Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.

[14,15-beta-oxido analog of proscillaridin (HOE 040). A new cardiac glycoside with low arrhythmic activity and greater absorption ratio]. / Das 14,15-beta-Oxido-Analoge des Proscillaridins (HOE 040) Ein neues Herzglykosid mit geringer Arrhythmie-Wirkung und hoher Resorptionsquote.

The 14,15-beta-oxido analogue of proscillaridin A (HOE 040), in a dose 4 times higher showed equally positive-inotropic effect on the isolated guinea pig heart as did proscillaridin A. In the dog in vivo HOE 040 was equally positive-inotropic, as measured by the increase of dp/dt of the left ventricle of the heart, as proscillaridin A. In combination with aconitine, HOE 040 also in 4fold higher dose caused less cardiac fibrillation on the isolated guinea pig heart than did proscillaridin A. The dose of HOE 040 which by infusion in the guinea pig in vivo precipitates cardiac arrhythmias was 4 times higher than that of proscillaridin A, the lethal dose was 5 times higher. In dogs in vivo the dose of HOE 040 by infusion causing prolongation of PQ or cardiac arrhythmias, resp., was twice the dose necessary of proscillaridin A, the lethal dose was nearly 5 times higher. The decrease of cardiac activity in Rhesus monkeys amounted to 69% in 24 h, whereas proscillaridin A decreased cardiac activity only by 41% in 24 h. The absorption of HOE 040 from the duodenum of dogs anesthetized with pentobarbital amounted to 72%, whereas proscillaridin A is observed by only between 14 and 25%. The concentration of the drug HOE 040 in hearts of rats was twice, in the hearts of dogs 3 fold that of proscillaridin A. The concentrations of both drugs in the brain of rats and dogs were not different. In the biochemical test system in vitro the blocking effect of both drugs on the Na+K+-ATPase of ox brain was not different.

Disposition rate of proscillaridin A in man after multiple oral doses.

After multiple oral doses, the disposition rate constant (beta) of proscillaridin was studied in 4 young healthy volunteers and 33 elderly patients with congestive heart failure. Glycoside activity in plasma was assayed by the 86Rb-technique. In the volunteers the beta averaged 0.0299 corresponding to a half-life (t 1/2) of 23 h. beta could be determined in 24 patients and was 0.0139 +/- 0.0077 (mean +/- SD). The SD of beta due to biological factors was estimated to be 0.0072. The total variation of beta was 10fold. The mean beta corresponded to a t 1/2 of 49 h with a range from 19 to 209 h. It is concluded that the great variation of beta means difficulty in obtaining adequate plasma levels of proscillaridin and that a rapid elimination of the glycoside cannot be presumed.

Biliary excretion and enterochepatic recycling of proscillaridin A after oral adminstration to man.

A single oral dose of proscillaridin A (1.0-1.5 mg) was given to six patients with T-tube drainage of the common bile duct, and simultaneous samples of bile and plasma were collected at various times during the following 24 hours. Glycoside activity was assayed by the 86Rb-uptake inhibition technique. Peak activities in plasma (mean 0.80 ng/ml) were attained after 0.5-2h, and in bile (mean 6.9 ng/ml) after 1-4h. Subsequently, proscillaridin activity in bile was less than 5 ng/ml for the remainder of the sampling period, and 10-100 times higher than that in plasma. Bile samples treated with beta-glucuronidase and sulphatase showed 100-200 fold increase in glycoside activity. Deconjugation was also produced by treatment with enteric contents. The results suggest that conjugation of unchanged proscillaridin is a major metabolic route. After excretion in the bile, the conjugates may be split in the intestine and reabsorbed as active glycoside.

Stability in vitro of methylproscillaridin.

The in vitro stability of methylproscillaridin has been compared with that of proscillaridin, the activities of the glycosides being assayed by the 86Rb-technique. After incubation in gastric juice at pH 1,2, and 3, the activity half life of each glycoside was proportional to pH and was approximately 0.25 h, 2.5 h, and 25 h, respectively. The inactivation rate in pure hydrochloric acid at pH 2 did not differ from that in gastric juice of the same pH. The glycosides were stable in bile and enteric juice. In faeces, methylproscillaridin was stable and proscillaridin was inactivated with a half life of 32 h. It is concluded that the difference in biological availability between the two glycosides cannot be explained by differences in gastrointestinal stability.

Activities of proscillaridin A in thoracic duct lymph after single oral doses in man.

In order to study the possibility that orally administered proscillaridin after absorption is transported by the lymph to the systemic circulation, the concentrations of the glycoside in thoracic duct lymph were analyzed in two patients with thoracic duct drainage. They received the drug as a single oral dose; plasma and lymph concentrations were measured by 86Rb-technique. Lymph was collected at various intervals for 24 hrs. The proscillaridin activity in thoracic duct lymph was low and followed closely that the plasma. During the sampling period, a total of 300 ng and 240 ng, respectively, was recovered in the lymph, corresponding to less than 0.03% of the administered dose. The results indicate that proscillaridin is not transported by the thoracic duct lymph.

On the absorption of proscillaridin A after single oral doses to normal and achlorhydric subjects.

