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Pharmacokinetics of intravenously administered digoxin and histopathological picture in rabbits with experimental bile duct obstruction
European Journal of Pharmaceutical Sciences 11 (2000) 215–222 www.elsevier.nl / locate / ejps
Pharmacokinetics of intravenously administered digoxin and histopathological picture in rabbits with experimental bile duct obstruction a b b, *, ´ ´ ´ Maciej Wojcicki , Marek Drozdzik , Tadeusz Sulikowski a , Jerzy Wojcicki b a c ´ ´ ´~ Barbara Gawronska-Szklarz , Stanil«aw Zielinski , Lidia Rozewicka a
Department of General and Transplantation Surgery, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland b Chair of Pharmacology and Toxicology, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland c Department of Histology and Embriology, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland Received 19 January 2000; received in revised form 10 March 2000; accepted 11 April 2000
Abstract This study aimed to examine the effect of obstructive cholestasis on the pharmacokinetics of digoxin. Eighteen male rabbits were randomly ascribed to the two study groups: the sham-operated control group and the examined group — with common and cystic bile duct ligations. Digoxin was administered intravenously as a single dose of 0.02 mg / kg, and blood samples were withdrawn for up to 24 h. Digoxin concentrations were determined by the FPIA method. The pharmacokinetic parameters were calculated using a noncompartmental analysis. During the whole observation period the blood serum concentrations of digoxin were statistically higher in animals with obstructive cholestasis versus the controls. A significant increase in the area under the plasma concentration–time curve, decrease in the total body clearance and in the volume of distribution on the 6th day after the bile ducts ligation as compared to the sham-operated controls, were observed. The obtained results suggest an impaired elimination of digoxin in obstructive cholestasis in rabbits. 2000 Elsevier Science B.V. All rights reserved. Keywords: Digoxin; Cholestasis; Pharmacokinetics
1. Introduction Extrahepatic, mechanical cholestasis is not a rare abnormality. It occurs in about 10% of patients suffering from cholelithiasis and in the majority of neoplasms affecting the pancreas and the common bile duct (Blamey et al., 1983). Less frequent cases of mechanical cholestasis include neoplasms and inflammatory processes of adjacent organs and tissues, papillitis scleroticans or ligation of the duct. In a state of extrahepatic cholestasis one can observe an altered absorption, distribution and elimination of drug depending on structural differences of drugs and the time elapsed since obstructing of the duct. Prolonged cholestasis may alter the liver function due to an impaired uptake, changed biotransformation and secretion as well as secondary abnormalities induced within the kidney (Ozawa et al., 1979; Moore et al., 1992). There are limited data on the pharmacokinetics of drugs in subjects with biliary obstruction. Moreover, the obtained *Corresponding author. Tel.: 148-91-482-0863; fax 148-91-4828539.
results are often conflicting, possibly due to different species used and interindividual differences (Harrison and Gibaldi, 1976; Ochs et al., 1978; Weidler et al., 1978; ´ Haustein, 1981; Fruncillo et al., 1982; Gawronska-Szklarz et al., 1983; Miyazawa et al., 1990). In the human about 15% of digoxin undergoes a metabolic transformation in the liver, and simultaneously some active metabolites are synthesized. The main route of elimination of digoxin belongs to the urinary tract, where as 60–80% of given dose is excreted as the unchanged form (Beveridge et al., 1978; Kramer and Reuning, 1978; Ben-Itzhak et al., 1985). Within the kidney digoxin undergoes glomerular filtration, and digoxin clearance parallels that of creatinine (Peters et al., 1978). The extent of the drug eliminated via the liver is small due to hepato-enteric circulation. However, there are data suggesting that 30% of digoxin or so, given intravenously may accumulate in the digestive tract within 24 h of administration (Doherty et al., 1961; Caldwell and Cline, 1976). The findings of Ochs et al. (1978) indicate that unlike the human, rat, and guinea pig, renal elimination of unchanged digoxin in rabbits contributes relatively little to the total clearance of the drug. So, in the rabbit, due to
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extensive biotransformation and subsequent fecal excretion of metabolic products, the drug can be applied as a model substance eliminated predominately via the liver. The aim of the study was to evaluate the pharmacokinetics of digoxin administered as a single intravenous dose in rabbits suffering from mechanical, extrahepatic cholestasis.
using TDx analyzer (Abbott, North Chicago, USA). The sensitivity and intra- and inter-assay variability were 0.2 mg / l and 1.4–2.9 and 1.4–4.1%, respectively, in the TDx digoxin assay. Abnormal levels of total bilirubin up to 342 mmol / l result in less than 10% error in quantitating a sample with the Digoxin II Abbott assay. In our experiment, the levels of total bilirubin in the group with ligated bile duct were from 0.7 to 21.7 mmol / l.
2. Material and methods
2.4. Pharmacokinetic analysis
2.1. Material
The digoxin serum concentration–time curve after intravenous injection was analyzed according to a two compartment open model. The half-life (t 1 / 2 lz ) was estimated from the total concentrations, by the relationship t 1 / 2 lz 5 ln 2 /lz , where lz was calculated on the basis on log-linear regression from the last three to six points. Noncompartmental method was used to estimate the total body clearance (CL / BW) (BW5body weight) as well as the volume of distribution at steady state (VDss ) and mean residence time (MRT). The total body clearance was calculated by equation: CL / BW5Dose /AUC?BW, where the area under the curve (AUC) was calculated by the linear trapezoidal rule up to the last data point (C 24 ), and the extrapolated area from the last data point to infinitive time was obtained by integration. The volume of distribution was calculated as VDss 5CL?MRT. The mean residence time was calculated according to the equation: MRT5AUMC /AUC, where AUMC is the area under the moment curve from zero to infinity.
