Europ. J.clin. Pharmacol. 9, 345-354 (1976) © by Springer-Verlag 1976

Metabolism of Digitoxin in Man and Its Modification by Spironolactone K. E. W~th*, J. C. Fr61ich, J. W. Hollffield, F. C. Falkner, B. S. Sweetman and J. A. Oates Department Vanderbilt

Received:

of M e d i c i n e University,

May

6, 1975,

and P h a r m a c o l o g y , D i v i s i o n Nashville, Tennessee, USA

and in revised

form:

August

of C l i n i c a l

6, 1975,

Pharmacology,

accepted:

August

18,

1975

Summary. The

effect of s p i r o n o l a c t o n e on the m e t a b o l i s m of i n t r a v e n o u s l y a d m i n i s t e r e d 3 H - d i g i t o x i n (80 ~Ci) was i n v e s t i g a t e d in eight patients. In three of them the labelled g l y c o s i d e was given on a second o c c a s i o n after s p i r o n o l a c t o n e treatment had been d i s c o n t i n u e d for at least 65 days. Of total u r i n a r y r a d i o a c t i v i t y 79 % was u n a l t e r e d drug and 12 % c o n s i s t e d of water soluble compounds. No d i g i t o x i g e n i n or d i g o x i g e n i n and only trace amounts ( < 2 %) of d i g o x i n and the bis- and m o n o g l y c o sides of d i g o x i g e n i n were found. After s p i r o n o l a c t o n e total u r i n a r y r a d i o a c t i v i t y was u n c h a n g e d but the fraction e l i m i n a t e d as u n c h @ n g e d d i g i t o x i n fell from 79 to 66 % and the water soluble compounds i n c r e a s e d from 12 to 26 % (p < 0.05). In a d d i t i o n spir o n o l a c t o n e caused a 20 % r e d u c t i o n in the h a l f - l i f e of serum r a d i o a c t i v i t y (p < O.O1) and a 16 % r e d u c t i o n in the volume of d i s t r i b u t i o n (p < 0.05). I n d u c t i o n of hepatic enz_vmes by s p i r o n o l a c t o n e is p r o p o s e d to e x p l a i n the a l t e r a t i o n in the m e t a b o l i s m of d i g i t o x i n in man. Both the altered m e t a b o l i c p a t t e r n and the r e d u c t i o n in the volume of d i s t r i b u t i o n appear to c o n t r i b u t e to the r e d u c t i o n in half-life.

Key words:

3H-digitoxin,

metabolism,

spironolactone,

Animal e x p e r i m e n t s have shown that spir o n o l a c t o n e reduces the t o x i c i t y of the cardiac g l y c o s i d e s d i g o x i n and d i g i t o x i n (5, 25, 28). R e c e n t l y it was found that s p i r o n o l a c t o n e increased the e l i m i n a t i o n and m e t a b o l i s m of d i g o x i n in the rat. This effect was seen even after spironolactone t r e a t m e n t had been d i s c o n t i n u e d for three weeks, s u g g e s t i n g that enzyme induction was of i m p o r t a n c e (36). In man s p i r o n o l a c t o n e shortens the h a l f - l i f e of antipyrine, an effect which is thought to be due to enzyme induction (33). SDir o n o l a c t o n e in large doses is f r e q u e n t l y used in the t r e a t m e n t of heart failure and m i n e r a l o c o r t i c o i d h y p e r t e n s i o n , and often given c o n c o m i t a n t l y with d i g i t o x i n (9). Therefore, the effect of large doses of s p i r o n o l a c t o n e on the m e t a b o l i s m of d i g i t o x i n in man has been studied.

enzyme

MATERIALS

induction,

man.

AND M E T H O D S

Tritium-Labelled Digitoxin

3 H - d i g i t o x i n (5.3 Ci/mM) was o b t a i n e d from A m e r s h a m / S e a r l e (Arlington Heights, Ill.). The p u r i t y was c o n f i r m e d by paper c h r o m a t o g r a p h y and by mass spectrometry. The mass spectrum of the 3 H - d i g i t o x i n used was compared with a sample of authentic d i g i t o x i n (Boehringer M a n n h e i m Corp., N e w York, N.Y.). The s t a b i l i t y of the label was tested by e v a p o r a t i n g aliquots of urine from 5 p a t i e n t s on the 5th, 12th and 18th day after a d m i n i s t r a tion of 3H-digitoxin. Less than 1.4 % of the total r a d i o a c t i v i t y was found in the d i s t i l l a t e s from these urines, w h i c h indicates that t r i t i u m exchange b e t w e e n water and d i g i t o x i n was negligible. Subjects and Procedures

~Fellow of the Paul Martini-Stiftung, Main, Germany

Frankfurt/

Eight p a t i e n t s with normal renal function and with m e d i c a l l y c o n t r o l l e d heart failure p a r t i c i p a t e d in the study. SGOT values were w i t h i n the normal range, ex-

346 Table I. Clinical details of the patients

Patient

sex

age

weight (kg)

Creatinine SGOT Clearance (KU) (ml/min)

BUN (mg%)

diagnosis

L.U. (i)

f

68

68.3

30

124.0

20.0

J.L. (i) E.P. (i)

m

67

99.1

25

98.3

14.5

Hypertension, ischemic heart disease, mild diabetes mellitus Hypertension

m

58

76.2

13

148.0

12.0

M.F.

f

57

49.5

28

90.7

16.O

daily medication

Ischemic heart disease, recent inferior myocardial infarction malignant pheochromocytoma

with metastases R.F. J.W.

m m

51 63

73.0 84.5

18 23

144.3 118.4

8.0 18.O

Cardiomyopathy Hypertension

L.U. (2)

f

68

68.2

20

93.0

22.5

J.L. (2) E.P. (2)

m

67

97.6

65

110.2

12.O

Hypertension, ischemic heart disease, mild diabetes mellitus Hypertension

m

58

75.0

18

131.1

13.5

A.M.

f

53

74.5

20

140.6

17.O

R.B.

