Naunyn-Schmiedeberg'sArch. Pharmacol. 287, 129--139 (1975) 9 by Springer-Verlag 1975

Influence of Spironolactone Pretreatment on Pharmacokinetics and Metabolism of Digitoxin in Rats* t t . - F . VShringer, L. Weller a n d N. Rietbroek Ins~itut fiir Klinische Pharmakologie, Klinikum Steglitz der Freien Universit~t Berlin Received October 23, 1974/Accepted January 6, 1975

Summary. The influence of pretreatment with spironolactone (84 mg/kg a t 3 days, twice per day) on the tritium levels in plasma, urine and feces of female SD-rats (n = 9) was investigated at various time periods after oral administration of 25 ~g/kg 3H-digitoxin. In plasma, the concentrations of total radioactivity are reduced in pretreated animals to about 20% of tritium levels in control rats, while the half-life of radioactivity in both groups is almost identical, 2.9 days in pretreated rats and 2.8 days in controls. The lower plasma levels of tritium in pretreated rats coincide with a six-fold decrease in the urinary SH-elimination and a corresponding increase in the fecal excretion. This is due to a higher biliary clearance of tritiated products in the early phase of elimination. The separation of the excretion products by TLC shows that spironolactone pretreatment enhances the splitting of the glycosidic bonds of digitoxin. The amount of digitoxigenin-bisdigitoxoside and of digitoxigenin-mono-digitoxoside excreted in urine and feces within 96 hrs is four and ten times greater than that recovered in control animals, respectively. The formation of the hydroxylation products digoxin and digoxigeninbis-digitoxoside is decreased from 50~ of the total excreted radioactivity in control to 15% in pretreated rats. The conjugation reactions with glucuronic and sulfuric acid are increased after pretreatment with spironolactone. Thus, the effect of spironolactone on digitoxin kinetics is apparently related to an enhancement of the hepatic excretory mechanism as well as to an enhanced metabolism. Key words: Spironolactone -- Digitoxin -- Half-Life in Plasma -- Renal and Fecal Excretion -- Metabolism. Several investigations have shown t h a t spironolactone p r e t r e a t m e n t protects the r a t against the anesthetic effects of certain steroids (Selye etal., 1969a), of h e x o b a r b i t a l a n d of p e n t o b a r b i t a l (Solymoss etal., 1969, 1970). This p r o t e c t i o n is suggested to be due to the i n d u c t i o n of d r u g - m e t a b o l i s i n g enzymes, whereby the n a t u r e of spironolactone i n d u c t i o n seems d e p e n d on sex (Stripp et al., 1971; Solymoss et al., 1971a; Leber st al., 1971). I n female rats, spironolactone p r e t r e a t m e n t

Send o]]print requests to: N. Rietbrock Klinikum Steglitz der Freien Universit~t Berlin, Institut fiir klinische Pharmakologie, D-1000 Berlin 45, Hindenburgdamm 30. * Supported by the Deutsche Forschungsgemeinschaft.

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increases t h e h e x o b a r b i t a l m e t a b o l i s m , t h e a c t i v i t y of e t h y l m o r p h i n e - N d e m e t h y l a s e a n d of I ~ A D P H - c y t o c h r o m c - c - r e d u c t a s e ; y e t t h e liver weight a n d t h e c o n t e n t of microsomal p r o t e i n are unaffected. The c o n t e n t of cytochrome-P450 is significantly decreased after p r e t r e a t m e n t w i t h spironolactone. S p i r o n o l a c t o n e also p r e v e n t s t h e toxic effects of digitoxin a n d of its h y d r o x y l a t e d p r o d u c t digoxin in female r a t s (Sclye et al., 1969b; Solymoss et al., 1971b). Thus, t h e faster h y d r o x y l a t i o n of d i g i t o x i n to digoxin does n o t p r o v i d e a n e x p l a n a t i o n for t h e influence of spironol a c t o n e on t h e digitoxin kinetics. Therefore, t h e e x p e r i m e n t s to be rep o r t e d here were p e r f o r m e d in female r a t s to d e t e r m i n e w h e t h e r t h e higher m e t a b o l i c r a t e or t h e higher r a t e of r e n a l a n d fecal e x c r e t i o n of t h e u n c h a n g e d d i g i t o x i n a n d its m e t a b o l i t e s was responsible for t h e prot e c t i v e effects of spironolactone.

