated spontaneously. Serum electrolytes, plasma levels of digoxine (1.76 ng/ml) and amiodarone (1.9 mcg/ml) were within therapeutic range. This case report is the first to describe induction of amiodarone-associated "torsade de pointes" tachycardia during concomitant beta-blocker and digitalis medication in a patient with atrial fibrillation and structural heart disease. This points towards an elevated risk for proarrhythmia under this triple therapy.

Complementary combining site contact residue mutations of the anti-digoxin Fab 26-10 permit high affinity wild-type binding.

Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 in a 26-10 mutant containing Arg-100 (26-10-RRALD). Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26-10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26-10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26-10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.

P-glycoprotein-mediated transport of digitoxin, alpha-methyldigoxin and beta-acetyldigoxin.

Digoxin is a drug with a narrow therapeutic index, which is substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidneys. Whereas particle size, dissolution rate and lipophilic properties have been identified as determinants for absorption of digitalis glycosides, little is known about P-glycoprotein transport characteristics of digitalis glycosides such as digitoxin, alpha-methyldigoxin, beta-acetyldigoxin and ouabain. Using polarized P-glycoprotein-expressing cell lines we therefore studied whether these compounds are substrates of P-glycoprotein. Polarized transport of digitalis glycosides was assessed in P-glycoprotein-expressing Caco-2 and L-MDR1 cells (LLC-PK1 cells stably transfected with the human MDR1 P-glycoprotein). Inhibition of P-glycoprotein-mediated transport of these compounds in Caco-2 cells was determined using the cyclosporine analogue PSC-833 (valspodar) as inhibitor of P-glycoprotein. No polarized transport was observed for ouabain. However, basal-to-apical transport of digitoxin, alpha-methyldigoxin and beta-acetyldigoxin was greater than apical-to-basal transport in Caco-2 and L-MDR1 cells. In Caco-2 cells net transport rates of these compounds were similar to those of digoxin (digoxin: 16.0+/-4.4%, digitoxin: 15.0+/-3.3%, beta-acetyldigoxin: 16.2+/-1.6%, alpha-methyldigoxin: 13.5+/-4.8%). Furthermore, polarized transport of these compounds could be completely inhibited by 1 microM PSC-833. In summary, these data provide evidence that not only digoxin, but also digitoxin, alpha-methyldigoxin and beta-acetyldigoxin are substrates of P-glycoprotein.

[The subcellular pathophysiology of heart failure due to toxic-allergic myocarditis and the action of refracterin on intracardiac hemodynamics and the functional state of the 3 subcellular cardiomyocyte systems responsible for the act of contraction-relaxation]. / Subkletochnaia patofiziologiia nedostatochnosti serdtsa, obuslovennoi toksiko-allergicheskim miokarditom, i deistvie refrakterina na vnutriserdechnuiu gemodinamiku i funktsional'noe sostoianie trekh subkletochnykh sistem kardiomiotsita otvetstvennykh za akt sokrashchenie--rasslablenie.

It is shown that cardiotropic drug refracterin promotes recovery of cardiac contraction and relaxation, their coordination destroyed in cardiac failure (CF) caused by 10-day toxico-allergic myocarditis (TAM). Pumping capacity of the heart returns to normal after normalization of functional activity of three systems of cardiomyocyte responsible for contraction-relaxation: contractile proteins, energy supply and calcium transport. The key process is refracterin-related reestablishment of normal content and proportion of adenyl nucleotides and creatininephosphate and regulation role of phosphorylation and energy of metabolic processes in the cells and their interaction. Thus, refracterin effectiveness lies in its ability to interfere in intracellular metabolic processes in the myocardium, to reestablish normal homeostasis of the systems responsible for contraction-relaxation function and eventually to remove left ventricular cardiac dysfunction.

Protection from digoxin-induced coronary vasoconstriction in patients with coronary artery disease by calcium antagonists.

Intravenous digoxin induces constriction of normal and stenotic coronary arteries in patients with coronary artery disease, which may lead to ischemic complications. We found that pretreatment with oral nisoldipine and intracoronary nitroglycerin neutralizes this digoxin-induced effect.

Separation of cardiac glycosides by micellar electrokinetic chromatography and microemulsion electrokinetic chromatography.

The interest of micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) for the resolution of four cardiac glycosides is demonstrated. First, the influence of some parameters on the resolution of the solutes in MEKC such as the concentration of the surfactant, pH, addition of organic modifiers and urea is discussed. Then, results are compared with those obtained in MEEKC using different microemulsion compositions. Results indicate that MEEKC possesses several advantages over MEKC for the separation of relatively hydrophobic compounds such as digitalic compounds. First, microemulsions allow a better manipulation of the migration time window and of the retention of the solutes. Moreover, efficiency is improved with shorter analysis time.

Extraction and quantitation of digoxin and acetyldigoxin from the Digitalis lanata leaf via near-supercritical methanol-modified carbon dioxide.

An extraction process is reported that employs a near-supercritical mixture of CO2 and MeOH to extract the cardiac glycoside, digoxin, from the Digitalis lanata leaf. The method development of the sample preparation procedure is presented in detail, and reasons for trends that occur in the natural products extraction are given.