m

68

78.4

24

134.3

16.5

cept for a t r a n s i e n t m a r g i n a l rise in p a t i e n t J.L. d u r i n g the second part of the trial (Table I). All of the p a t i e n t s had t a k e n an oral m a i n t e n a n c e dose of d i g i t o x i n O.1 m g / d a y (Parke, D a v i s & Co., Detroit, Mich.) for at least 8 w e e k s prior to c o m m e n c e m e n t of the study. O t h e r p e r t i n e n t p a t i e n t i n f o r m a t i o n a b o u t the s u b j e c t s is shown in T a b l e I. P a t i e n t M.F. r e q u i r e d a d d i t i o n a l d a i l y t r e a t m e n t w i t h 80 mg p r o p r a n o l o l , 20 mg p h e n o x y b e n z a m i n e and 130 m g p r o p o x y p h e n e . P a t i e n t A.M. also r e c e i v e d 37.5 mg guan e t h i d i n e daily. T h e s e m e d i c a t i o n s were m a i n t a i n e d u n c h a n g e d t h r o u g h o u t the study. T r i t i a t e d d i g i t o x i n was i n j e c t e d int r a v e n o u s l y (80 pCi; 11.6 pg) in 1.6 ml 50 % e t h a n o l d u r i n g 2 m i n u t e s . S p i r o n o lactone (Se~rle & Co., Chicago, Ill.) was a d m i n i s t e r e d o r a l l y in a dose of 400 m g / d a y to 5 p a t i e n t s (Table 1) for at least 10 days prior to a single injection of 3H-digitoxin. In 3 of the pat i e n t s (L.U., J.L., E.P.) a d m i n i s t r a t i o n of 3 H - d i g i t o x i n was r e p e a t e d after 65

Ischemic heart disease, recent inferior myocardial infarction Hypertension, recent congestive heart failure Ischemic heart disease

O.I mg digitoxin

O.i mg digitoxin O.i mg digitoxin

O.i 80.0 20.0 130.0 O.i O.i

mg mg mg mg mg mg

digitoxin propranolol phenoxybenzamine propoxyphene digitoxin digitoxin

O.I mg digitoxin 400.0 mg spironolactone

O.i 400.0 O.i 400.0

mg mg mg mg

digitoxin spironolactone digitoxin spironolactone

0. I 400.0 37.5 O.i 400.0

mg mg mg mg mg

digitoxin spironolactone guanethidine digitoxin spironolactone

to 85 days. At that time no r a d i o a c t i v i ty from the p r e v i o u s i n j e c t i o n was det e c t a b l e in the urine. In this way it was p o s s i b l e to e x a m i n e the m e t a b o l i s m of 3 H - d i g i t o x i n in 3 p a t i e n t s b e f o r e and d u r i n g t r e a t m e n t w i t h s p i r o n o l a c t o n e . 24-hour u r i n e s were c o l l e c t e d for 20 days s t a r t i n g w i t h the i n j e c t i o n of 3Hdigitoxin. The samples of day 4 and day 9 were a n a l y z e d for d i g i t o x i n and its metabolites. V e n o u s blood samples were c o l l e c t e d at 15 and 30 m i n u t e s and at I, 2, 3, 4, 5, 7, 9, 11, 14, 17, 24 hours after inj e c t i o n of the l a b e l l e d g l y c o s i d e and d a i l y t h e r e a f t e r for 20 days. P a r t i c i pants in the study gave their i n f o r m e d consent. The study had been a p p r o v e d by the V a n d e r b i l t C l i n i c a l I n v e s t i g a t i o n Committee. Determination

of Total R a d i o a c t i v i t y

in Serum

and Urine

A l i q u o t s Qf s e r u m and u r i n e added to 10 ml U n o g e l ® (Schwarz/Mann, O r a n g e b u r g , N.Y.) were a n a l y z e d for total r a d i o a c -

347

tivity by liquid s c i n t i l l a t i o n c o u n t i n g (Packard 3575). Q u e n c h c o r r e c t i o n was achieved by the external standard ratios method.

