Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs.

Cardiac glycosides have been reported to exhibit cytotoxic activity against several different cancer types, but studies against colorectal cancer are lacking. In a screening procedure aimed at identifying natural products with activity against colon cancer, several cardiac glycosides were shown to be of interest, and five of these were further evaluated in different colorectal cancer cell lines and primary cells from patients. Convallatoxin (1), oleandrin (4), and proscillaridin A (5) were identified as the most potent compounds (submicromolar IC50 values), and digitoxin (2) and digoxin (3), which are used in cardiac disease, exhibited somewhat lower activity (IC50 values 0.27-4.1 microM). Selected cardiac glycosides were tested in combination with four clinically relevant cytotoxic drugs (5-fluorouracil, oxaliplatin, cisplatin, irinotecan). The combination of 2 and oxaliplatin exhibited synergism including the otherwise highly drug-resistant HT29 cell line. A ChemGPS-NP application comparing modes of action of anticancer drugs identified cardiac glycosides as a separate cluster. These findings demonstrate that such substances may exhibit significant activity against colorectal cancer cell lines, by mechanisms disparate from currently used anticancer drugs, but at concentrations generally considered not achievable in patient plasma.

Determination of proscillaridin in plasma by radioimmunoassay.

The development of a sensitive and selective radioimmunoassay for determination of proscillaridin in plasma is described. Antiserum against proscillaridin was obtained from guinea pig immunized with an immunogen prepared by conjugating methylproscillaridin, 14 beta-hydroxy-3 beta-[(4-O-methyl-alpha-L-rhamnosyl)oxy]bufa-4,20,20, 22-trienolid to bovine serum albumin. Methyl N-[3-[(14 beta-hydroxybufa-4,20,22-trienolid-3 beta-yl)oxy-carbonyl] propanoyl]-L-[3',5'-125I2] diiodotyrosinate ([125I] SST) was used as a radioactive ligand. [125I] SST and antiserum were added to the recovered sample from plasma using a Bond Elut column. After overnight incubation, the antibody-bound and free [125I] SST were separated using polyethylene glycol. The mean coefficients of variation of intra and interassay at four different plasma concentrations were 4.0 and 5.0%, respectively. The assay was able to determine as little as 125 pg/ml of proscillaridin in plasma by using 1.5 ml of sample. Beagle dogs were orally administered one tablet (Talusin) containing 0.25 mg of proscillaridin. Proscillaridin was rapidly absorbed, exhibiting plasma maximum concentration of 2.06 ng/ml at 20 min. Thereafter, the plasma level declined biphasically with half-lives of 0.6 and 25.4 h.

[Plasma concentration and elimination behavior of the cardiac glycoside meproscillarin in patients with liver cirrhosis]. / Plasmakonzentration und Eliminationsverhalten des Herzglykosids Meproscillarin bei Patienten mit Leberzirrhose.

During a one week period patients with liver cirrhosis and a control group were treated with a repeated dosage of the new heart glcoside Meproscillarin. After achieving a steady state in plasma level the same Meproscillarin plasma levels were found among both groups. Compared with the control group no difference was detected in the elimination rate of Meproscillarin in patients with liver cirrhosis, which means a complex disturbed liver function. Nevertheless the greater variance of the Meproscillarin plasma levels in the patients with liver cirrhosis in comparison with the controls means a diminished predictability of the therapeutic success in the cirrhosis group. With this limitation Meproscillarin can be used therapeutically in patients with liver cirrhosis, because a toxic accumulation is not to be expected.

[Meproscillarin in patients with renal failure and concomitant heart failure (author's transl)]. / Meproscillarin bei gleichzeitiger Nieren- und Herzinsuffizienz. Wirkungund Plasmakonzentration bei Dauernwendung.