Proscillaridin A was given in single oral doses (1.5-2.5 mg) to normal and achlorhydric subjects. Plasma activities of the glycoside were analysed by 86Rb-technique. The absorption pattern was similar in both groups. A marked first peak of proscillaridin activity was seen after about 30 min. After a first minimum, a second peak of activity was registered within 6-12 hrs. An estimate of the amount of active glycoside absorbed during the first 12 hrs after the administration was obtained by calculating the areas under the plasma activity curves (AUC). When corrected for differences in dose per kg body weight, the mean AUC in the achlorhydric group was about 60 per cent greater than in the normal group. The results suggest that proscillaridin is rapidly absorbed; gastric acidity seems to contribute to inter-individual differences in the bio-availability of the glycoside.

[Enteral availability and therapeutic activity of proscillaridin-4'-methyl ether (author's transl)]. / Enterale Verfügbarkeit und therapeutische Wirksamkeit von Proscillaridin-4' methyläther

20 cardiac patients with the clinical and radiological signs of heart failure were treated alternately intravenously and orally with proscillaridin-4'-methyl ether in a 2-period change-over procedure. With medium rate full saturation a steady state was maintained under observation of electrocardiographic parameters. The maintenance dose was determined to be 1.00 mg intravenously and 1.71 mg orally. Thus, relative enteral availability was 60%. In 70% of patients cardiac recompensation could be reached only by treatment with proscillaridin-4'-methyl ether. In 9 of 20 patients undesirable side effects consisted of laxative effects.

[Investigations on the Stability of Proscillaridine, Proscillaridine-3'-methylether and Proscillaridine-4'-methylether in Artificial Gastric and Intestinal Fluids (author's transl)]. / Untersuchungen zur Stabilität von Proscillaridin, Proscillaridin-3'-methyläther und Proscillaridin-4'-methyläther in künstlichen Verdauungsflüssigkeiten

The stability of bufadienolides in artificial gastric and intestinal fluids was investigated at different pH-values as a function of incubation time using proscillaridine, proscillaridine-3'-methylether and proscillaridine-4'-methylether. These glycosides were completely stable on pH 3.0--8.5 during the investigated time interval of 1 h. At lower pH-values cleavage of the glycosidic bond occurred. At pH 1.0 73% of proscillaridine, 33% of proscillaridine-3'-methylether and 41% of proscillaridine-4'-methylether were decomposed. At pH 2.0 only about 10% decomposition of these compounds could be detected. These experiments show that the stability of proscillaridine in gastric juice is comparable with the stability of digitalis glycosides and digoxin derivatives. The stability of methylated proscillaridine derivatives in artificial digestion fluids, however, proved to be superior to any orally administered cardiac glycoside.

Excretion of methylproscillaridin in patients with a biliary fistula.

Four patients with a biliary fistula received 0.5 mg 3H-methylproscillaridin as a single oral dose. 55% of the dose was excreted in bile, 16% in urine and 3% in faeces. More than 60% of the radioactivity excreted in bile and urine appeared within the first 24 hours. 78% of the radioactivity in bile consisted of CHCl3-insoluble metabolites of methylproscillaridin, incubation of which with beta-glucuronidase led to splitting off glucuronic acid from almost 80%. Methylproscillaridin can be regarded as the principal conjugated compound. TLC-separation of CHCl3-soluble compounds from bile showed identical running of radioactivity with methylproscillaridin, proscillaridin and scillarenin and three unknown metabolites P1, P2, and P3. In urine the CHCl3-soluble fraction averaged 16% to 34% of the total amount and was identified as methylproscillaridin, proscillaridin, scillarenin, P2 and P3. The relative composition of the total radioactivity in faeces amounted to 77% methylproscillardidin, 4% scillarenin and 12% polar metabolites.

On the pharmacokinetics of proscillaridin A in man.

The plasma concentration of proscillaridin was measured by a modified 86Rb method during treatment with multiple doses of a commercial preparation of proscillaridin. Despite high doses, very low plasma levels were found, and there were only minute amounts of glycoside activity in urine and faeces. Administration of an enteric-coated proscillaridin preparation gave higher plasma levels, which raises the possibility of inactivation of the glycoside by acid gastric juice. The results suggest that proscillaridin has low biological availability when given orally, and that it is extensively metabolised in the body.

Metabolism and excretion of methylproscillaridin by man.

0.5 mg 3H-proscillaridin-4-methylether was administered orally to 5 healthy males. Maximum plasma levels of total radioactivity were reached after one to two hours. In two subjects a second peak was observed between 6 and 12 hours. The plasma half life of total radioactivity was 51 hours. 20% and 56% respectively of the dose were eliminated in urine and faeces during the following 7 days. 55% of the total radioactivity in plasma, 80% in urine and 20% in faeces consisted of CHC13-insoluble compounds. 50-60% of the latter in plasma and urine could be hydrolysed by beta-glucuronidase. More than 90% of the split products were identified as conjugates of methylproscillaridin. TLC-separation of the CHC13-soluble fractions of plasma and urine yielded two unidentified metabolites, P2 and P3, as the main compounds, besides methylproscillaridin, proscillaridin and scillarenin. In faeces more than 90% of the non-polar fraction was identified as methylproscillaridin. Shortly after administration of 3H-methylproscillaridin, the radioactivity in plasma consisted mainly of CHC13-insoluble conjugates and of the metabolite P2.

In vitro stability of proscillaridin A.

In an in vitro study, proscillardin A was found to be rapidly inactivated at low pH. More than 50 per cent of its activity, measured by 86Rb assay, was lost after incubation for 15 minutes at pH 1 and 37 degrees C. Compared with proscillaridin, the rate of inactivation of digoxin was lower in these experiments. The rapid inactivation of proscillaridin might be of clinical importance when treating patients with this glycoside.