The study was carried out on 18 male rabbits, randomly ascribed to two equal groups. Group 1, control — shamoperated, and group 2, experimental — comprising the animals submitted to common bile duct ligation, weighing prior to surgery: 3.360.2 kg and 4.060.3 kg, respectively. Both groups were housed identically, in a controlled temperature (228C), and 12-h light–12-h dark cycle environment. During the 1-week of acclimation period, food (LSM, Motycz) and water were given ad libitum.
2.2. Surgery The operation was performed under 12 mg / kg pentobarbital (Vetbutal, Biowet) anaesthesia. In the experimental group the common bile duct as well as the cystic bile duct were ligated. In the controls the same surgical procedure, except for ligation of the ducts, was carried out.
2.5. Anatomo–pathological procedure 2.3. Experimental design Body weight of the animals was measured prior to the operation and at autopsy, i.e. on the 7th day following surgery. In all animals, 4 days prior to and 6 days after the operation, the following laboratory parameters were estimated: serum total bilirubin, creatinine, glucose and albumin concentrations, as well as activities of alanine, aspartate aminotransferases and alkaline phosphatase, using a standard laboratory analyser-RA-1000 (Technicon). Digoxin (Digoxin, Polfa) was administered into the ear marginal vein of the 18-h fasting animals 4 days before the operation and 6 days after the surgery, at a dose of 0.02 mg / kg in 0.3 ml saline solution, within 5 min, followed by a 1-ml saline injection. All animals were restrained from chow for 3 h following the drug application. Blood samples were collected from the contralateral ear marginal vein before and then 15 min, 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after the drug administration. Clotted blood samples were immediately centrifuged, and sera were stored at 2208C, until analysis. The blood serum of digoxin was determined by a fluorescence polarization immunoassay
All animals were sacrified at the end of the study, i.e. on the 7th day after the bile duct ligation. Kidneys and livers were weighed, and examined macro- and microscopically. For microscopic studies a part of the liver pieces was freeze-sectioned and stained with oil red 0 for the presence of neutral lipid. Other liver samples were placed into the Schafer’s solution (Romeis, 1968), and afterwards the prepared slices were stained with hematoxilin, eosin and PAS. Slices of the kidney were placed into Carnoy’s solution (Romeis, 1968), and in a further step stained with hematoxilin and eosin.
2.6. Statistical analysis Statistical analysis was performed using Wilcoxon ’ s signed rank test, with a P value of ,0.05 being statistically significant.
3. Results The first symptoms of jaundice appeared in animals from the experimental group 3 days after the operation.
´ et al. / European Journal of Pharmaceutical Sciences 11 (2000) 215 – 222 M. Wojcicki
Motor activity of animals was depressed and there was a yellow colour detectable on the ears and the conjunctivas. The intensity of these symptoms increased as the study period progressed. Body weight of the jaundiced rabbits decreased by 11% (P,0.01). There were no significant changes in the laboratory tests in the control animals, except for a drop in albumin concentration from 41.960.6 to 38.861.0 g / l (P,0.02), whereas in rabbits with the bile duct ligated, the following alterations in the blood serum were observed on the 6th day after the operation: a 32-fold rise in bilirubin concentration from 0.760.1 to 21.763.2 mmol / l, as well as an increase in activities of aminotransferases: alanine from 36.263.2 to 169.2632.3 U / l (P,0.01), and aspartate from 36.663.4 to 136.2632.3 U / l (P,0.01) as well as alkaline phosphatase from 95.0623.3 to 212.6633.4 U / l (P,0.01). Creatinine concentration in the sham-operated control group did not changed markedly. It was 168.6612.2 mmol / l before surgery, and 150.467.9 mmol / l after the operation, while in the animals with ligated bile ducts the value increased insignificantly from 164.7619.3 to 178.1 627.8 mmol / l. As in controls, a pronounced drop in albumin content from 40.761.2 to 32.661.1 g / l (P, 0.01) was seen in the jaundiced animals. The average blood serum concentrations of digoxin are outlined in Figs. 1 and 2. Concentrations of the drug in rabbits with experimental bile duct ligation were significantly elevated as compared to the results in the same
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animals prior to surgery (Fig. 1), whereas in the control group no statistical difference in the blood serum concentration of digoxin was detected (Fig. 2). The calculated pharmacokinetic parameters are presented in Table 1. There were no significant alterations in digoxin parameters of animals from the control group measured prior to and after the operation, and as compared to group 2 at the onset of the study. In contrast, most pharmacokinetic parameters were significantly changed in animals subjected to the bile ducts ligation, as compared with the preoperative values. The area under the plasma concentration–time curve (AUC) increased by 219% (P, 0.01), the volume of distribution decreased by 47% (P, 0.01), and total body clearance dropped by 59% (P,0.01). At the end of the study all animals were autopsied. The livers from the animals with obstructive cholestasis were enlarged and more fragile, with yellowish colour and strained capsules. Mean relative weight of the livers from the jaundiced group (31.361.4 g / kg) was significantly higher (P,0.05) in comparison with controls (26.361.5 g / kg), whereas the same parameter of the kidney was similar in both groups coming to 5.760.5 g / kg in controls and 6.760.5 g / kg in animals with cholestasis. The structure of the liver from the control group was of normal morphology (Fig. 3), whereas in animals with common bile duct obstruction several abnormalities were detected. Some regions of hepatocytes of these animals contained small yellow droplets of bile located predominantly around
Fig. 1. Blood serum concentrations (6S.E.) vs. time of digoxin in rabbits before (h-h-h) and after (^-^-^) bile duct ligation (*, P,0.05; **, P,0.01; ***, P,0.001).