Meproscillarin is a glycoside with a high bioavailability (about 70%) and an elimination independent of the renal function. It was to be investigated whether a good cardiac effectiveness can be demonstrated during oral long-term application of meproscillarin to patients with renal failure. 29 patients with renal failure of varying degree and concomitant heart failure were daily given an oral dose of 0.75 mg of meproscillarin over 14 days. The effectiveness of the glycoside was measured as change of the electromechanical systole (QS2c) and the quotient of the diameter of heart and thorax (C/T) from the 1st--15th day. The plasma levels of the glycoside were determined on the 1st, 8th, and 15th day. There was a significant shortening of QS2c (by mean = 27 ms, P less than 0.005) and a marked decrease in the size of the heart (P less than 0.0025); heart rate and PQ-interval were only insignificantly influenced. Plasma levels of 0.95 ng/ml were found after 8 days of treatment compared to 1.25 ng/ml after 15 days. As the pharmacokinetics of the glycoside is practically not influenced by the renal function, meproscillarin represents an alternative in the treatment of patients with heart failure and impaired renal function.

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.

Proscillaridin activity in portal and peripheral venous blood after oral administration to man.

The absorption of proscillaridin A was studied in four patients undergoing catheterization of the portal vein for diagnostic purposes. Proscillaridin 1.5 mg was given as a single oral dose and plasma glycoside activity was analyzed by the 86Rb-uptake inhibition technique. Proscillaridin appeared rapidly in the portal blood, peak activity being found after 15 min in three and after 30 min in one patient. In peripheral blood the peak activity occurred after approximately 35 min. Despite rapid passage across the gut wall, porto-peripheral differences in glycoside activity were small; they were zero after 4h. The mean amount absorbed as active porscillaridin during the first 4h after the dose was calculated to be only 7.1% of the given amount. Late porto-peripheral differences, probably due to enterohepatic recycling, appeared after 6h in three patients; The results suggest that proscillaridin undergoes first pass inactivation in the gut wall. Enterohepatic recirculation may contribute to the amounts of active glycoside that reach the systemic circulation.

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.

Pharmacokinetics and pharmacodynamics of methyl proscillaridin in healthy man.

The aim of this study was to obtain data about the pharmacological properties of a new glycoside derivative in man. Plasma concentrations and ECG parameters were measured after oral and intravenous administration of a single dose of 1.2 mg methyl proscillaridin in 16 healthy volunteers, using a strictly randomized, two-period change-over design. Glycoside concentrations were measured using a modified 86Rb-erythrocyte-assay. QT-duration, corrected for frequency (QTc), was the principal variable measured in the ECG. By either route, there was a maximum plasma level after 1 hour, which had decreased to a minimum at 3 hours, followed by a second peak at 4 to 10 hours (orally greater than iv). From 10 to 72 hours the concentrations decreased with a median t 1/2 of 23.3 hours (iv) and 33.0 hours (orally). Comparison of the ratio of plasma concentrations following oral and iv administration resulted in a bioavailability of 69% using the 48 hour plasma levels, and 59% using the areas under the concentration-time curves. The mean QTc was maximally shortened to 28 msec at 1 hour after iv and to 19 msec at 10 hours after the oral dose. A distinct similarity between time-concentration and time-QTc curves was seen after the initial distribution phase, both after oral and intravenous administration. The new derivative shows a rapid elimination. Its bioavailability is reasonably high.

Plasma concentrations during repeated intravenous and oral methyl-proscillaridin application in man.

The aim of this study was to establish data on the plasma levels of the glycoside derivative methyl-proscillaridin (MP) following repeated intravenous and oral doses. The study was carried out on healthy male volunteers. Each received 0.5 mg of MP daily at 8 a.m. for 7 days. 6 volunteers received the drug i.v., 6 p.o. as tablets and 5 p.o. as elixir. Plasma glycoside concentrations were measured utilizing a 86Rb-erythrocyte assay. The mean plasma concentrations on the 6th and 7th days of application were: 752.9 (Sx = 303.5) pg/ml for the i.v. route, 432.9 (Sx = 115.5) pg/ml for the tablets and 473.1 (Sx = 321.5) pg/ml for the elixir. The mean ratio between plasma concentrations with tablets and i.v. injection averaged 63%. It is concluded that the therapeutic activity of oral MP is about 60 to 70% that of the i.v. application.