Materials and Methods Female Sprague-Oawley rats (SPF-rats from MUS-Rattus AG, Germany) weighing about 200 g were used. The animals were kept under normal laboratory conditions and fed with an Altromin| standard diet. Each animal was placed in a separate metabolic cage. Food was withdrawn 14 hrs before the animals received the glycoside, water being available ad libitum. Spironolactone was administered by stomach tube (84 mg/kg) twice daily for three consecutive days. Control animals received an equal volume of a vehicle (an aqueous micronised suspension of 1 ~ (v/v) gummi arabicum). The volume of all administered substances was 2.5 ml/kg. The randomly-labelled tritiated digitexin (New England Nuclear, ]3oston) was given by stomach tube (25 tzg/kg, spec. activity 26.2 mCi/mg) 14 hrs after the last pretreatment. At different times after 0.3--0.5 ml blood was withdrawn heparinized, weighed, centrifuged transferred to a vial and counted for

administration of the glycoside, approximately by cardiac puncture. The blood samples were in plastic tubes, and the plasma layers were total radioactivity. Specimens of urine and feces

were collected twice during the first 24 hrs and once daily for the following 7 days. The samples were extracted, purified and subjected to thin-layer chromatography as described previously (Rietbrock and VShringer, 1974). Additionally, the CHC13insoluble material was separated on an A1203-column (VShringer and Rietbrock, 1974). The thin-layer chromatography was performed on silica gel plates (Fa. Merck, Darmstadt, West-Germany), 0.25 mm layer, flow distance 15 cm. Solvent system A: Chloroform/acetone (1 : 1), four times development; solvent system ]3: Diisopropylcther/methanol (9:1), five times development (Watson etal., 1972). The radioactive bands were located with a radiochromatogram scanner (Thin-layer scanner II, ]3erthold, West-Germany). Quantitative analysis was performed by automatic integration of the activity peaks (Integrator ]3erthold LB 2437, WestGermany). Calculations and Statistics. The half-life times were calculated with a program In2 (Olivetti programma) based on the equation tl/2 = - ~ - - . Statistical analysis of the data was done using arithmetic means and standard deviations.

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Results A. Plasma Tritium Levels and Excretion Rate~ As illustrated in Fig. 1, tritium levels in plasma of spironolactonepretreated rats are lower t h a n the levels observed in control animals already 1 hr after administration of ~H-digitoxin. The m a x i m u m of radioactivity is reached in both groups at nearly the same time period, after 3 hrs. Between 4 and 12 hrs, pretreatment with spironolactone causes an 8 fold lower level of tritium in the plasma than in the control group. After the first day, the concentrations of radioactivity in both groups differ b y a constant relationship at all time periods tested and decline with an almost identical half-life time of 2.9 days in pretreated and of 2.8 days in control animals, respectively. The decreased levels of radioactivity in the plasma of spironolactonepretreated rats m a y be the result of an increased elimination of unchanged digitoxin and/or its metabolites. This mechanism can be ruled out b y the determination of the cumulative excretion of tritium in urine and feces (Fig.2). Spironolactone pretreatment causes a six-fold decrease in the urinary excretion of tritiated compounds at all collection periods. The fecal excretion of radioactivity is correspondingly increased. However, the most striking difference in the fecal elimination of tritium

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between the two groups is observed in the first two collection periods. The feces of pretreated rats contain 51~ as compared to 31~ of the administered radioactivity in control animals at the 24 hrs time period. After 48 hrs, spironolactone pretreatcd animals have excreted via feces 61 ~ and the controls 51~ of the given dose, respectively. B. Characterization o/the CHCla-Soluble and CHCl~-Insoluble Fractions in Urine and Feces Table 1 gives the quantitative evaluation of the metabolites excreted in urine and feces after pretreatment with spironolactone for ibur time intervals. From this it becomes evident that the main components in urine are the CHCla-insoluble fraction and the unchanged substance digitoxin. The remaining radioactivity includes the bis- and monodigitoxosides of digitoxigenin as well as the hydroxylated products digoxin and digoxigenin-bis-digitoxoside. In contrast, most of the radioactivity excreted in feces is associated with digitoxigenin-bis-digitoxoside and digitoxigenin-mono-digitoxoside, besides smaller amounts of digitoxin and epi-digitoxigenin, of the hydroxylated compounds digoxin and digoxigenin-bis-digitoxoside, and of the CttCla-insoluble fraction. The relative distribution of the metabolites identified in the different collection periods is shown in Table 2. As compared to controls, the excretion of digitoxin in urine is considerably increased at 48 hrs in treated rats but there are no differences at 24, 72 and 96 hrs. The relative portion of the bis- and mono-digitoxosides of digitoxigenin is slightly increased in all collection periods. A major difference between control