porated to dryness and r e d i s s o l v e d in 50 ul chloroform. To this final phase were added 10 ~g each of digoxin, digoxigenin b i s d i g i t o x o s i d e and m o n o d i g i t o x o side, digoxigenin, digitoxin, d i g i t o x i genin b i s d i g i t o x o s i d e and m o n o d i g i t o x o side and d i g i t o x i g e n i n (Boehringer MannIdentification of Digitoxin and Its Metabolites heim Corp., N e w York, N.Y.) to serve as from Urine carriers and markers. An aliquot of the final c h l o r o f o r m solution c o n t a i n i n g Paper c h r o m a t o g r a p h i c s e p a r a t i o n of digiabout 105 dpm was applied to the approtoxin and its m e t a b o l i t e s was p e r f o r m e d priate f o r m a m i d e - i m p r e g n a t e d strip. The by a m e t h o d similar to that employed by strip was then p o s i t i o n e d so that the Lage and Spratt (16) and Castle and Lage end touched a petri dish placed at the (6). For each analysis two strips of bottom of the c h r o m a t o g r a p h y tank. After W h a t m a n No. 3 paper (47 x 8.75 cm) were 8.5 h of d e v e l o p m e n t in d e s c e n d i n g used, one to separate d i g i t o x i n and its fashion with solvent system I digitoxin, lower glycosides. Three solvent systems its mono- and b i s g l y c o s i d e s and its were used to separate and identify digigenin were c o m p l e t e l y eluted from the toxin and its metabolites. Solvent system paper into the petri dish. The contents I was a b i p h a s i c paper system c o n s i s t i n g of the petri dish were e v a p o r a t e d to of c h l o r o f o r m : f o r m a m i d e (90 : 60, v/v). dryness, d i s s o l v e d in 50 ~i chloroform, After e q u i l i b r a t i o n for 12 hours the applied q u a n t i t a t i v e l y to the second f o r m a m i d e phase was used to saturate paper strip and d e v e l o p e d in d e s c e n d i n g filter paper lining the c h r o m a t o g r a p h y fashion in solvent system II for 8 hours. tank. The c h l o r o f o r m phase was the deThe spots on the paper strips were viv e l o p i n g phase for the paper c h r o m a t o sualized w i t h Kedde reagent, Spray A grams. S o l v e n t system II was also a bi(29). The f o l l o w i n g Rf values were obphasic system c o n s i s t i n g of isopropylserved on the first strip : digoxin, 0.64 ether : t e t r a h y d r o f u r a n e : f o r m a m i d e 0.04; digoxigenin bisdigitoxoside, (54 : 36 : 30, v/v/v) e q u i l i b r a t e d for 12 0.33 ± 0.02; d i g o x i g e n i n m o n o d i g i t o x o hours. The upper layer was used for deside, O.21 ± 0.03; digoxigenin, 0.28 ± v e l o p m e n t of the paper chromatogram, 0.02 (n = 9; mean ~ SE). The Rf values while the lower layer was used to satuof the second strip were : digitoxin, rate the filter paper lining the walls 0.29 ± 0.02; d i g i t o x i g e n i n b i s d i g i t o x o of the c h r o m a t o g r a p h y tank. Solvent sysside, O.41 ± 0.03; d i g i t o x i g e n i n monotem III was e t h y l a c e t a t e : l-butanol (9 : digitoxoside, 0.67 ± 0.04; and d i g i t o I, v/v) and was used to d e v e l o p the aluxigenin, 0.44 ± 0.02 (n = 7; mean ± SE) . mina TLC plates (F-254, type T, E. M e r c k Because of u n s a t i s f a c t o r y s e p a r a t i o n L a b o r a t o r i e s , Inc., Elmsford, N.Y.). The of d i g o x i g e n i n and d i g o x i g e n i n bisdigiW h a t m a n No. 3 paper used for d e s c e n d i n g toxoside, as well as of d i g i t o x i g e n i n c h r o m a t o g r a p h y was i m p r e g n a t e d with and d i g i t o x i g e n i n b i s d i g i t o x o s i d e , the f o r m a m i d e before use. The strips used to following TLC m e t h o d was used for their separate the d i g o x i n series of c o m p o u n d s separation. An aliquot of the final were placed in a solution of 30 % forc h l o r o f o r m phase c o n t a i n i n g about 40.000 m a m i d e in a c e t o n e and those used for dpm was applied to a l u m i n u m oxide plates. s e p a r a t i o n of the d i g i t o x i n series were The plates were d e v e l o p e d for 3 hours placed in 25 % formamide in acetone. in solvent system III when a d i s t i n c t After soaking for 10 m i n u t e s the strips s e p a r a t i o n of d i g i t o x i g e n i n and d i g o x i were allowed to a i r - d r y for 10 minutes. genin from all the other m e t a b o l i t e s Urine was assayed for d i g i t o x i n and and the parent c o m p o u n d s was observed. its m e t a b o l i t e s on the 4th and 9th days The Rf values of d i g o x i g e n i n and digiafter i n j e c t i o n of the labelled drug. t o x i g e n i n were 0.24 ± O.O1 and 0.41 ± A 1OO ml a l i q u o t from a 24 hour urine 0.03, r e s p e c t i v e l y (n = 8; mean ± SE). c o l l e c t i o n was shaken four times w i t h All other r a d i o a c t i v e m a t e r i a l r e m a i n e d 200 ml of e t h a n o l - f r e e chloroform. The at the origin. For q u a n t i t a t i o n of radiototal r a d i o a c t i v i t y in the water and a c t i v i t y the paper strip was cut into c h l o r o f o r m phases was d e t e r m i n e d separa0.5 cm segments and each segment was tely. The c h l o r o f o r m phase was passed cut into small pieces and placed in a through an a c t i v a t e d alumina column counting vial. D i s t i l l e d water (0.4 ml) (neutral, activity grade I, Woelm, W a t e r and three hours later 10 m l ~ U n o g e l ® Assoc. Framingham, Mass.) 1.O cm diam. were added. This p r o c e d u r e y i e l d e d the and 5 cm high (2, 7). The column was same results as e x t r a c t i o n of the paper washed with 20 ml carbon t e t r a c h l o r i d e followed by 20 ml c h l o r o f o r m : ethanol with m e t h a n o l : c h l o r o f o r m (I : I) (16). For q u a n t i f i c a t i o n of label on the TLC (2 : I). No r a d i o a c t i v i t y was d e t e c t a b l e plates strips 0.5 cm wide were scraped in the carbon t e t r a c h l o r i d e fraction. The c h l o r o f o r m : ethanol f r a c t i o n was eva- from the plates and t r a n s f e r r e d d i r e c t -

348

ly to s c i n t i l l a t i o n vials. D i s t i l l e d water (0.5 ml) was added to d e a c t i v a t e the s o r b e n t at least 3 hours b e f o r e add i t i o n of 10 ml U n o g e l ® s c i n t i l l a t i o n fluid. A f t e r the latter the v i a l s w e r e s o n i c a t e d for 10 m i n u t e s to a c h i e v e better d i s p e r s i o n . The o v e r a l l r e c o v e r y of r a d i o a c t i v i t y from urine by the above scheme was 94.31 ± 3.18 % (n = 5) for the paper c h r o m a t o g r a p h i c t e c h n i q u e and 93.48 ± 4.33 % (n = 4) for the TLC technique. I d e n t i f i c a t i o n of d i g i t o x i n e x c r e t e d in urine was done by m a s s s p e c t r o m e t r y in a d d i t i o n to c h r o m a t o g r a p h y (35). For this purpose, d i g i t o x i n was e x t r a c t e d from urine and c h r o m a t o g r a p h e d as des c r i b e d above. S u b s e q u e n t l y it was h y d r o lyzed (14) and d e r i v a t i z e d w i t h N , O - b i s (trimethylsilyl)-trifluoroacetamide-1% t r i m e t h y l c h l o r o s i l a n e (10 ~i), m e t h y l e n e c h l o r i d e (10 ul) and N - t r i m e t h y l s i l y l i m i d a z o l e (10 ul) at room t e m p e r a t u r e for 18 hours to y i e l d a m i x t u r e of d i g i t o x i g e n i n - t r i m e t h y l s i l y l - 2 and d i g i t o x i g e n i n trimethylsilyl-1 (10). The m a t e r i a l was i n t r o d u c e d into an LKB 9000 m a s s s p e c t r o m e t e r via an 0.5 m (4 m m i.d.) c o l u m n of 1 % D e x s i l 300 on C h r o m o s o r b G 80 / 1OO m e s h at 265 ° , with h e l i u m c a r r i e r gas at 30 ml/min. The i n j e c t i o n port t e m p e r a t u r e was 270 ° , the ion source 250 ° and the s e p a r a t o r 260 ° . T r a p c u r r e n t was 60 ~A and the e l e c t r o n e n e r g y 70 eV. The m a g n e t + w a s set to m o n i t o r the ion m / e 356 (M -90 of d i g i t o x i g e n i n - t r i m e thylsilyl-1) u s i n g the t e c h n i q u e of single ion m o n o t o r i n g . Evaluation of Kinetic Data

