Naunyn-Schmiedeberg's

Archivesof

Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 281- 287 (1977)

Pharmacology 9 by Springer-Verlag 1977

Effect of Enterohepatic Circulation on the Pharmacokinetics of Spironolactone in Man U. ABSHAGEN*, U. VON GRODZICKI, U. HIRSCHBERGER**, and H. RENNEKAMP Institut ffir Klinische Pharmakologie und Medizinische Klinik, Klinikum Steglitz, Freie Universitfit Berlin, Hindenburgdamm 30, D-1000 Berlin 45

Summary. 100 gCi 3H-spironolactone together with

Key words." Spironolactone - Pharmacokinetics -

300 mg unlabeled drug were administered orally to 8 patients, 24 h after choledochotomy with subsequent complete drainage of bile for a further 96 h by means of a special tube. To evaluate the role of entero-hepatic circulation, the excretion of tritiated substances in bile was investigated and their kinetics in plasma and urine were compared with that of controls. The 3H-activity in plasma declined monoexponentially with tl/2:1.80 + 0.12 d from 12 h after patients received the drug. This half life was not different from that of controls in the terminal slope, 96-144 h after administration. However, between 2 4 - 9 6 h the elimination was significantly delayed (tl/2" 2.79 _+ 0.29 d) in controls. This was the result of entero-hepatic cycling of spironolactone metabolites with high biliary clearance. 5.4-32.7 ~o of the dose was excreted in bile within 4 days; of this 5 0 - 70 ~ consisted of polar material, 1 0 - 20 ~ of canrenone, 5 - 15 ~ of 6/~-OH-7~-methylsulfinyl-spirolactone and 3 - 1 0 % of 6/~-OH-7e-thiomethyl-spirolactone. In urine, identical percentages of the dose were excreted in patients and controls. TLC-examination of the lipophilic fraction revealed less sulfoxidized metabolites while, at the same time, significantly higher amounts of 6/LOH-Te-thiomethylspirolactone and canrenone were eliminated in patients with biliary fistula. Thus, interruption of enterohepatic circulation had resulted in a shift of metabolic pathways of this drug in spite of an unchanged overall metabolic rate. Furthermore, the experiments allow a logical interpretation of the kinetics of 3H-activity in plasma after administration of 3H-spironolactone in normal man.

Enterohepatic circulation Metabolism.

S e n d offprint requests to." U. Abshagen, Abteilung Klinische Pharmakologie, Boehringer Mannheim GmbH, Sandhofer Strasse 116, D-6800 Mannheim 31, Federal Republic of Germany ** This paper includes parts of the thesis of U. Hirschberger *

Urinary excretion -

INTRODUCTION During studies on the pharmacokinetics of spironolactone in normal subjects (Abshagen et al., 1976b), a delayed elimination of total radioactivity from plasma up to 96 h after administration of 3H-spironolactone was observed, with a subsequent striking acceleration. This kinetic phenomenon could not be ascribed to self-induction or to saturation processes. Since we know from animal experiments that spironolactone or its metabolites are cleared via the biliary route to a considerable extent (Sadae et al., 1972, 1974), the delayed elimination of the drug from plasma within the first days might occur as a consequence of an enterohepatic circulation. To test this hypothesis, we studied the influence of interruption of the enterohepatic circulation by complete biliary drainage on the pharmacokinetics of spironolactone in patients.

METHODS AND MATERIALS Procedure. Eight patients who had to undergo cholecystectomy and choledochotomy in order to remove gallstones, gave their informed consent to the following investigation. After revision of the common bile duct a Fogarty-catheter (Nr. 2 - 3) was placed in the distal part of the duct before the papilla Vateri, while another simple catheter was placed in the proximal part of the duct for biliary drainage. Twentyfour hours after surgery, just before starting the experiments, the balloon of the Fogarty-catheter was filled with water (0.751.25 ml) to block the flow towards the duodenum. The tightness of the system was checked by retrograde X-ray cholangiography through the proximal catheter (Fig. i). Then, approximately 100 gCi 3H-spironolactone together with 300 mg unlabeled spironolactone (6 dragees Aldactone 50| Boehringer, Mannheim) were given orally

282

Nannyn-gchmiedeberg's Arch. Pharmacol. 300 (1977)

Fig. 1 X-ray of the Fogarty-catheter in typical position, which was filled with Angiografin| (Schering AG, Berlin) for visualisation (left) and additional retrograde cholegraphy via the proximal catheter (right): the flow into the duodenum is blocked by the balloon of the Fogartycatheter

Table 1. Initials, age, sex and preoperative laboratory data of patients investigated. Abbreviations for histological diagnosis: C = cholostasis; CN = celt necrosis; CP = chronic pericholangitis; FD = fatty degeneration; PI = periportal infiltration; + = slight; + + = moderate; + + + = pronounced. In E.A- and G.D. the liver appeared normal with respect to macroscopical criteria and histological examination was not performed Patient

Age (yrs.)