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Fig. 2. Blood serum concentrations (6S.E.) vs. time of digoxin in the control rabbits before (h-h-h) and after (^-^-^) sham operation.
the portal space (Fig. 4). Bile deposits were observed in the bile canaliculi. An enlargement of portal spaces, hypertrophy of bile ducts as well as dilatation of interlobular ducts were seen. Hyperplasia of the connective tissue in the portal spaces was observed (Fig. 5). Some lymphoidal cell infiltrations were also noted. Hepatocytes in the rabbits with obstructive jaundice contained small quantities of grains of glycogen. Macroscopic examination of the kidney did not reveal any abnormality in both study groups, whereas microscopic presentation was normal only in the control animals (Fig. 6). In animals with mechanical cholestasis a contraction of the glomeruli, dilatation of the Bowman’s space as well as thickening of the Bowman’s capsules were observed (Fig. 7). Within the kidney a lot of the tubules presented flat epithelium and dilated lumen, in
some of them total absence of epithelium was detected (Fig. 8). Degeneration of the renal bodies were also seen in the jaundiced rabbits.
4. Discussion All kinetic studies concerning elimination of drugs under mechanical cholestasis have been scarce and referred to the drugs mostly being metabolised in the liver and / or excreted into the bile, i.e. tetracycline antibiotics (Gaw˜ ronska-Szklarz et al., 1983), procainamide (Basseches and Digregorio, 1982), theophylline (Fruncillo et al., 1982), pentobarbital (Carulli et al., 1975) and phenazone (Elfstrom and Lindgren, 1974; Hepner and Vesell, 1975). The
Table 1 Mean (6S.E.) values of the pharmacokinetic parameters of digoxin in the control rabbits (group 1) prior to (1A) and after the surgery (1B), and in animals with obstructive cholestasis (group 2) prior to (2C) and after the bile ducts ligation (2D)a Parameter (unit)
AUC (ng / ml?h) MRT (h) lz (h 21 ) t 1 / 2 lz (h) VDss (L) CL / BW (L / h?kg) a
Group 1
Group 2
A
B
C
D
22.3764.39 21.9668.69 0.0960.01 9.9562.36 91.82611.41 1.2460.27
23.1464.21 23.9968.49 0.1060.03 10.0161.65 84.4068.64 1.1160.19
19.4261.89 17.0264.09 0.0860.01 10.2561.90 72.74612.24 1.1160.11
61.94614.59** 1 22.5466.01 0.0660.01 11.7561.99 38.8466.82** 11 0.4660.09** 1
BW, body weight (kg); C vs. D, **P,0.01; B vs. D, 1 , P,0.05;
11
, P,0.01.
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Fig. 3. Normal morphology of the rabbit’s liver from the control group. H–E350.
data, although in some cases equivocal, suggest an impaired elimination in subjects with mechanical, extrahepatic cholestasis. Digoxin applied in the present study fulfils criteria of a model drug excreted via the liver in the rabbit. Ochs et al. (1978) reported that 75% of radioactivity of digoxin labelled with [ 3 H] was recovered in feces, and only 15% was measured in urine. Ligation of the bile
ducts leads towards characteristic changes both in laboratory tests (increased concentration of bilirubin and activity of alkaline phosphatase) and microscopic findings. The changes are consistent with other authors’ findings (Aronson et al., 1993). In our study prolonged elimination of digoxin was showed. The observed changes most probably result from impaired liver function and interruption of the
Fig. 4. Small droplets of bile in hepatocytes in rabbits with obstructive jaundice (arrows). The darker droplets belong to lipids (arrow head). Freeze section. Oil Red O3670.
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Fig. 5. Liver of rabbit with obstructive jaundice. Enlargement of portal space and hyperplasia of connective tissue occur. H–E3160.
hepatoenteric circulation. However, abnormal kidney function might contribute to an altered behaviour of digoxin to some extent, due to reduced renal excretion of the drug. An impaired liver function may induce some abnormalities in the kidney. Renal function in the course of obstructive jaundice has been the subject of great interest ever since the association between jaundice and renal failure was described (Sitprija et al., 1990). In the present study an
altered microscopic morphology of the kidneys from animals with extrahepatic cholestasis paralleled changes in serum creatinine concentration which were elevated. Some other abnormalities like decreased activity of adrenergic system (Zambraski and Dibona, 1983; Dibona and Sawin, 1991), increased content of thromboxan together with a decreased concentration of prostaglandins (Epstein, 1987), and a rise in endothelin (Moore et al., 1992), increased
Fig. 6. Normal morphology of the kidney in rabbit from the control group. H–E350.
´ et al. / European Journal of Pharmaceutical Sciences 11 (2000) 215 – 222 M. Wojcicki
Fig. 7. Cortex of kidney in rabbit with obstructive jaundice. Contraction of glomeruli and dilatation of Bowman space are seen. PAS3160.
activity of renin–angiotensin system leading to a reduced glomerular filtration (Epstein and Norisk, 1983; Ichikawa and Brenner, 1984), and damaging properties of constituents of bile, i.e. deoxycholic acid (McLuen and Fouts, 1961), might all be responsible for the observed alteration of kidney morphology, involving a possible impact on the
Fig. 8. Kidney in rabbit with obstructive jaundice. Some tubules with lack of epithelium or with flat epithelium. PAS3160.