The serum levels of r a d i o a c t i v i t y were fitted by n o n l i n e a r r e g r e s s i o n a n a l y s i s on a d i g i t a l c o m p u t e r to a t r i p h a s i c curve, as shown in Fig. 3. The k i n e t i c terms m e a s u r e d are d e f i n e d in the following equations : terminal -

in 2 Y

serum half-life

tl/2

~ = t e r m i n a l rate constant (= slope x in 10)

total b o d y c l e a r a n c e C1 D

D = dose of 3H-digitoxin. AUC = area under the serum level curve, c a l c u lated by t r a p e z o i d a l rule on an IBM 360 computer

AUC

renal

clearance

= Cu x V u Cs

Clre n

Cu = 3H-concentration urine

in

C s = 3 H - c o n c e n t r a t i o n in serum V u = v o l u m e of u r i n e / m i n v o l u m e of d i s t r i b u t i o n

Vd~-~

C1 Y P r o t e i n b i n d i n g of r a d i o a c t i v i t y in serum from all the p a t i e n t s was d e t e r m i n e d by e q u i l i b r i u m d i a l y s i s on the 5th day, and in two p a t i e n t s (L.U., R.F.) also on the 2nd and 15th days after i n j e c t i o n of 3H-digitoxin. S e r u m 3 ml was d i a l y z e d at room t e m p e r a t u r e t h r o u g h D i a l y z e r T u b i n g s (Thomas & Co., P h i l a d e l p h i a , PA) a g a i n s t 6 ml p h o s p h a t e buffer, pH 7.4 for 48 hours. P r o t e i n b i n d i n g was c a l c u l a t e d by the formula: % radioactivity =

bound

inside - o u t s i d e inside

x 1OO

Statistical Analysis

In the three p a t i e n t s (L.U., J.L., E.P.) in w h o m the study was done twice data were c o m p a r e d by S t u d e n t ' s t-test for p a i r e d o b s e r v a t i o n s . D i f f e r e n c e s were c o n s i d e r e d to be s i g n i f i c a n t at the 5 % level. RESULTS 3 H - D i g i t o x i n s p o t t e d d i r e c t l y on a paper strip or e x t r a c t e d from urine to w h i c h it had been d e l i b e r a t e l y added showed only one d i s t i n c t peak of r a d i o a c t i v i t y on paper c h r o m a t o g r a p h y . An i d e n t i c a l peak could be seen on c h r o m a t o g r a m s of urine e x t r a c t s from p a t i e n t s who had r e c e i v e d 80 ~Ci 3H-digitoxin. Fig. I shows the mass s p e c t r u m of the 3 H - d i g i t o x i n used. It is c h a r a c t e r i z e d by p r o m i n e n c e of ions m/e 374 (digitoxigenin), 357 ( d i g i t o x i g e n i n 17) and 339, and was t h e r e f o r e almost i d e n t i c a l both w i t h the m a s s s p e c t r u m of the pure d i g i t o x i n from B o e h r i n g e r - M a n n h e i m and the m a s s s p e c t r u m of d i g i t o x i n r e p o r t e d by B r o w n et al. (4). This e s t a b lishes the c h e m i c a l and r a d i o c h r o m a t o g r a p h i c i d e n t i t y of the l a b e l l e d comp o u n d w i t h digitoxin. A t y p i c a l radioc h r o m a t o g r a m of a c h l o r o f o r m e x t r a c t of urine from a p a t i e n t t r e a t e d w i t h 3H-dig i t o x i n is shown in Fig. 2. There is a p r o m i n e n t peak w i t h an Rf v a l u e identical to d i g i t o x i n , as well as a m i n o r peak. M a t e r i a l w i t h the same c h r o m o t o g r a p h i c b e h a v i o u r as d i g i t o x i n was i s o l a t e d from the u r i n e of one p a t i e n t t r e a t e d w i t h 3H-digitoxin. It was h y d r o l y z e d , d e r i v a t i z e d and a n a l y z e d by single ion m o n i t o r i n g of m / e 356. A p r e d o m i n a n t peak at a r e t e n t i o n time i d e n t i c a l to that of the

349

I00 ~5 8o

r131

357

147 >

203((5-203) 246(G-128)

339 (6-17) (0-35) [ /,01

O2

I/0' 160' JO' 2(]0'2{~0'240'2GO ' 280' 300 ~ 320 "" 340 "' ~-360 380 600 ~20 m/e Fig.

i. P a r t i a l m a s s spectrum of 3 H - d i g i t o x i n

Fig. 2. R a d i o c h r o m a t o g r a m of 3 H - d i g i t o x i n e x t r a c t e d from urine from a p a t i e n t (L.U. I) who received 80 uCi 3H-digitoxin: I first paper strip; II second paper strip with e f f l u e n t of first strip; III t h i n - l a y e r chromatogram. S = origin; F = solvent front; A = u n i d e n t i f i e d metabolites; B = digitoxin; C = d i g i t o x i n and the bis- and m o n o g l y c o s i d e s . A t b o t t o m s of radiochromatograms: C o - c h r o m a t o g r a p h e d u n l a b e l l e d reference compounds, a = digoxigenin m o n o d i g i t o x o s i d e ; b = digoxigenin; c = d i g o x i g e n i n b i s d i g i t o x o s i d e ; d = digoxin; e = digitoxin; f = d i g i t o x i g e n i n bisdigitoxoside; g = digitoxigenin; h = d i g i t o x i g e n i n m o n o d i g i t o x o s i d e ; k = digoxin, digitoxin, bis- and m o n o g l y c o s i d e s .