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Spironolactone and the Pharmacokineticsof Digitoxin

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and treated groups is found in the relative distribution of the hydroxylated excretion products. Almost 10 times less digoxin is eliminated in urine of pretreated animals during four collection periods than in control animals. During the same time the relative portion of digoxigeninbis-digitoxoside does not differ between the two groups, whereas digoxigenin-mono-digitoxoside is only found in the urine of control animals. Furthermore the polar, CHCla-insoluble fraction constitutes at all time periods a greater portion of total radioactivity in pretreated than in control rats. In feces, on the basis of percentage of the total radioactivity excreted in the respective time intervals, almost ten times more digitoxigenin-mono-digitoxoside and nearly three times more digitoxigenin-bisdigitoxoside are eliminated in the pretreated than in the control group. There are no significant differences between the two groups in the relative amount of unchanged digitoxin, whereas a small amount of epi-digitoxigenin can only be observed in the pretreated group. The excretion of the hydroxylated products digoxin and digoxigenin-bis-digitoxoside is considerably decreased after spironolactone pretreatment in all collection periods. After 96 hrs the pretreated animals have eliminated altogether 3.3 times less hydroxylated compounds in feces than did the control animals. Finally, the relative portion of polar metabolites is slightly reduced in the pretreated group. To characterize the CHCt3-insohible fraction in the urine and feces of the pretreated animals, the total hydrophilic radioactivity was separated on alumina column. With urine and feces, this procedure revealed the greatest percentage (for each 693/0 of the CHC13-insohible material) as non-conjugated polar metabolites. Furthermore, the sulfuric-acid conjugates accounted for 60/0 of the hydrophilic fraction in urine and for i0 ~ in feces, respectively. The third fraction was expected to contain glueuronides, 25~ in urine and 21~ in feces. The enzymatic cleavage of the last two fractions in urine and feces with fl-glueuronidase and aryl-sulfatase showed that significant amounts of radioactivity for chromatography were obtained only from the glucuronic-aeid conjugates in feces. This fraction was hydrolysed to 53~ by incubation ~or 24 hrs with fi-ghieuronidase. The thin-layer chromatographic analysis of the lipophilic splitting products revealed the bisdigitoxoside of digitoxigenin as the main conjugation partner of glucurohie-acid besides smaller amounts of digitoxigenin-mono-digitoxoside, digoxin and digoxigenin-bis-digitoxoside. However, there is a striking difference in the metabolic pattern of the CHC13-insoluble fraction in feces between spironolactone pretreated and control animals. As could be shown in a preliminary report (Rietbrock and VShringer, 1974), 3-epi-digitoxigenin was obtained almost exclusiveiO *

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ly, besides smaller amounts of digoxin and of digoxigenin-bis-digitoxoside, in non-pretreated rats after splitting theOHCt~-insoluble fecal material with ~-glucuronidase and aryl sulfatase. After pretreatment with spironolactone the main conjugation reaction takes place on the step of digitoxigenin-bis- digitoxoside. Discussion The presented data which are in accordance with the results of Solymoss et al. (1971) and Castle and Lage (1972) show that prior to 1 hr after the administration of the glycoside the concentration of radioactivity is reduced to almost 20 ~ of the tritium level in control animals. After reaching the maximum level of tritium at nearly the same time period (3 hrs) in the two groups, the radioactivity drops more rapidly in the pretreated animals indicating a faster disappearance of tritium from the blood. These findings may reflect a higher biliary clearance of tritium in rats after spironolactone pretreatment than in control animals. This assumption is confirmed by observations of Castle and Lage (1973a) who reported a significantly increased biliary excretion of aH-digitoxin in rats after pretreatment with spironolactone between 15 rain and 8 hrs time periods. Furthermore, an enhancement of the hepatic excretory mechanism under the influence of spironolactone is known to occur with other substrates such as bilirubin (Solymoss and Zsigmond, 1972), sulfobromophthalein (Zsigmond and Solymoss, 1972), fl-methyldigoxin (Abshagen, 1973), digoxin (Wirth and FrSlich, 1974) and ouabain (Klaassen, 1974). The disappearance of digitoxin from the plasma of spironolactone-pretreated rats may not only be attributed to increased biliary excretion alone. Since it seems evident that spironolaetone induces the drugmetabolising enzymes (Stripp et al., 1971, Leber eta/., 1971 ) the metabolie pattern of digitoxin in urine and feces was investigated. The enzymatic degradation of digitoxin in rats involves splitting of glycosidic bonds, 12fl-hydroxylation and conjugation reactions with glueuronic and sulfuric acid, respectively. On the basis of percentage of the total radioactivity excreted in urine and feces during 96 hrs, spironolactone pretreated rats eliminate 4 times more digitoxigenin-bisdigitoxoside and 10 times more digitoxigenin-mono-digitoxoside than do the control animals. Thus, in accordance with Castle and Lage (1973b) the cleavage of the sugar side chains attached at carbon three is apparantly enhanced after pretreatmeut with spironolactone. Furthermore, there is evidence that spironolactone pretreatment reduces the elimination of hydroxylated compounds. 15~ of the total excreted radioactivity is associated with digoxin and digoxigenin-bisdigitoxoside in contrast to control animals in which 50 ~ of the labelling