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pharmacokinetics of digoxin. As was stated by Moore (1997) in hepatorenal syndrome functional renal failure (as one form of the syndrome) may evolve to acute tubular necrosis. Some tubular damage was also seen in our study. Although, serum creatinine concentrations were not significantly elevated in the jaundiced animals, but disturbed hemodynamics induced by cirrhosis and possibly cholestasis could contribute to alterations of creatinine clearance, and may be at least partly responsible for the observed creatinine levels (Orlando et al., 1999). The present results show a pronounced increase in the area under the plasma concentration time curve and a decrease in volume of distribution and total body clearance. Thus digoxin, that is eliminated also via the liver in the rabbit, undergoes prolonged elimination in the rabbit organism. This is due to impairment of liver function induced by the common bile duct obstruction, and may be due to impaired to a degree renal secretion. A pronounced deterioration of digoxin elimination was consistent with an elevation in serum creatinine concentration. The results by Ochs et al. (1978) suggest that the fraction of digoxin metabolised by the liver in rabbits depends on the time of the drug administration. Their data indicate that repeated application of digoxin appears to stimulate digoxin biotransformation. So, in the present study where digoxin was given as a single dose the contribution of the liver to elimination of digoxin is expected to be less pronounced, as compared to multiple administration of the drug. On the other hand, data reported by Tateishi et al. (1998) and Basseches and Digregorio (1982) suggest that in animals with extrahepatic biliary obstruction the activity of liver enzymes can be either increased or decreased. Altered activity of enzymes that take part in the metabolism of digoxin might also contribute to the extent of liver clearance of the studied drug. To further clarify the role of the kidney in the digoxin elimination, assays of secreted digoxin in the urine would be worthwhile. An increase in serum bilirubin concentration, and possibly some endotoxins contribute to the decreased tissue binding of digoxin, leading in turn to some reduction of apparent volume of distribution, to which an interrupted enterohepatic recirculation may also contribute (Basseches and Digregorio, 1982). As reported by Ochs et al. (1978), tissue uptake of digoxin in rabbits was more extensive than in humans. An elevated concentration of digoxin is not efficiently excreted via the impaired liver, and may be abnormal renal function, and is accumulated in the circulation, leading to an increase in the blood serum concentrations of digoxin. In the light of the present study it can be concluded that obstructive cholestasis alters the pharmacokinetics of intravenously administered digoxin, a drug predominantly eliminated in humans via the kidney. Taking into account the present data, as well as published sources concerning the rabbit, it seems that changes of digoxin clearance in obstructive cholestasis result mainly from the liver function impairment.
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References Aronson, D.C., Chamuleau, R., Frederiks, W.M., Gooszen, H.G., Heijmans, H., James, J., 1993. Reversibility of cholestatic changes following common bile duct obstruction: Fact or fantasy? J. Hepatol. 18, 85–95. Basseches, P.J., Digregorio, G.J., 1982. Pharmacokinetics of procainamide in rats with extrahepatic biliary obstruction. J. Pharmaceut. Sci. 71, 1256–1259. Ben-Itzhak, J., Bassan, H.M., Shor, R., Lanir, A., 1985. Digoxin quinidine interaction: a pharmacokinetic study in the isolated perfused rat liver. Life Sci. 37, 411–415. Beveridge, T., Nuesch, E., Ohnhaus, E.E., 1978. Absolute bioavailability of digoxin tablets. Arzneimittelforsch. 28, 701–703. Blamey, S.L., Fearon, K.C., Gilmour, W.H., Osborne, D.H., Carter, D., 1983. Prediction of risk in biliary surgery. Br. J. Surg. 70, 535–538. Caldwell, J.H., Cline, C.T., 1976. Biliary excretion of digoxin in man. Clin. Pharmacol. Ther. 19, 410–415. Carulli, N., Maneti, E., de Leon, P., 1975. Alteration of drug metabolism during cholestasis in man. Eur. J. Clin. Invest. 5, 455–462. Dibona, G.F., Sawin, L.L., 1991. Role of renal nerves in sodium retention in cirrhosis and congestive heart failure. Am. J. Physiol. 260, R298– R305. Doherty, M.D., Perkins, W.H., Mitchel, G.K., 1961. Tritiated digoxin studies in human subjects. Arch. Intern. Med. 108, 531–536. Elfstrom, J., Lindgren, S., 1974. Disappearance of phenazone from plasma in patients with obstructive jaundice. Eur. J. Pharmacol. 7, 467–471. Epstein, M., 1987. Renal eikosanoids as determinants of renal function in liver disease. Hepatology 7, 1359–1367. Epstein, M., Norisk, P., 1983. In: Epstein, M. (Ed.), The Kidney in Liver Disease, 3rd Edition. Williams and Wilhins, Baltimore, p. 331. Fruncillo, R.J., Deangelis, P., Digregore, J., 1982. Pharmacokinetics of theophylline in rats with biliary stasis. J. Pharm. Pharmacol. 34, 741. ´ ´ ´ B., Gawronska-Szklarz, B., Wojcicki, J., Kardel-Mizerska, T., Kustron, 1983. Influence of biliary stasis and surgical procedure on the pharmacokinetics of tetracycline given intragastrically (in Polish). Pol. Przegl. Chir. 35, 543–549. Harrison, L.I., Gibaldi, M., 1976. Pharmacokinetics of digoxin in the rat. Drug Metab. Res. 4, 88–93. Haustein, K.O., 1981. Interindividual differences in the pharmacokinetics of digoxin and digitoxin during long-term treatment. Eur. J. Clin. Pharmacol. 19, 45–51. Hepner, G.W., Vesell, E.S., 1975. Normal antipyrine metabolism in