Sex

Liver histology

GOT (U/l)

Bilirubin (gmol/1)

Quick (~)

Q.G.

61

fem.

CP++, PI+, FD++

12

15

155

18.93

96

77

L.B.

52

fern.

FD+, PI+

34

101

200

10.31

100

61

D.O.

70

male

C + +, C P + + , CN+, PI+

37

62

323

72.26

100

87

H.K.

50

fern.

F D + +, PI+

10

22

139

9.46

100

90

I.H.

71

male

C+, CP+ + +, CN+, FD+

21

26

656

103.01

E.A.

83

male

-

R.A.

66

male

Ct, FD+, CN+, PI+ + , CP+ + +

G.D.

64

fem.

-

GPT (U/l)

Alcal. phosph. (U/I)

170

34

84

134.36 881

20.45

1098

36.00

Creatinine (gmol/1)

103 83

85 85

100

92

to the subjects who had eaten no breakfast. The labeled spironolactone was dissolved in 2 ml abs. ethanol which was subsequently diluted with 200 ml warm tea. The dose of the tritiated substance that was actually ingested was determined by subtraction of losses in the drag container. Venous blood was drawn in heparinized tubes at 10, 15, 30 and 60 rain and at 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84 and 96 h after administration. Urine was collected after spontaneous micturition at short intervals during the first 24 h and later on at daily intervals. Bile was collected from 0 - 4 , 4 - 8 , 8 - I 2 , 12-36, 36-48, 4 8 - 7 2 and 72--96 h after administration. Clinical and laboratory data of the patients are given in Table 1. Control subjects, who were also given 100 gCi ~H-spironolactone, but 500 mg unlabeled drug, are taken from Abshagen et al. (1976b).

Native bile (0.2 ml) was mixed with 0.2 ml of H202 (30 v/v). After bleaching for 24 - 4 8 h, 10 ml of scintillation fluid (10 ~ naphthalene, 0.98 diphenyloxazole, 0.02~o 2,2-p-phenylene-bis-(5-phenyloxazole) in dioxane) was added and the total tritium activity was determined. Another 5 ml of native bile was extracted 3 times with twice its volume of CH_~Clz and aliquots of both phases were taken for counting. The remaining main portions of the bile specimens were dialyzed through latex membranes (preservative 'Blausiegel R 3') against 3 times their volume of CH2Cla for 24 h, after which time the final partition-as determined by direct extraction-was reached. The CH2C12 layers were subsequently evaporated to dryness in vacuo at 35~ the residues were taken up with methanol: dichloromethane (1 : 1) and aliquots of 50 p.l were applied to thin layer plates for TLC.

Analyses. The measurement of telal radioactivity in plasma and urine, the separation into a CHzClz extractable and a polar fi'action in plasma and urine and the thin-layer-chromatographic (TLC) analysis of the CH2C12 extractable fraction in urine were performed as described in detail by Abshagen et al. (1976b).

Calculations. The half-lives of the log linear phases of the concentration curves in plasma were calculated (Olivetti programma) using a program which ascertained the best fitting value for hi2 = ln2: k~ by means of the linear regression procedure. Analysis of

U. Abshagen et al. : Spironolactone Kinetics in Biliary Fistula Patients

283

Table 2. Pharmacokinetic parameters of total radioactivity after oral administration of 3H-spironolactone in biliary fistula patients. Controls (n = 5) from Abshagen et al. (1976b) Patients

Urinary recovery 0-96 h

Biliary recovery 0-96 h

Q.G. L.B. D.O. H.K. I.H. E.A. R.A. G.D.