Sv.ronolaetone and the Pharmacokinetics of Digitoxin

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is represented by hydroxylated products. This observation leads to the assumption of a diminished hepatic hych'oxylation under the influence of spironolaetone. Additional experiments in our laboratory show t h a t the content of the hydroxylating enzyme Cyt-P450 in the liver is decreased to about 50~ after pretreatment with spironolactone (from 1.1 to 0.6 nmoles/mg protein). However, the observed reduction of the hydroxylation rate seems to be involved only in the case of digitoxin in rats. In contrast, Solymoss et al. (1969, 1970) have reported that spironolactone pretreatment in female rats enhance the aliphatie hydroxylation of hexobarbital and pentobarbital. Human studies concerning the influence of spironolactone on drug metabolism have been also inconsistent : Leber et al. (1972) did not observe a faster metabolising rate of aminopyrine after pretreatment with spironolactone; studies of Huffman et al. (1973) gave evidence that spironolaetone is an inducer of 4'hydroxylation of antipyrine. Spironolaetone pretreatment also alters the excretion of the CHC13insoluble metabolites of digitoxin. These polar compounds constitute a much larger portion of the total radioactivity in the urine of spironolaetone pretreated rats than in the control group. In feces, the relative amount of the polar compounds is slightly diminished. These findings suggest a divergency of metabolic pattern on the CHCla-insoluble fraction between bile and feces. Experiments of Castle and Lage (1973c) gave evidence that the bile of spironolaetone pretreated rats contained larger amounts of water-soluble metabolites than did the feces. A great percentage of the polar metabolites excreted in bile seemed to be converted to non-polar compounds during the colon transfer. Thus, from the relative high level of CHC13-insoluble products in urine and the diminished portion in feces reported here, it is concluded that conjugation reactions with glucuronic and sulfuric acid are enhanced after pretreatment with spironolaetone. Apparently, the conjugations take place mainly on the step of digitoxigenin-mono-digitoxoside (Castle and Lage, 1974) and of digitoxigenin-bis-digitoxoside. In conclusion the data presented indicate that the accelerated elimination of digitoxin after pretreatment with spironolactone in rats is the result of an enhanced cleavage of the digitoxose sugar moieties as well as of an enhanced formation of polar metabolites. References Abshagen, U.: Effects of pretreatment with spironolactone on pharmacokinetcis of 4"-methyldigoxin in rats. Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 91--100 (1973) Castle, M.C., Lage, G. L. : Effect of pretreatment with spironolactone, phenobarbital or fi-diethylaminoethyl diphenylpropylacetate (SICF 525-A) on tritium levels in blood, heart and liver of rats at various times after administration of (aI-I)digito:xin.Biochem. Pharmacol. 21, 1449--1455 (1972)

