digoxin and 16-acetylgitoxin

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.

Topics: Acetyldigoxins; Animals; Antibodies, Monoclonal; Arginine; Binding Sites; Crystallography, X-Ray; Digoxin; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Haptens; Kinetics; Mice; Models, Chemical; Mutagenesis, Site-Directed; Mutation; Protein Binding; Serum Albumin; Valine

In isolated, spontaneously beating guinea-pig atria the uptake, liberation and inotropic effect of [3H]16-acetyl-gitoxin were studied. At a concentration of 53.5 nmol/l 16-acetyl-gitoxin caused an increase in contractile force of 21% within 45 min. Within 60 min, 190.7 pmol per g tissue were taken up. The tissue-to-medium radioactivity ratio (T/M ratio) was 4.7. In the wash-out experiments, 40% of the accumulated radioactivity was liberated into the medium within 30 min, while the glycoside-dependent increment of inotropy ceased to continue. The kinetic properties of 16-acetyl-gitoxin were found to resemble more closely those of digitoxin than those of ouabain.

Topics: Acetyldigoxins; Animals; Digoxin; Female; Guinea Pigs; In Vitro Techniques; Kinetics; Male; Myocardial Contraction; Myocardium

The systolic time intervals (STI) corrected for changes in the heart rate, electromechanical systole (QS2c) and left ventricular ejection time (LVETc), and the ECG-derived PQ-time and QT-interval were measured in five female and four male healthy subjects. Each volunteer took 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg pengitoxin over six days, with a glycoside-free interval of two or three weeks between two doses. The glycoside plasma level was measured radioimmunologically. Linear correlations were found between the shortening of QS2, LVET, and QT (delta QS2c, delta LVETc, delta QTc) and the plasma level of 16-acetyl-gitoxin. The PQ-time showed a flat dose-dependent increase. The shortening of STI observed after therapeutic and subtoxic doses of pengitoxin was in accordance with that after intake of digitoxin and digoxin in corresponding doses. The efficacy of pengitoxin estimated by shortening of STI justifies the administration of daily maintenance doses between 0.30 and 0.45 mg.

Topics: Acetyldigoxins; Adult; Digoxin; Dose-Response Relationship, Drug; Electrocardiography; Female; Heart Rate; Humans; Male; Radioimmunoassay

In ten patients suffering from moderate to severe disturbances of liver function (hepatic cirrhosis, carcinomatous metastases of the liver) the elimination of 16-acetyl-gitoxin from plasma was measured after a single oral dose of 1.2 mg pengitoxin. In 9 of 10 patients the mean half-life amounted to 67.3 h (SD 14.2 h). In one patient with carcinomatous infiltration of the liver and concomitant cirrhosis, the half-life was prolonged to 165.3 h. The results point to an unchanged elimination of 16-acetyl-gitoxin in patients suffering from liver cirrhosis.

Topics: Acetyldigoxins; Adult; Aged; Biotransformation; Digoxin; Half-Life; Humans; Liver Diseases; Middle Aged

In six volunteers the pharmacokinetics of 16-acetyl-gitoxin (16AG, 0.5 mg) administered intravenously (A1) and as an oral solution (A2) and of pengitoxin (PAG, 0.6 mg) administered intravenously (A3) was evaluated. In six volunteers the bioavailability of 16AG from two PAG tablet formulations (1.2 mg) (B2, B3) was measured by comparison with the absorption after administration of a pengitoxin solution (1.2 mg) (B1). In both studies the test was performed using a crossover design. After a single i.v. injection of equimolar doses, 16AG and PAG showed similar mean kinetic parameters: t1/2 = 51.6 hr (16AG) and 60.8 hr (PAG), CL = 11.7 ml min-1 (16AG) and 12.7 ml min-1 (PAG), CLR = 4.1 ml min-1 (16AG) and 4.2 ml min-1 (PAG). The 16AG was absorbed from solution with a mean half-life of 0.2 hr to an extent of 98.6%. The mean urinary excretion/Ae (0,4)/of 16AG amounted to 24.6% (A1), 20.8% (A2) and 28.1% (A3). On the basis of AUC values, the mean bioavailability of PAG from either tablet formulation amounted to 79.6% (B2) and 89.6% (B3). The pharmacokinetic parameters of 16AG (PAG) are closer to those of digitoxin than those of digoxin. In general, 16AG is characterized as a digitoxin with a digoxin-like elimination half-life.