patients with cholesterol cholelithiasis evidence that the disease is not due to generalized hepatic microsomal dysfunction. Am. J. Digest. Dis. 20, 9–12. Ichikawa, I., Brenner, B.M., 1984. Glomerular actions of angiotensin II. Am. J. Med. 76, 43–49. Kramer, W.G., Reuning, R.H., 1978. Use of area under the curve to estimate absolute bioavailability of digoxin. J. Pharm. Sci. 64, 141– 142. McLuen, E.F., Fouts, J.R., 1961. The effect of obstructive jaundice on drug metabolism in rabbits. J. Pharmacol. Exp. Ther. 131, 7–12. Miyazawa, Y., Sato, T., Kobayashi, K., Akahori, F., Suzuki, T., Miyashita, H., 1990. Influence of induced cholestasis on pharmacokinetics of digoxin and digitoxin in dogs. Am. J. Vet. Res. 51, 605–610. Moore, K., Wendon, J., Frazer, M., Karani, J., Williams, R., Badr, K., 1992. Plasma endothelium immunoreactivity in liver disease and the hepatorenal syndrome. New Engl. J. Med. 327, 1774–1778. Moore, K., 1997. The hepatorenal syndrome. Clin. Sci. 92, 433–443. Ochs, H., Bodem, G., Bales, G., Greenblatt, D., Smith, T., 1978. Increased clearance of digoxin in rabbits during repeated administration. J. Pharmacol. Exp. Ther. 204, 262–270. Orlando, R., Floreani, M., Padrini, R., Palatini, P., 1999. Evaluation of measured and calculated creatinine clearances as glomerular filtration markers in different stages of liver cirrhosis. Clin. Nephrol. 51, 341–347. Ozawa, K., Yamada, T., Tanaka, J., Ukiksua, M., Tobe, T., 1979. The mechanism of suppression of renal function in patients and rabbits in jaundice. Surg. Gynecol. Obstet. 149, 54–60. Peters, U., Falk, L.C., Kakman, S.H., 1978. Digoxin metabolism in patients. Arch. Intern. Med. 138, 1074–1076. Romeis, B., 1968. In: Mikroskopische Technik. R. Oldenbourg Verlag, Muchen-Vien, p. 59. Sitprija, V., Kashemsant, U., Sriratanban, A., Arthachinta, S., Poshyachinda, V., 1990. Renal function in obstructive jaundice in man: cholangiocarcinoma model. Kidney Int. 38, 948–955. Tateishi, T., Watanabe, M., Nakura, H., Tanaka, M., Kumai, T., Kobayashi, S., 1998. Liver damage induced by bile duct ligation affects CYP isoenzymes differently in rats. Pharm. Toxocol. 82, 89–92. Weidler, D.J., Jallad, N.S., Movahhed, H.S., Sakmar, E., Wagner, J.G., 1978. Pharmacokinetics of digoxin in the cat and comparisons with man and the dog. Res. Commun. Chem. Pathol. Pharmacol. 19, 57–66. Zambraski, E.J., Dibona, G.F., 1983. In: Epstein, M. (Ed.), The Kidney in Liver Disease, 3rd Edition. Williams and Wilkins, Baltimore, p. 469.
Pharmacokinetics of intravenously administered digoxin and histopathological picture in rabbits with experimental bile duct obstruction a b b, *, ´ ´ ´ Maciej Wojcicki , Marek Drozdzik , Tadeusz Sulikowski a , Jerzy Wojcicki b a c ´ ´ ´~ Barbara Gawronska-Szklarz , Stanil«aw Zielinski , Lidia Rozewicka a
Department of General and Transplantation Surgery, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland b Chair of Pharmacology and Toxicology, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland c Department of Histology and Embriology, Pomeranian Academy of Medicine, 70 -111 Szczecin, Poland Received 19 January 2000; received in revised form 10 March 2000; accepted 11 April 2000
Abstract This study aimed to examine the effect of obstructive cholestasis on the pharmacokinetics of digoxin. Eighteen male rabbits were randomly ascribed to the two study groups: the sham-operated control group and the examined group — with common and cystic bile duct ligations. Digoxin was administered intravenously as a single dose of 0.02 mg / kg, and blood samples were withdrawn for up to 24 h. Digoxin concentrations were determined by the FPIA method. The pharmacokinetic parameters were calculated using a noncompartmental analysis. During the whole observation period the blood serum concentrations of digoxin were statistically higher in animals with obstructive cholestasis versus the controls. A significant increase in the area under the plasma concentration–time curve, decrease in the total body clearance and in the volume of distribution on the 6th day after the bile ducts ligation as compared to the sham-operated controls, were observed. The obtained results suggest an impaired elimination of digoxin in obstructive cholestasis in rabbits. 2000 Elsevier Science B.V. All rights reserved. Keywords: Digoxin; Cholestasis; Pharmacokinetics
1. Introduction Extrahepatic, mechanical cholestasis is not a rare abnormality. It occurs in about 10% of patients suffering from cholelithiasis and in the majority of neoplasms affecting the pancreas and the common bile duct (Blamey et al., 1983). Less frequent cases of mechanical cholestasis include neoplasms and inflammatory processes of adjacent organs and tissues, papillitis scleroticans or ligation of the duct. In a state of extrahepatic cholestasis one can observe an altered absorption, distribution and elimination of drug depending on structural differences of drugs and the time elapsed since obstructing of the duct. Prolonged cholestasis may alter the liver function due to an impaired uptake, changed biotransformation and secretion as well as secondary abnormalities induced within the kidney (Ozawa et al., 1979; Moore et al., 1992). There are limited data on the pharmacokinetics of drugs in subjects with biliary obstruction. Moreover, the obtained *Corresponding author. Tel.: 148-91-482-0863; fax 148-91-4828539.