350

Table 2. Urinary excretion of metabolites in per cent of total urinary radioactivity after intravenous injection of 80 zCi 3H-digitoxin before and during oral therapy with 400 mg/day spironolactone. All the 8 patients, of whom 3 were investigated twice, received a daily maintenance dose of O.I mg digitoxin Digitoxin Patient

water soluble metabol,

digitoxin

bisdigit,

LU(1) JL(1) EP(1) MF RF JW

11.87 13.6 8.26 14.47 16.11 iO.O1

76.32 72.9 80.99 81.78 78.14 83.45

< 2 3.93 2.74 < 2 < 2 2.44

SE

12.39 1.19

78.93 1.9

monodigit,

genin

digoxin

bisdigit,

monodigit,

< 2 < 2 < 2 < 2 < 2

< 2

genin

unidentified metabolites 8.67 7.98 6.28 3.41 2.64 3.22 5.37 1.07

Digitoxin and spironolactone LU(2) JL(2) EP(2) AM RB

23.98 27.64 32.1 25.29 19.68

67.91 60.14 62.98 68.72 69.86

SE

25.74 2.05

65.92 1.86

< 0.O5

0.05

p

2.86 2.45 < 2 < 2 < 2

< 2 < 2 < 2 < 2

digitoxigenin-trimethylsilyl-1 standard w a s o b s e r v e d . T h i s f i n d i n g c o n f i r m s the i d e n t i t y of the m a t e r i a l isolated from urine with digitoxin. A n a l y s i s of u r i n e f r o m the p a t i e n t s not treated with spironolactone revealed t h a t o n a v e r a g e 79 % of t h e t o t a l u r i n a r y radioactivity c o n s i s t e d of d i g i t o x i n . In addition, patients excreted small amounts of d i g i t o x i g e n i n bisdigitoxoside. About 5 % of t h e r a d i o a c t i v i t y of t h e c h l o r o f o r m p h a s e c o u l d n o t be i d e n t i f i e d a n d 12 % of the r a d i o a c t i v i t y r e m a i n e d in t h e a q u e o u s p h a s e (Table 2). T h i s m e t a b o l i c p a t t e r n w a s t h e s a m e on d a y s 4 a n d 9 a f t e r i n j e c t i o n of 3 H - d i g i t o x i n . Spironolactone treatment significantly a l t e r e d the m e t a b o l i c p a t t e r n of t h e labelled glycoside: t h e e x c r e t i o n of d i gitoxin was reduced significantly from 79 % to 66 %, a n d the e x c r e t i o n of w a t e r s o l u b l e c o m p o u n d s c l e a r l y r o s e f r o m 12 % to 26 %. T h e r e w a s no a l t e r a t i o n in the a m o u n t of u n i d e n t i f i e d compounds eliminated, n o r in the a m o u n t s of t h e b i s - or monoglycosides (Table 2). T h i s p a t t e r n , too, w a s t h e s a m e on d a y s 4 a n d 9 a f t e r i n j e c t i o n of 3 H - d i g i t o x i n . The terminal h a l f - l i f e of s e r u m r a d i o a c t i v i t y was 256.4 ± 8.4 h (n = 6) b e f o r e a n d 204.7

< 2 < 2

2.13

2.41 8.35 3.68 4.31 7.28 5.21 1.12

±7.8 h (n = 5) d u r i n g t r e a t m e n t w i t h spironolactone. In the t h r e e p a t i e n t s in w h o m the s t u d y w a s d o n e t w i c e t h e d i f f e r e n c e in the h a l f - l i f e w a s s t a t i s t i cally significant (p < 0 . O 1 ) . T h e t i m e c o u r s e of s e r u m r a d i o a c t i v i t y in o n e of these patients before and during spiron o l a c t o n e t r e a t m e n t is s h o w n in Fig. 3. A s c a n be s e e n f r o m T a b l e 3 t h e r e w a s n o difference in t h e m e a n d a i l y u r i n e v o l ume, t h e c u m u l a t i v e u r i n a r y e x c r e t i o n o f t r i t i u m or t h e r e n a l c l e a r a n c e of t r i t i u m before and during treatment with spironolactone. By the 2Oth day after inject i o n of t h e l a b e l l e d d r u g 4 4 . 8 % of t h e radioactivity h a d b e e n e x c r e t e d in u r i n e of w h i c h 35.4 % c o n s i s t e d of u n a l t e r e d digitoxin (n = 3). T h e l a t t e r v a l u e agrees well with that reported by Lukas (18), w h o f o u n d t h a t 28 % of a s i n g l e o r a l d o s e of d i g i t o x i n w a s e x c r e t e d in u r i n e as u n a l t e r e d d r u g o v e r a 21 d a y period. Spironolactone d i d n o t a l t e r the c u m u l a t i v e u r i n a r y e x c r e t i o n of r a d i o a c t i v ity, w h i c h a m o u n t e d to 46.2 % (n = 2) of the d o s e of t r i t i u m , d u r i n g t h e 19 d a y s a f t e r its i n j e c t i o n . T h e r e n a l c l e a r a n c e of t r i t i u m , w h i c h w a s in t h e s a m e r a n g e as the d i g i t o x i n

351 Table 3. Summary of pharmacokinetic results Dig±toxin Patient

n.u.

(i) J.L.

(I) E.P.

t i/2 a t i/2 a mean daily serum urine urine excretion (h) (h) (ml, x ± SE)

n

256.7

81.9

234.1

288.8

cummulative urinary excretion of tritium (% of dose) ist day 7th day iOth day

renal tritium clearance ist day

7th day

iO day min)

14

7.8

35.0

43.4

1.6

1.4

1.4

1.5

1921.0 ± 142.9

16

4.1

19.7

30.0

1.3

1.4

1.5

1.6

4.0

25.7

35.0

1•i

1.2

2175.4 f

--C1b (ml/

266.5

312.8

2038.4 _+ 103.3

ii

M.F.

231.O

236.3

1718.7 ±

69.4

10

R.F.