44.50 73.29 59.00 61.32 54.75 54.42 33.29

21.00 19.23 5.35 12.64 7.36 ~19.30 11.93 32.70

2 __+S~

54.42 4.76

16.19 3.11

Plasma yma• (% dose/1 plasma)

/max (min)

tl/2 12--96 h (days)

2.95 6.03 3.48 2.08 2.95 4.36 2.14 1.82

60 30 60 120 45 30 240 240

2.38 1.47 1.69 1.73 1.44 1.67 2.17 1.79

3.56 0.53

94.29 37.71

1.80 0.]2

2.44 0.17

32.00 2.55

2.79 0.26

Controls : 9~ + S~

52.80 2.28

statistical differences were made with Student's t-test for unpaired experiments. Substances. Aldactone 50 | tablets (Boehringer, Mannheim) and 3H-20,21-spironoIactone, specific activity 3.5 Ci/mmol (Searle & Co., Chicago) were used. The original radiochemical purity of aH-spironolactone was approx. 75 %. After chromatographic cleanup in 2 solvent systems the radiochemical purity was more than 95 %. Reference siabstances were spironolactone, canrenone, and canren9 K, (crist., purissimum; Boehringer, Mannheim). The 3-(3-oxo7~-methylthio-17~-hydroxy-4-androsten-17c~-yl)-propionic acid 7lactone was prepared according to Karim and Brown (1972). 3-(3oxo- 7c~-methylsulfinyl- 17fl-hydroxy-4-androsten- 17c~-yl)-propionic acid v-lactone; 3-(3-oxo-7c~-methylthio-6fl,17fl-dihydroxy-4-androsten-17e-yl)-propionic acid "t-lactone and 3-(3-oxo-7e-methylsulfinyl6/3,17fl-dihydroxy-4-androsten-17c~-yl)-propionicacid y-lactone were prepared microbiologically 1 according to Marshek and Karim (1973). By oxidation of the latter compound with m-chlorobenzoic acid, the 3-(3-oxo-7~-methylsulfonyl-6fl,17fl-dihydroxy-4-androsten-i7c~-yl)-propionic acid 7-1actone was synthesized (Abshagen et al., 1976a).

dosing onwards, tritium concentrations fell re9 exponentially with a half life of 1.796 + 0.12 days which was not statistically different (P < 0.30) from that of normal persons during the terminal slope, 96-144 h after dosing. In contrast, between 2 4 - 9 6 h, the elimination of total radioactivity was significantly (P < 0.0025) delayed in normal subjects as compared to biliary fistula patients (Fig. 2). For ethical reasons, the balloon of the Fogarty-catheter could only be held in a blocking position for a maximum of 96 h. In 2 patients, blood concentrations were followed up to 144 h and they declined with a half life similar to that determined between 12-96 h. In urine, 54.4 + 4.8 % of the given dose was excreted within 4 days-almost the same amount as in

4,~

RESULTS

A. Kinetics of Total Radioactivity in Plasma, Urine and Bile After oral administration of 3H-spironolactone maximum concentrations of tritium activity in plasma were reached at 94 _+ 38 rain amounting to 3.56 _+ 0.53 % of the dose/1 (Table 2). The later mean peak observed in patients and the larger deviations as compared to normals might be due to different degrees of postoperative paralysis of the intestine. From 12 h after 1 Thanks are due to Dr. Ktichler, Department of Microbiology, F U Berlin, for his support in performing the microbiological procedures

"O

t112 =1,80

2,0

1,0

.

._~...,~~LI,,~ i " ' ' Z L k=. . ~ ~

.tl/2 =1,70

0,4-

t112 = 2,79

~, ~6_~

0,2

"-... 12

;'4

36

48

72

96

120

144 h

Fig.2. Kinetics of total radioactivity in plasma of biliary fistula patients (n = 8) ( O - - O ) and of normal test subjects (n = 5) (9 - 9 (Abshagen et al., 1976b) after oral administration of 3H-spironolactone. Means _+ S.E.M.

Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

284 60

_

~5

504O "5

9

3C

t~

~5

20 10

O

5

0

6 12

2/.

36

48

72

h

96

e~

Fig.3.