results are often conflicting, possibly due to different species used and interindividual differences (Harrison and Gibaldi, 1976; Ochs et al., 1978; Weidler et al., 1978; ´ Haustein, 1981; Fruncillo et al., 1982; Gawronska-Szklarz et al., 1983; Miyazawa et al., 1990). In the human about 15% of digoxin undergoes a metabolic transformation in the liver, and simultaneously some active metabolites are synthesized. The main route of elimination of digoxin belongs to the urinary tract, where as 60–80% of given dose is excreted as the unchanged form (Beveridge et al., 1978; Kramer and Reuning, 1978; Ben-Itzhak et al., 1985). Within the kidney digoxin undergoes glomerular filtration, and digoxin clearance parallels that of creatinine (Peters et al., 1978). The extent of the drug eliminated via the liver is small due to hepato-enteric circulation. However, there are data suggesting that 30% of digoxin or so, given intravenously may accumulate in the digestive tract within 24 h of administration (Doherty et al., 1961; Caldwell and Cline, 1976). The findings of Ochs et al. (1978) indicate that unlike the human, rat, and guinea pig, renal elimination of unchanged digoxin in rabbits contributes relatively little to the total clearance of the drug. So, in the rabbit, due to
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extensive biotransformation and subsequent fecal excretion of metabolic products, the drug can be applied as a model substance eliminated predominately via the liver. The aim of the study was to evaluate the pharmacokinetics of digoxin administered as a single intravenous dose in rabbits suffering from mechanical, extrahepatic cholestasis.
using TDx analyzer (Abbott, North Chicago, USA). The sensitivity and intra- and inter-assay variability were 0.2 mg / l and 1.4–2.9 and 1.4–4.1%, respectively, in the TDx digoxin assay. Abnormal levels of total bilirubin up to 342 mmol / l result in less than 10% error in quantitating a sample with the Digoxin II Abbott assay. In our experiment, the levels of total bilirubin in the group with ligated bile duct were from 0.7 to 21.7 mmol / l.
2. Material and methods
2.4. Pharmacokinetic analysis
2.1. Material
The digoxin serum concentration–time curve after intravenous injection was analyzed according to a two compartment open model. The half-life (t 1 / 2 lz ) was estimated from the total concentrations, by the relationship t 1 / 2 lz 5 ln 2 /lz , where lz was calculated on the basis on log-linear regression from the last three to six points. Noncompartmental method was used to estimate the total body clearance (CL / BW) (BW5body weight) as well as the volume of distribution at steady state (VDss ) and mean residence time (MRT). The total body clearance was calculated by equation: CL / BW5Dose /AUC?BW, where the area under the curve (AUC) was calculated by the linear trapezoidal rule up to the last data point (C 24 ), and the extrapolated area from the last data point to infinitive time was obtained by integration. The volume of distribution was calculated as VDss 5CL?MRT. The mean residence time was calculated according to the equation: MRT5AUMC /AUC, where AUMC is the area under the moment curve from zero to infinity.
The study was carried out on 18 male rabbits, randomly ascribed to two equal groups. Group 1, control — shamoperated, and group 2, experimental — comprising the animals submitted to common bile duct ligation, weighing prior to surgery: 3.360.2 kg and 4.060.3 kg, respectively. Both groups were housed identically, in a controlled temperature (228C), and 12-h light–12-h dark cycle environment. During the 1-week of acclimation period, food (LSM, Motycz) and water were given ad libitum.
2.2. Surgery The operation was performed under 12 mg / kg pentobarbital (Vetbutal, Biowet) anaesthesia. In the experimental group the common bile duct as well as the cystic bile duct were ligated. In the controls the same surgical procedure, except for ligation of the ducts, was carried out.
2.5. Anatomo–pathological procedure 2.3. Experimental design Body weight of the animals was measured prior to the operation and at autopsy, i.e. on the 7th day following surgery. In all animals, 4 days prior to and 6 days after the operation, the following laboratory parameters were estimated: serum total bilirubin, creatinine, glucose and albumin concentrations, as well as activities of alanine, aspartate aminotransferases and alkaline phosphatase, using a standard laboratory analyser-RA-1000 (Technicon). Digoxin (Digoxin, Polfa) was administered into the ear marginal vein of the 18-h fasting animals 4 days before the operation and 6 days after the surgery, at a dose of 0.02 mg / kg in 0.3 ml saline solution, within 5 min, followed by a 1-ml saline injection. All animals were restrained from chow for 3 h following the drug application. Blood samples were collected from the contralateral ear marginal vein before and then 15 min, 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after the drug administration. Clotted blood samples were immediately centrifuged, and sera were stored at 2208C, until analysis. The blood serum of digoxin was determined by a fluorescence polarization immunoassay
All animals were sacrified at the end of the study, i.e. on the 7th day after the bile duct ligation. Kidneys and livers were weighed, and examined macro- and microscopically. For microscopic studies a part of the liver pieces was freeze-sectioned and stained with oil red 0 for the presence of neutral lipid. Other liver samples were placed into the Schafer’s solution (Romeis, 1968), and afterwards the prepared slices were stained with hematoxilin, eosin and PAS. Slices of the kidney were placed into Carnoy’s solution (Romeis, 1968), and in a further step stained with hematoxilin and eosin.
2.6. Statistical analysis Statistical analysis was performed using Wilcoxon ’ s signed rank test, with a P value of ,0.05 being statistically significant.