256.7

1908.5 +_ 98.7

17

6.7

31.2

39.9

1.8

1.4

1.6

1.5

J.W•

238.9

2214.3 ± 166.2

8

8.2

29.5

38.6

1.5

1.6

1.4

1.7

SE

256.4 + 8.4

6.2 + 0.9

28.2 _+ 2.6

37.5 + 2.4

1.5 ± 0.i

1.4 ± O.1

1.5 ± O.i

1.5 + O.i

(1)

1991.2 ± 111.4

1.4 1.5

Dig±toxin + Spironolactone n.u.

(2) J.L.

(2) E.P.

(2)

190.9

216.3

210.O 182.4

198.3

2310.3 i

81.i

17

5.9

33.7

42.7

1.4

1.6

1.6

1.8

2034.4 ± 277.2

13

6.1

32.7

33.5

1.6

1.7

1.5

1.7

1880.3 ±

40.6

16

5.1

27.6

38.0

i.O

0.8

I.o

1.9

12

2.8

23.5

39.3

I.I

1.3

1.3

1.6

6

8.8

32.1

1.8

1.7

5.7 ± 1.O

29.9 ± 1.9

1.4 ± 0.2

I .4 ± 0.2

A.M.

223.6

1985.6 ± 104.9

R•B•

216.6

1690• 4 ±

x

204.7 ± 7.8

SE

1974.4 +- 124.8

ahalf-life of radioactivity

46.2

38.4 ± 1.9

1.8 1.4 ± 0.2

i .8 ± O.i

btotal body clearance of radioactivity

clearance calculated by S t o r s t e i n (31), and the total body clearance of r a d i o activity was not altered significantly by s p i r o n o l a c t o n e treatment. However, there was a trend for total body cleara n c e to be i n c r e a s e d by spironolactone. T h e v o l u m e of d i s t r i b u t i o n of t r i t i u m was significantly decreased by s p i r o n o l a c t o n e f r o m 3 5 . 5 7 ± 2 . 2 1 (n = 3) to 3 0 . 0 3 ± 0 . 8 3 1 (n : 3) (p < 0 . 0 5 ) . The protein binding of radioactivity determined after 5 days remained cons t a n t , i.e. 9 3 . 3 ± 0 . 2 4 % (n = 6) b e f o r e a n d 9 3 . 0 ± 0 . 4 % (n = 5) d u r i n g s p i r o n o lactone treatment• T h i s d e g r e e of b i n d i n g is in r a n g e r e p o r t e d by others (15, 19). It w a s f o u n d to be t h e s a m e (93 %) on t h e 2nd and 15th days after injection of t h e label• Total protein (7.19 ± 0.31 g%) a n d albumen (3.82 ± 0 . 2 3 g%) r e m a i n e d constant throughout the study. The serum potassium, w h i c h w a s in t h e n o r m a l r a n g e in a l l p a t i e n t s , remained unchanged during treatment with spironolactone.

nCi/ml 8.0~- . _ _ . 3H_digitoxin 5.0~ × x 3H- dig±toxin + spironolactone

1.0L

"~

*"~-, ~.q~--~ . . . . . .

I

I

t

I

2/,/,B 96

I

I

]&&

I

t

l

I

I

I

I

I

.

I

I

I

f

I

l

192 240 288 335 38/, 432h&80

Fig. 3. Serum levels of radioactivity after intravenous administration of 80 ~Ci 3H-dig±toxin in a patient (L.U.) before (-) and during treatment with spironolactone (*--~)

352 DISCUSSION

There is an apparent discrepancy between the results of Okita et al. (21) and The metabolism of digitoxin has been the present findings. Besides digitoxin studied by several investigators (3, 21, itself, only unknown chloroform-soluble 23, 34). Repke (22) demonstrated that the compounds (about 5 %) and water soluble predominant pathway in rats led from the metabolites (about 12 %) in the urine parent glycoside by stepwise hydrolysis could be demonstrated in the studies via the bis- and monodigitoxoside to the described here. The nature of the water genin. In addition, there appeared to be soluble compounds was not established. activity of 12-~-hydroxylase leading to They might have been unconjugated polar digoxin-related compounds and digoxin itmetabolites and conjugation products self (3). The metabolic conversion of with glucuronic and sulfuric acids, as digitoxin to digoxin was thought to have suggested by V~hringer and Rietbrock important pharmacokinetic implications (34), although Lukas and Peterson (20), (8, 12, 13), since the half-life of using a highly specific double dilution digoxin, in contrast to digitoxin, is method, were unable to find digitoxigenin markedly prolonged in renal failure. after acid hydrolysis of water soluble Quantitative aspects of digitoxin metametabolites from the urine of patients treated with digitoxin. bolism in man were first investigated by Okita et al. (21), who found that There is a suggestive evidence that only 6 - 10 % of a dose of biosynthetienzyme induction can affect digitoxin cally labelled 14C-digitoxin was excreted disposition in man. Administration of as unaltered drug. There are indications phenylbutazone, a known inducer of certhat digitoxin itself may be the major tain hepatic hydroxylation enzymes, led urinary excretory product (17) but the to a reduction in steady state digitoxin methods employed have not permitted chemi- levels measured by 86Rb uptake into red cal identification of digitoxin nor the cells (26). Spironolactone has been found demonstration of small amounts of digoxin, to increase pentobarbital oxidation, Using a highly specific and sensitive ethylmorphine N-demethy!ation, hexobardouble isotope dilution method Lukas bital oxidation and 3,4-benzpyrene (18) examined urine from three patients hydroxylation by the drug metabolyzing treated with a single dose of digitoxin system in liver microsomes from rats (11, and found only digitoxin itself and no 27, 32). Experiments in ~ v o in rats redigoxin-related compounds during daily vealed that spironolactone increased collections lasting for more than two hepatic metabolism of digitoxin (6). In weeks. Interestingly, the rat produced the same species we have shown that the digoxin and digoxigenin bisdigitoxoside metabolic pattern of digoxin was affected (4 % and 7 % of total urinary radioacfor as long as 25 days after cessation tivity, respectively) when it received of spironolactone treatment (36). This the same 3H-digitoxin employed in the long time span makes it extremely unlikepresent study I. This demonstrates that ly that the observed effect was caused by conversion of digitoxin to digoxin can the remaining portion of the drug or its take place in some species and so conmetabolites, since their half-life in the firms an earlier report by Repke (22). rat is only a few hours (24). The present results indicate that afIn man spironolactone has been found ter administration of 3H-digitoxin, 79 % to reduce the half-life of antipyrine, of the urinary radioactivity consisted which was interpreted as evidence of enof the parent compound. This finding is zyme induction (33). On the other hand based on two major lines of evidence: spironolactone seemed not to alter terI. The 3H-digitoxin administered to minal plasma half-life, rate of urinary the patients was analyzed by chromatoexcretion or metabolic pattern of digoxin graphy and found to be a homogenous suband 4'''-methyldigoxin in man (1, 36). stance. More importantly, the substance However, the large differences in kinetwas identified as digitoxin by mass specics from one glycoside to another pretrometry. clude utilization of these data as pre2. Material obtained from urine and dictors for the present study. Steiness chromatographically identical to digi(30) showed in patients with chronic toxin was found by mass spectrometric congestive heart failure that spironoanalysis to yield digitoxigenin after lactone significantly increased the hydrolysis. From these observations it plasma concentration and decreased the is clear that the labelled compound was renal clearance of digoxin. digitoxin and that the predominant uriThe results presented here show that nary excretory product was digitoxin. spironolactone decreased the half-life of serum radioactivity and the urinary elimination of unchanged digitoxin in man. The excretion of water soluble i Unpublished observation metabolites was correspondingly in-