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Castle, M. C., Lage, G. L. : Enhanced biliary excretion of digitoxin following spironolactone as it relates to the prevention of digitoxin toxicity. Res. Comm. Chem. Path. Pharmaeol. 5, 99--108 (1973a) Castle, M. C., Lage, G. L. : Excretion of 8H-digitoxin and its metabolites following spironolaetone pretreatment in rats. Drug Metab. Disp. 1, 590--598 (1973b) Castle, ~ . C., Lage, G. L. : 3H-digitoxin and its metabolites following spironolactone pretreatment of rats. Res. Comm. Chem. Path. Pharmaeol. 6, 601--612 (1973c) Castle, M. G., Lage, G. L. : Cleavage by fi-glueuronidase of the water-soluble metabolites of digitoxin excreted in the bile of control and spironolactone-pretreated rats. Toxicol. appl. Pharmacol. 27, 641--647 (1974) Huffman, D. H., Shoeman, D. W., Pentikiiinen, P., Azarnoff, D. L. : The effect of spironolactone on antipyrine metabolism in man. Pharmacology 10, 338--344 (1973) Klaassen, C. D.: Stimulation of the development of the hepatic excretory mechanism for ouabain in newborn rats with mierosomal enzyme inducers. Toxieol. appl. Pharmacol. 29, 146 (1974) Leber, H . W . , Harders, P., Schiitterle, G.: Untersuchungen zum EinfluB yon Aldactone auf Arzneimittel abbauende Enzyme im endoplasmatisehen Retieulum der Leber der Ratte und des Mensehen. Vcrh. dtsch. Ges. inn. Med. 78, 1358--1362 (1972) Leber, H. W., Rawer, P., Sehiitterle, G. : Beeinflussung mikrosomaler Enzyme der Rattenleber durch Spironolacton, Etacryns~ure und Furosemid. Klin. Wsehr. 49, 116--118 (1971) Rietbrock, N., VShringer, H.-F.: Metabolism and excretion of aH-digitoxin in the rat. Bioehem. Pharmacol. 28, 2567--2575 (1974) Selye, H., M6cs, I., Savoie, L. : Inhibition of anesthetics and sedative actions by spironolaetone. Anesthesiology 81, 261--264 (1969a) Selye, H., M6cs, I., Tamura, T. : Effect of spironolactone and norbolethone on the toxicity of digitalis compounds in the rat. Brit. J. Pharmacol. 87, 485--488 (1969b) Solymoss, B., Classen, H. G., Varga, S.: Increased hepatic microsomal activity induced by spironolactone and other steroids. Proe. Soe. exp. Biol. (N.Y.) 182, 940--942 (1969) Solymoss, B., T6th, S., Varga, S., Selye, H.: Protection by spironolactone and oxandrolone against chronic digitoxin or indomethaein intoxication. Toxicol. appl. Pharmaeol. 18, 586--592 (1971b) Solymoss, B., Varga, S., Classen, It. G. : Effect of various steroids on microsomal aliphatic hydroxylation and N-dealkylation. Europ. J. Pharmacol. 10, 127--130 (1970) Solymoss, B., Werringloer, J., T6th, S.: The influence of pregnenolone-16cc-earbonitrile on hepatic mixed-function oxygenases. Steroids 17, 427--433 (19719) Solymoss, B., Zsigmond, G. : Enhanced excretion of bilirubin induced by spironolactonc and pregnenolone-16ac-carbonitrile. Fifth International Congress on Pharmacology, Volunteer Abstracts 1306 (1972) Stripp, B., Hamrick, M., Zampagli~)ne, N., Gilette, J. : The effect of spironolactone on drug metabolism by hepatic mierosomes. J. Pharmacol. exp. Ther. 176, 766--771 (1971) VShringer, tI.-F., Rietbrock, 5I.: Metabolism and excretion of aH-digitoxin in man. Clin. Pharmacol. Ther. 16, 796--806 (1974)

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Watson, E., Tramell, P., Kalman, S. M. : Identification of submicrogram amounts of digoxin, digitoxin, and their metabolic products. Isolation by chromatography and preparation of derivatives for assay by electron capture detector. J. Chromatogr. 69, 157--163 (1972) Wirth, K. E., FrSlich, J. C. : The influence of spironolactone on the excretion and metabolism of 8H-digoxin in the rat. Naunyn-Sehmiedeberg's Arch. Pharmacol. 282, R107 (i974) Zsigmond, G., Solymoss, B. : Effect of spironolactone, pregnenolone-16cc-carbonitrile and cortisol on the metabolism and biliary excretion of sulfobromophthalein and pheno|-3,6-dibromophthalein disulfonate in rats. J. Pharmacoh exp. Ther. 188, 499'--507 (1972)