The effect of meloxicam on the pharmacokinetics of beta-acetyl-digoxin.

The influence of multiple-dose administration of meloxicam on the pharmacokinetics of oral beta-acetyl-digoxin was studied in 12 healthy male volunteers in a randomized double-blind two-way crossover study. The primary endpoint, Cminss, was within the accepted range for bioequivalence, as were Cmaxss and AUCss. The 90% confidence interval and the point estimator of 98.7 for Cminss were within the equivalence range of 0.8-1.25. MRT and tmax were also unchanged, while the elimination rate constant was decreased slightly by 12%, which is of no therapeutic relevance. It is concluded that co-treatment with meloxicam has no effect on the pharmacokinetics of oral digoxin.

Photostability studies of ouabain, alpha-acetyldigoxin and digoxin in solid state.

Ouabain, alpha-acetyldigoxin and digoxin were subjected to irradiation using different light sources in crystalline state and their respective yields of photoproducts were determined densitometrically. alpha-Acetyldigoxin was found to be less stable than digoxin yielding a higher percentage of photoproducts under each light source examined. Ouabain showed photostability under the conditions of investigation.

Digoxin and digoxin derivative induced arrhythmias: in vitro binding and in vivo abolition of arrhythmias by digoxin immune Fab (DIGIBAND).

OBJECTIVE: The aim was to compare the binding characteristics of a highly purified digoxin specific antigen binding fragment (digoxin immune Fab: DIGIBIND) with digoxin and with two commonly used derivatives of digoxin, beta methyl digoxin and beta acetyl digoxin, and to assess its ability to abolish the arrhythmogenic effects of these digitalis glycosides. METHODS: The binding characteristics of DIGIBIND with digoxin, beta methyl digoxin, and beta acetyl digoxin were assessed in vitro by measuring their ability to inhibit the binding of DIGIBIND to 3H-digoxin. From these studies the affinities of the interactions between DIGIBIND and these glycosides, and the binding capacity of DIGIBIND for each of these glycosides, could be measured. The ability of DIGIBIND to abolish the arrhythmogenic effects of digoxin, beta methyl digoxin, and beta acetyl digoxin was assessed using an in vivo anaesthetised guinea pig model (n = 36, weight 300-400 g), in which these glycosides were infused intravenously (50 micrograms.kg-1 x min-1) until the onset of ventricular arrhythmias, at which point the total amount of glycoside given was calculated. A single bolus dose of either vehicle or DIGIBIND was then given intravenously, and the time to restoration of normal cardiac rhythm noted. After the administration of DIGIBIND, a second infusion of the same glycoside was given to reinitiate the ventricular arrhythmias. The time to onset of the arrhythmias was noted, and the additional amount of glycoside given calculated. RESULTS: In vitro studies showed the binding of DIGIBIND to 3H-digoxin to be inhibited by digoxin and by the two derivatives. The affinities of these interactions with DIGIBIND were significantly different, that for digoxin being some twofold greater than that for beta methyl digoxin and beta acetyl digoxin. The ED50 concentrations were 14.1 (95% CI 12.2, 15.2), 29.2(26.1, 32.7), and 36.2(33.0, 39.8) nM, respectively. However, there were no significant differences between these glycosides in their binding capacities. The in vivo studies showed that intravenous infusion of digoxin, beta methyl digoxin, or beta acetyl digoxin induced similar ventricular arrhythmias. The onset of the arrhythmias was clearly discernible, and required a significantly lower dose of digoxin compared with that of beta methyl digoxin and beta acetyl digoxin. These doses were 667(SEM 55), 868(33), and 854(40) nmol.kg-1, respectively. Termination of the infusion had no effect on the arrhythmias, and in those animals which received a bolus intravenous injection of saline there was no return to normal cardiac rhythm. By contrast, in animals which received a bolus intravenous injection of DIGIBIND, there was complete abolition of the arrhythmias within 4-6 min. Although the dose of DIGIBIND given to abolish digoxin induced arrhythmias was approximately 25% less than that given to abolish beta methyl digoxin and beta acetyl digoxin induced arrhythmias (p < 0.05), the time to restoration of normal cardiac rhythm after DIGIBIND was not significantly different for digoxin compared with beta methyl digoxin and beta acetyl digoxin, at 4.6(0.9), 4.9(0.8), and 5.7(0.8) min, respectively. To reinitiate the arrhythmias in those animals which had received DIGIBIND, a dose of glycoside was required which was not significantly different from that given prior to the DIGIBIND. This observation therefore confirmed the stoichiometric relationship between DIGIBIND and each of the glycosides in respect of the neutralising action of DIGIBIND in abolishing the arrhythmogenic effects of these agents. CONCLUSIONS: Although there is some small difference in the affinities of the binding interactions, there is no difference in the binding capacities of DIGIBIND for digoxin, beta methyl digoxin, or beta acetyl digoxin in vitro. These binding interactions are manifest as the ability of DIGIBIND to abolish the arrhythmogenic effects of digoxin and the two derivatives in vivo.