3. Results The first symptoms of jaundice appeared in animals from the experimental group 3 days after the operation.
´ et al. / European Journal of Pharmaceutical Sciences 11 (2000) 215 – 222 M. Wojcicki
Motor activity of animals was depressed and there was a yellow colour detectable on the ears and the conjunctivas. The intensity of these symptoms increased as the study period progressed. Body weight of the jaundiced rabbits decreased by 11% (P,0.01). There were no significant changes in the laboratory tests in the control animals, except for a drop in albumin concentration from 41.960.6 to 38.861.0 g / l (P,0.02), whereas in rabbits with the bile duct ligated, the following alterations in the blood serum were observed on the 6th day after the operation: a 32-fold rise in bilirubin concentration from 0.760.1 to 21.763.2 mmol / l, as well as an increase in activities of aminotransferases: alanine from 36.263.2 to 169.2632.3 U / l (P,0.01), and aspartate from 36.663.4 to 136.2632.3 U / l (P,0.01) as well as alkaline phosphatase from 95.0623.3 to 212.6633.4 U / l (P,0.01). Creatinine concentration in the sham-operated control group did not changed markedly. It was 168.6612.2 mmol / l before surgery, and 150.467.9 mmol / l after the operation, while in the animals with ligated bile ducts the value increased insignificantly from 164.7619.3 to 178.1 627.8 mmol / l. As in controls, a pronounced drop in albumin content from 40.761.2 to 32.661.1 g / l (P, 0.01) was seen in the jaundiced animals. The average blood serum concentrations of digoxin are outlined in Figs. 1 and 2. Concentrations of the drug in rabbits with experimental bile duct ligation were significantly elevated as compared to the results in the same
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animals prior to surgery (Fig. 1), whereas in the control group no statistical difference in the blood serum concentration of digoxin was detected (Fig. 2). The calculated pharmacokinetic parameters are presented in Table 1. There were no significant alterations in digoxin parameters of animals from the control group measured prior to and after the operation, and as compared to group 2 at the onset of the study. In contrast, most pharmacokinetic parameters were significantly changed in animals subjected to the bile ducts ligation, as compared with the preoperative values. The area under the plasma concentration–time curve (AUC) increased by 219% (P, 0.01), the volume of distribution decreased by 47% (P, 0.01), and total body clearance dropped by 59% (P,0.01). At the end of the study all animals were autopsied. The livers from the animals with obstructive cholestasis were enlarged and more fragile, with yellowish colour and strained capsules. Mean relative weight of the livers from the jaundiced group (31.361.4 g / kg) was significantly higher (P,0.05) in comparison with controls (26.361.5 g / kg), whereas the same parameter of the kidney was similar in both groups coming to 5.760.5 g / kg in controls and 6.760.5 g / kg in animals with cholestasis. The structure of the liver from the control group was of normal morphology (Fig. 3), whereas in animals with common bile duct obstruction several abnormalities were detected. Some regions of hepatocytes of these animals contained small yellow droplets of bile located predominantly around
Fig. 1. Blood serum concentrations (6S.E.) vs. time of digoxin in rabbits before (h-h-h) and after (^-^-^) bile duct ligation (*, P,0.05; **, P,0.01; ***, P,0.001).
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Fig. 2. Blood serum concentrations (6S.E.) vs. time of digoxin in the control rabbits before (h-h-h) and after (^-^-^) sham operation.
the portal space (Fig. 4). Bile deposits were observed in the bile canaliculi. An enlargement of portal spaces, hypertrophy of bile ducts as well as dilatation of interlobular ducts were seen. Hyperplasia of the connective tissue in the portal spaces was observed (Fig. 5). Some lymphoidal cell infiltrations were also noted. Hepatocytes in the rabbits with obstructive jaundice contained small quantities of grains of glycogen. Macroscopic examination of the kidney did not reveal any abnormality in both study groups, whereas microscopic presentation was normal only in the control animals (Fig. 6). In animals with mechanical cholestasis a contraction of the glomeruli, dilatation of the Bowman’s space as well as thickening of the Bowman’s capsules were observed (Fig. 7). Within the kidney a lot of the tubules presented flat epithelium and dilated lumen, in
some of them total absence of epithelium was detected (Fig. 8). Degeneration of the renal bodies were also seen in the jaundiced rabbits.
4. Discussion All kinetic studies concerning elimination of drugs under mechanical cholestasis have been scarce and referred to the drugs mostly being metabolised in the liver and / or excreted into the bile, i.e. tetracycline antibiotics (Gaw˜ ronska-Szklarz et al., 1983), procainamide (Basseches and Digregorio, 1982), theophylline (Fruncillo et al., 1982), pentobarbital (Carulli et al., 1975) and phenazone (Elfstrom and Lindgren, 1974; Hepner and Vesell, 1975). The
Table 1 Mean (6S.E.) values of the pharmacokinetic parameters of digoxin in the control rabbits (group 1) prior to (1A) and after the surgery (1B), and in animals with obstructive cholestasis (group 2) prior to (2C) and after the bile ducts ligation (2D)a Parameter (unit)
AUC (ng / ml?h) MRT (h) lz (h 21 ) t 1 / 2 lz (h) VDss (L) CL / BW (L / h?kg) a
Group 1
Group 2
A
B
C
D
22.3764.39 21.9668.69 0.0960.01 9.9562.36 91.82611.41 1.2460.27
23.1464.21 23.9968.49 0.1060.03 10.0161.65 84.4068.64 1.1160.19
19.4261.89 17.0264.09 0.0860.01 10.2561.90 72.74612.24 1.1160.11
61.94614.59** 1 22.5466.01 0.0660.01 11.7561.99 38.8466.82** 11 0.4660.09** 1
BW, body weight (kg); C vs. D, **P,0.01; B vs. D, 1 , P,0.05;
11
, P,0.01.