353

creased. Since the cumulative urinary excretion of t r i t i u m w a s n o t i n c r e a s e d , even though the half-life of s e r u m r a d i o activity was decreased, greater fecal excretion of d i g i t o x i n and/or its metabolites seemed very likely. Since both the decrease in t h e v o l u m e of d i s t r i b u t i o n (Vd~) and the increased r a t e of m e t a b o l i s m could have produced the decrease in t h e h a l f - l i f e of s e r u m radioactivity, an a t t e m p t w a s m a d e to analyze the relative contribution of e a c h of t h e m to t h e s e a l t e r a t i o n s . For this analysis, tl/2 was calculated as tl/2

:

in

2 x VdC--T C1

It w a s 2 7 3 . 5 ± 1 3 . 9 h o u r s b e f o r e a n d 193.3 ±15.9 hours during spironolactone treatment in p a t i e n t s L.U., J.L. and E.P. The values were identical when tl/2 was calculated as tl/2

-

in 2 y

and showed that spironolactone reduced t l / 2 b y 2 9 . 3 %. To evaluate the relative contribution of Vd~- ~ to t h e d e c r e a s e in t i / 2 , t h e latter was calculated using___VdC-f of t h e spironolactone p e r i o d a n d C1 ( t o t a l b o d y clearance) of t h e c o n t r o l p e r i o d . T h i s t l / 2 w a s 232 h o u r s , i.e. 15 % s h o r t e r than the control tl/2 of 273.5 hours. T h u s , t h e c h a n g e in VdC--~ a c c o u n t e d for a b o u t h a l f of t h e c h a n g e in t l / 2 b e t w e e n the control and spironolactone periods. Conversely, when tl/2 was calculated by u s i n g t h e VdC--[ of t h e c o n t r o l p e r i o d a n d C1 of t h e s p i r o n o l a c t o n e period, tl/2 w a s 2 3 0 h o u r s . It a p p e a r s t h e r e f o r e that t h e c h a n g e s in V d ~ and C1 after adminis t r a t i o n of s p i r o n o l a c t o n e each contrib u t e d a b o u t h a l f of t h e o v e r a l l d e c r e a s e in t l / 2 . Induction of h e p a t i c m e t a b o l i s m by spironolactone accounts for the altered pattern of urinary metabolites and part of the decrease in t l / 2 . T h e i n t e r a c t i o n between spironolactone and digitoxin app e a r s to be of m i n o r c l i n i c a l importance, since the changes observed were within t h e r a n g e of i n t e r - i n d i v i d u a l variability of d i g i t o x i n kinetics.

Acknowledgements.

We gratefully acknowledge support for one of us (KEW) by the Paul MartiniStiftung, Frankfurt/Main, Germany. The work was supported in part by NIH Grant GM 15431.

REFERENCES i. Abshagen, U., Rennekamp, H., Kuhlmann, J.: Pharmacokinetics of 4'''-methyldigoxin after pretreatment with spironolactone in man.