Aplicaçäo de Metildigorcina, alfa-e-Acetildigoxina em cäes sadios: concentraçöes séricas, período de meia-vida e efeitos sobre o eletrocardiograma / Methyl-, alfa-and B-acetyl-digoxin administration in healthy dogs: blood levels, half-life and effect on the electrocardiogram

A aplicaçäo de derivados de digoxina em cäes forneceu concentraçöes séricas médias, no platô, de 1,43ng/ml para metildigoxina e de 1,08 a 1,02ng/ml para B- e alfa-acetildigoxina, respectivamente. O período de meia-vida sérica foi de 53,6 h para o metilderivado, de 21,5h e de 23,4h, respectivamente, para os acetilderivados alfa e B. Alteraçöes eletrocardiográficas limitaram-se ao prolongamento do intervalo PQ, quando os níveis séricos mantiveram-se nos limites terapêuticos. Bloqueio atrioventricular de primeiro grau e inversäo de ondas T ocorrem em animais com concentraçöes digitálicas superiores a 0,2 ng/ml

[Absolute bioavailability of beta-acetyldigoxin from tablets and drops in healthy subjects]. / Absolute Bioverfügbarkeit von beta-Acetyldigoxin aus Tabletten und Tropfen bei gesunden Probanden.

In a crossover design in random order 12 healthy male volunteers were given either beta-acetyldigoxin (Novodigal, CAS 5511-98-8) tablets, oral solution or i.v. application at a digoxin equivalent dose of 0.284 mg. To reach steady state each preparation was given for 10 days on a once-daily schedule. On days 8, 9 and 10 of each observation period blood was sampled to determine trough concentrations of digoxin in steady state. In addition, on day 10 blood was collected repeatedly at appropriate time intervals and urine was sampled concomitantly for 24 h. Trough values during steady state and 24 h AUC were used to calculate digoxin bioavailability for tablets and oral solution. From trough values, the mean bioavailability for beta-acetyldigoxin tablets was 91.2% (range 73.1-118.1) and for solution 93.8% (range 65.7-114.8). Using the AUCs 0-24 h at steady state bioavailability was calculated 77.7% for the tablets and 84.5% for the solution. Since trough values in steady state represent the body burden of digoxin which is supposed responsible for the therapeutic effect, trough values should be given priority for the determination of digoxin bioavailability from beta-acetyldigoxin tablets and solution. All formulations were well tolerated. No clinically relevant side effects were observed.

[Differences in mechanisms of action of beta-acetyldigoxin, strophanthin K and ouabain]. / O razlichii mekhanizmov deistviia beta-atsetildigoksina, strofantina K i uabaina.

In vitro on skinned myocardial fibers (SMF) with extracted or functionally inactivated enzymes and membranes of mitochondria, longitudinal sarcoplasmic reticulum, triads and sarcolemma, new evidence of beta-acetyldigoxin and strophanthin K direct stimulating effects on contractile protein system of myocardium has been obtained. It has been revealed in energy release stimulation and force generation, in quantitative (beta-acetyldigoxin) or quantitative and qualitative (strophanthin K) stimulation of energy transduction, in the increase of contractile process cooperativity and Ca-sensitivity of SMF as well as in the SMF relaxation time extension (in the case of strophanthin K). It is suggested that different effects of beta-acetyldigoxin and strophanthin K are due to the differences in the conformations of actomyosin ensembles formed by strong bound (AMESB), which are induced by the influence of these cardiac glycosides. It has been demonstrated that ouabain (strophanthin K) has no direct effect on functioning of AMESB.

Effects of calcium antagonists in patients with coronary disease and heart failure: left ventricular function following nisoldipine measured by radionuclide ventriculography.

Thirty-two patients with chronic heart failure were investigated with radionuclide ventriculography at rest and during exercise. The global ejection fraction (EF), ejection rate, ejection time, and filling rate were measured. The patients were subdivided into three subgroups: patients with extremely (< 25%), moderately (25-33%), and mildly decreased EF (33-40%). All patients were investigated by thallium-201 myocardial scintigraphy and the correlation between ejection parameters and infarct size was investigated. In a random protocol 17 patients (14 men, 3 women, mean age of 54.6 years) received beta-acetyldigoxin at a dose of 0.3 mg daily over 4 weeks, 15 patients (13 men, 2 women, mean age of 58.2 years) received nisoldipine at a dose of 20 mg daily. Patients on digitalis showed a further lowering of the extremely decreased EF (-1%), but in patients with moderately decreased EF, digoxin produced a marked increase in EF of +8% (p < 0.001). In mildly decreased EF, there was no significant change (+0.3%, n.s.). Nisoldipine produced an increase in pump function (+2%) in the group with extremely decreased EF up to 9% in some individual cases. A significant increase (+5%) was achieved in moderately decreased EF, while patients with mildly decreased EF did not respond. Thus, nisoldipine may be indicated in the treatment of heart failure after repeated myocardial infarctions, in particular in patients with severely decreased ejection fraction.