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Fig. 3. Normal morphology of the rabbit’s liver from the control group. H–E350.
data, although in some cases equivocal, suggest an impaired elimination in subjects with mechanical, extrahepatic cholestasis. Digoxin applied in the present study fulfils criteria of a model drug excreted via the liver in the rabbit. Ochs et al. (1978) reported that 75% of radioactivity of digoxin labelled with [ 3 H] was recovered in feces, and only 15% was measured in urine. Ligation of the bile
ducts leads towards characteristic changes both in laboratory tests (increased concentration of bilirubin and activity of alkaline phosphatase) and microscopic findings. The changes are consistent with other authors’ findings (Aronson et al., 1993). In our study prolonged elimination of digoxin was showed. The observed changes most probably result from impaired liver function and interruption of the
Fig. 4. Small droplets of bile in hepatocytes in rabbits with obstructive jaundice (arrows). The darker droplets belong to lipids (arrow head). Freeze section. Oil Red O3670.
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´ et al. / European Journal of Pharmaceutical Sciences 11 (2000) 215 – 222 M. Wojcicki
Fig. 5. Liver of rabbit with obstructive jaundice. Enlargement of portal space and hyperplasia of connective tissue occur. H–E3160.
hepatoenteric circulation. However, abnormal kidney function might contribute to an altered behaviour of digoxin to some extent, due to reduced renal excretion of the drug. An impaired liver function may induce some abnormalities in the kidney. Renal function in the course of obstructive jaundice has been the subject of great interest ever since the association between jaundice and renal failure was described (Sitprija et al., 1990). In the present study an
altered microscopic morphology of the kidneys from animals with extrahepatic cholestasis paralleled changes in serum creatinine concentration which were elevated. Some other abnormalities like decreased activity of adrenergic system (Zambraski and Dibona, 1983; Dibona and Sawin, 1991), increased content of thromboxan together with a decreased concentration of prostaglandins (Epstein, 1987), and a rise in endothelin (Moore et al., 1992), increased
Fig. 6. Normal morphology of the kidney in rabbit from the control group. H–E350.
´ et al. / European Journal of Pharmaceutical Sciences 11 (2000) 215 – 222 M. Wojcicki
Fig. 7. Cortex of kidney in rabbit with obstructive jaundice. Contraction of glomeruli and dilatation of Bowman space are seen. PAS3160.
activity of renin–angiotensin system leading to a reduced glomerular filtration (Epstein and Norisk, 1983; Ichikawa and Brenner, 1984), and damaging properties of constituents of bile, i.e. deoxycholic acid (McLuen and Fouts, 1961), might all be responsible for the observed alteration of kidney morphology, involving a possible impact on the
Fig. 8. Kidney in rabbit with obstructive jaundice. Some tubules with lack of epithelium or with flat epithelium. PAS3160.
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pharmacokinetics of digoxin. As was stated by Moore (1997) in hepatorenal syndrome functional renal failure (as one form of the syndrome) may evolve to acute tubular necrosis. Some tubular damage was also seen in our study. Although, serum creatinine concentrations were not significantly elevated in the jaundiced animals, but disturbed hemodynamics induced by cirrhosis and possibly cholestasis could contribute to alterations of creatinine clearance, and may be at least partly responsible for the observed creatinine levels (Orlando et al., 1999). The present results show a pronounced increase in the area under the plasma concentration time curve and a decrease in volume of distribution and total body clearance. Thus digoxin, that is eliminated also via the liver in the rabbit, undergoes prolonged elimination in the rabbit organism. This is due to impairment of liver function induced by the common bile duct obstruction, and may be due to impaired to a degree renal secretion. A pronounced deterioration of digoxin elimination was consistent with an elevation in serum creatinine concentration. The results by Ochs et al. (1978) suggest that the fraction of digoxin metabolised by the liver in rabbits depends on the time of the drug administration. Their data indicate that repeated application of digoxin appears to stimulate digoxin biotransformation. So, in the present study where digoxin was given as a single dose the contribution of the liver to elimination of digoxin is expected to be less pronounced, as compared to multiple administration of the drug. On the other hand, data reported by Tateishi et al. (1998) and Basseches and Digregorio (1982) suggest that in animals with extrahepatic biliary obstruction the activity of liver enzymes can be either increased or decreased. Altered activity of enzymes that take part in the metabolism of digoxin might also contribute to the extent of liver clearance of the studied drug. To further clarify the role of the kidney in the digoxin elimination, assays of secreted digoxin in the urine would be worthwhile. An increase in serum bilirubin concentration, and possibly some endotoxins contribute to the decreased tissue binding of digoxin, leading in turn to some reduction of apparent volume of distribution, to which an interrupted enterohepatic recirculation may also contribute (Basseches and Digregorio, 1982). As reported by Ochs et al. (1978), tissue uptake of digoxin in rabbits was more extensive than in humans. An elevated concentration of digoxin is not efficiently excreted via the impaired liver, and may be abnormal renal function, and is accumulated in the circulation, leading to an increase in the blood serum concentrations of digoxin. In the light of the present study it can be concluded that obstructive cholestasis alters the pharmacokinetics of intravenously administered digoxin, a drug predominantly eliminated in humans via the kidney. Taking into account the present data, as well as published sources concerning the rabbit, it seems that changes of digoxin clearance in obstructive cholestasis result mainly from the liver function impairment.
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