Cumulative excretion of total radioactivity in urine (9 and bile (O . . . . O) after oral administration of ~Hspironotactone in biliary fistula patients. Means • S.E.M.

controls without biliary drainage (Table 2, Fig. 3). The half life of the urinary excretion rate which decreased monoexponentially with hi2 of 1.23 __ 0.24 d was not significantly longer (P < 0.1) than in controls. Large differences between individuals in the extent of biliary excretion of the drug were observed. A mean fraction of 16.2 4_-_3.1% of the dose could be recovered in bile during the experimental period (Fig. 3). Thereby the excretion of tritium activity proved to be largely independent of bile flow (Table 3). The patient with the highest flow rate (I.H.) excreted only 7.36 % within 4 days, whereas the highest biliary output of the drug was 32.7% of the dose (G.D.) excreted in less than half the volume of bile in I.H. Obviously, the extent of biliary excretion was inversely correlated to the (histologically proven) severity of liver damage. Thus the patients with histological signs of cholostasis (D.O., I.H., R.A.) showed the lowest biliary excretion of tritiated material. The different excretory capacity of the liver is also demonstrated by the ratios of tritium concentrations in bile and plasma. Maximum bile/plasma ratios differ by a factor of more than 10 between patients. In 2 patients, more than 30-fold concentrations as compared to plasma were measured in bile during the first day after dosing, with subsequent decline of the respective ratios (Fig. 4). The mean half life of the biliary excretion rate was 0.92 + 0:16 days and thus was distinctly shorter than the half life of tritium concentrations in plasma.

B. Composition of Radioactivity Eliminated by Bile and Urine Approx. 60 ~ of the radioactive material in urine and bile (Figs. 5, 6) and 50 % of that in plasma consisted

T d

9

9

u.Z

_d

22 e~ (D

I / l l l l l l

U. Abshagen et al. : Spironolactone Kinetics in Biliary Fistula Patients

285

3H-c~r~centration bile plasma 4O

3

i Fig. 4 Time course of the quotients of concentrations bile/plasma after oral administration of 3H-spironolactone in biliary fistula patients. (O O) mean curve

L.B. .G. G.D. HK. R.A.

1'2

2~4

3'6

~8

7'2

E,A. D.Q I.H ~6 h

almost identical in biliary fistula patients and in normal subjects within 24 h of the administration of the drug. In contrast, statistically significant differences were observed in the lipophilic fraction (Fig. 6). Apparently, all the sulfoxidation reactions were diminished in biliary fistula patients, whereas the formation of 6/LOH-7~-thiomethyl-spirolactone and the dethioacetylation yielding canrenone were enhanced. Thus, the interruption of the enterohepatic circulation had resulted in a shift of the metabolic pathways of spironolactone in spite of the fact that the overall metabolic rate was unchanged.

[}--

o

(D

IX_

2 1

DISCUSSION I

0 /~ 8 12 h 2/~ Fig. 5. Cumulative excretion of total radioactivity (O O)0 CH2C12 non-extractablefraction (v1 ---D), canrenone (e O), 6]LOH-7~-methyl-sulfinyl-spirolactone(A A) and 6]?-OH-7c~thiomethyl-sI0irolactone(O O) in bile within 24 h after administration of aH-spironolactone.Mean + S.E.M. (n = 8)

of polar material which was not extractable with CH2C12. The remaining constituents in bile were canrenone, 6B-OH-7c~-methylsulfinyl-spirolactone and 6/3-OH-Tc~-thiomethyl-spirolactone. Of these constituents the canrenone portion decreased while 6/3-OH7~-methylsulfinyl-spirolactone increased during the time in which TLC analysis was possible ( 0 - 2 4 h). The 6/?-OH-7~-methylsulfonyl-spirolactone and the non-hydroxylated sulfoxide could not be detected in bile (Fig. 5). In urine, the excretion of total radioactivity as well as the amount of the polar fraction were

In order to investigate the influence of enterohepatic circulation on the pharmacokinetics of spironolactone, a method was designed which allowed complete interruption of the enterohepatic circulation. Results of this procedure, of course, do not allow any conclusion to be drawn about the dynamic process of enterohepatic circulation. Furthermore, it must be borne in mind that the experiments could only be performed in patients whose liver function was impaired due to extrahepatic cholostasis of different degree and duration. This might account for the large differences found between individuals in biliary concentration power and excretion of the drug. It also may be assumed that the biliary excretion capacity for spironolactone and its metabolites will be higher in normal subjects. A high concentration gradient against plasma was established during biliary excretion. The fact that the

286

Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

O -O 15

0a

total 3H-activity

40-

~

C H2C12 non extractable

H

3

10 i~ 6

5

4

3

2

1

5'

p< 0,10

p

Effect of entekohepatic circulation on the pharmacokinetics of spironolactone in man.

Naunyn-Schmiedeberg's Archivesof Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 281- 287 (1977) Pharmacology 9 by Springer-Verlag 1977 Effect of Ente...
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