Naunyn Schmiedeberg's Arch. Pharmacol. Suppl. 285, R i (1974) 2. Anton, A.H., Sayre, D.F.: A study of the factors effecting the aluminium oxide-trihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. exp. Ther. 138, 360 - 375 (1962) 3. Ashley, J.J., Brown, B.T., Okita, G.T., Wright, S.E.: The metabolites of cardiac glycosides in human urine. J. biol. Chem. 232, 315 322 (1958) 4. Brown, P., Br~schweiler, F., Pettit, G.R., Reichstein, T.: Field ionization mass spectrometry. III: cardenolides. Org. Mass. Spectrum 5, 573 - 597 (1971) 5. Buck, S.H., Lage, G.L.: Possible mechanism of the prevention of digitoxin toxicity by spironolactone in the mouse. Arch. int. Pharmacodyn. 189, 192 - 197 (1971) 6. Castle, H.C., Lage, G.L.: Excretion of 3Hdigitoxin and its metabolites following spironolactone pretreatment in rats. Drug Metab. Dis. i, 590 - 597 (1973) 7. Doherty, J.E., Perkins, W.H., Mitchell, G.K.: Tritiated digoxin studies in human subjects. Arch. int. Med. 108, 531 - 539 (1961) 8. Doherty, J.E., Hall, W.H., Murphy, M.L., Beard, O.W.: New information regarding digitalis metabolism. Chest 59, 433 - 437 (1971) 9. Douglas, J.G., Hollifield, J.W., Liddle, G.W.: Treatment of low-renin essential hypertension. J. Amer. med. Ass. 227, 518 - 527 (1974) io. Falkner, F.C., Fr61ich, J.C., Watson, J.T.: Mass spectrometry of the trimethylsilyl derivatives of some cardiac aglycones and monoglycosides. Org. Mass. Spectrum 7, 141 - 153 (1973) ii. Hamrick, M.E., Zampaglione, N.G., Stripp, B., Gilette, J.R.: Investigation of the effect of methyltestosterone, cortisone and spironolactone on the hepatic microsomal mixed function oxydase system in male and female rats. Biochem. Pharmacol. 22, 293 - 310 (1973) 12. Jelliffe, R.W., Buell, J., Kalaba, R., Sridhar, R., Rockwell, R., Wagner, J.G.: An improved method of digitoxin therapy. Ann. int. Med. 72, 453 - 464 (1970) 13. Jelliffe, R.W.: Discussion of the paper: Some aspects of the distribution and disposition of digitoxin in man (Lukas, D.S.). Ann. N.Y. Acad. Sci. 179, 359 - 361 (1971) 14. Katzung, B.G., Meyers, F.H.: Biotransformation of digitoxin in the dog. J. Pharmacol. exp. Ther. 154, 575 - 580 (1966) 15. Kuschinsky, K.: Bestimmung der EiweiBbindung verschiedener Herzglykoside mittels Sephadex ®Gelfiltration. Naunyn schmiedeberg's Arch. Pharmak. exp. Path. 259, 394 - 399 (1968) 16. Lage, G.L., Spratt, J.L.: 3H-Digoxin metabolism by adult male rat tissues in vitro. J. Pharmacol. exp. Ther. 149, 248 - 256 (1965) 17. Lahrtz, Hg., Reinold, H.M., Van Zwieten, P.A.: Serumkonzentration und Ausscheidung von 3HDigitoxin beim Menschen unter normalen und pathologischen Bedingungen. Klin. Wschr. 47, 695 - 700 (1969) 18. Lukas, D.S.: Some aspects of the distribution

354

19.

20.

21.

22.

23.

24.

25.

26.

27.

and disposition of digitoxin in man. Ann. N. Y. Acad. Sci. 179, 338 - 359 (1971) Lukas, D.S., DeMartino, A.G.: Binding of digitoxin and some related cardenolides to human plasma proteins. J. clin. Invest. 48, 1041 - 1053 (1969) Lukas, D.S., Peterson, R.E.: Double isotope dilution derivative assay of digitoxin in plasma, urine and stool of patients maintained on the drug. J. clin. Invest. 45, 782 - 795 (1966) Okita, G.T., Kelsey, F.E., Talso, P.J., Smith, L.B., Geiling, E.M.K.: Studies on the renal excretion of radioactive digitoxin in human subjects with cardiac failure. Circulation 7, 161 - 168 (1953) Repke, K.: Die Bis- und Mono-digitoxoside des Digitoxigenins und Digoxigenins: Metaboliten des Digitoxins. Naunyn Schmiedeberg's Arch. exp. Path. Pharm. 237, 155 - 170 (1959) Repke, K.: Metabolism of cardiac glycosides. Proc. First. Internat. Pharmacol. Mtg., Stockholm 1961, 3, 47 - 73 Oxford: Pergamon Press 1963 Sad@e, W., Abshagen, U., Finn, C., Rietbrock, N.: Conversion of spironolactone to canrenone and disposition kinetics of spironolactone and canrenoate-potassium in rats. Naunyn Schmiedeberg's Arch. Pharmacol. 283, 303 318 (1974) Selye, H., Krajny, M., Savoie, L.: Digitoxin poisoning: prevention by spironolactone. Science 164, 842 - 843 (1969) Solomon, H.M., Reich, S., Spirt, N., Abrams, W.B.: Interactions between digitoxin and other drugs in vitro and in vivo. Ann. N.Y. Acad. Sci. 179, 362 - 368 (1971) Solymoss, B., Classen, H.G., Varga, S.: Increased hepatic microsomal activity induced by spironolactone and other steroids. Proc. Soc. exp. Biol. (N.Y.) 132, 940 - 942 (1969)

28. Solymoss, B., Toth, S., Varga, S., Selye, H.: Protection by spironolactone and oxandrolone against chronic digitoxin or indomethacin intoxication. Toxicol. appl. Pharmacol. 18, 586 - 592 (1971) 29. Stahl, E.: Thin-layer chromatography, p. 870, 2nd ed., Berlin, Heidelberg, New York: Springer-Verlag 1969 30. Steiness, E.: Renal tubular secretion of digoxin. Circulation 50, 103 - 107 (1974) 31. Storstein, L.: Studies on digitalis. I. Renal excretion of digitoxin and its cardioactive metabolites. Clin. Pharmacol. Ther. 16, 14 - 24 (1974) 32. Stripp, B., Hamrick, M.E., Zampaglione, N.G., Gilette, J.R.: The effect of spironolactone on drug metabolism by hepatic microsomes. J. Pharmacol. exp. Ther. 176, 766 - 771 (1971) 33. Taylor, S.A., Rawlins, M.D., Smith, S.E.: Spironolactone - a weak inducer in man. J. Pharm. Pharmacol. 24, 578 - 579 (1972) 34. VShringer, H.F., Rietbrock, N.: Metabolism and excretion of digitoxin in man. Clin. Pharmacol. Ther. 16, 796 - 806 (1974) 35. Watson, J.T., Pelster, T.R., Sweetman, B.J., FrSlich, J.C., Oates, J.A.: A display oriented data system for multiple ion detection with gas chromatography-mass spectrometry in quantifying biomedieally important compounds. Anal. Chem. 45, 2071 - 2078 (1973) 36. Wirth, K.E., Fr61ich, J.C.: Effect of spironolactone on excretion of 3H-digoxin and its metabolites in rats. Europ. J. Pharmacol. 29, 43 - 51 (1974) Dr. K.E. Wirth Pharmakologisches Institut der Univ. MoorenstraBe 5 D - 4000 D~sseidorf Federal Republic of Germany

Metabolism of digitoxin in man and its modification by spironolactone.

Europ. J.clin. Pharmacol. 9, 345-354 (1976) © by Springer-Verlag 1976 Metabolism of Digitoxin in Man and Its Modification by Spironolactone K. E. W~t...
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