Topics: Acetyldigoxins; Administration, Oral; Adult; Biological Availability; Digitoxin; Digoxin; Female; Humans; Injections, Intravenous; Kinetics; Male; Tablets

The pharmacokinetics of pengitoxin has been studied in 28 healthy subjects after intravenous and oral administration. The mean plasma concentration 24 h after 0.5 mg i.v. was 5.2 ng X ml-1. Following an open two-compartment model a mean elimination half-life of 60.5 h (24.9 to 103.5 h) and a mean volume of distribution (Vdarea) of 66.91 (31.8 to 109.61) were calculated. Absorption calculated by comparison of the AUC0-infinity-values amounted to 99%. Within 4 days, 16.7% (11.7 to 21.1%) or 27.8% (18.4 to 33.7%) (0.5 mg i.v. or 1.2 mg p.o.) was excreted in urine. After pengitoxin 0.5 mg i.v. total body clearance and renal clearance were 13.3 ml X min-1 (7.0 to 18.6 ml X min-1) and 3.0 ml X min-1 (1.9 to 3.9 ml X min-1) respectively. The elimination half-life of pengitoxin is longer than that of digoxin and distinctly shorter than that of digitoxin, whilst its distribution volume and clearance are closer to those of digitoxin than of digoxin.

Topics: Acetyldigoxins; Adult; Digoxin; Female; Humans; Kinetics; Male; Radioimmunoassay

The plasma protein binding of 16-acetyl-gitoxin (16-AG) was estimated in comparison with that of digoxin by the use of equilibrium dialysis, ultracentrifugation, and gel filtration techniques. Depending on temperature, the binding of 16-AG reached values between 81 and 90%. Calculations of the reaction enthalpy and entropy show that hydrophobic interaction results. The corresponding values of digoxin amount to 13 and 15%.

Topics: Acetyldigoxins; Blood Proteins; Cardiac Glycosides; Digoxin; Humans; Protein Binding; Temperature

The potency of 17 derivatives of 16 alpha-gitoxin was tested in the isolated atrium and heart of the guinea pig and the contractility-increasing activity of the 16 alpha-gitoxin 16 alpha-acetate was compared with that of 16 alpha-gitoxin in the anesthetized dog. The potency of 16 alpha-gitoxin was increased by the substitution of 16 alpha-OH for 16 alpha-methyl ether, 16 alpha-acetate and 16 alpha-nitrate. Substitution of the 16 alpha-acetyl group for substituents with a higher molar volume diminished this enhanced potency. Variation in the digitoxose moiety caused an increase or decrease in potency depending on the position and number of the substituted OH groups. In spite of changes in 16 alpha-OH, the low influence on rhythmicity persisted, as was found in experiments in the dog.

Topics: Acetyldigoxins; Animals; Arrhythmias, Cardiac; Cardiac Glycosides; Digoxin; Dogs; Dose-Response Relationship, Drug; Female; Guinea Pigs; Heart; Heart Rate; Isomerism; Male; Myocardial Contraction; Stimulation, Chemical; Structure-Activity Relationship

A simple and reliable radioimmunoassay procedure for the specific determination of 16-acetyl-gitoxin, the main cardioactive metabolite of penta-acetyl-gitoxin, in serum is described. Antisera raised against 15-acetyl-gitoxin-bovine serum albumin conjugates proved to be highly specific, thus permitting direct analysis of serum. The assay is capable of detecting 0.4 ng of 16-acetyl-gitoxin. This covers the therapeutic and toxic range when 0.1 ml serum is analyzed. A single worker can assay about 100 serum samples per day in triplicate.

Topics: Acetyldigoxins; Animals; Digoxin; Humans; Radioimmunoassay