Different Effects of Microsomal Enzyme Inducers on the Biliary Excretion of Digoxin1

Abstract The effects of pretreatment with spironolactone, phenobarbital and 3,4–benzpyrene on biliary excretion of digoxin was studied in the isolated perfused rat liver system after a single dose of ³H–digoxin. After spironolactone pretreatment (100 mg/kg intraperitoneally for 4 days) the treated group excreted into bile in 45 min. 67 % of the administered dose compared to about 27 % in the control group. Since there was no significant increase in bile flow at any of the time periods, the enhanced biliary excretion of digoxin was entirely due to the increase in the bile level of digoxin or its metabolites or both. Pretreatment with phenobarbital (75 mg/kg intraperitoneally for 4 days) resulted in a slight increase in bile level of tritium at 15 min. and in bile flow, which could account for the doubled amount of digoxin excreted by the treated group, as compared to the control group at 45 min. Bile flow and biliary excretion of digoxin were unaffected by pretreatment with 3,4–benzpyrene (20 mg/kg intraperitoneally for 4 days). The results suggest that the enhancing effect of spironolactone and phenobarbital on biliary excretion of digoxin is a result of different mechanisms of action, which can not be directly related to the induction of microsomal drug metabolism.     

Effects of Quinuronium Sulfate Pre-exposure on Hepatic Excretion of Sulfobromophtalein (BSP) as Measured by Isolated Perfused Rat Liver

Abstract: Effects of quinuronium sulphate pre-exposure on hepatic excretion of sulfobromophthalein (BSP) as measured by isolated perfused rat liver was investigated. Seventeen rats were used in the study. Rats were divided into controls (Group 1, n = 5), low quinuronium treated animals (Group 2, n = 6), and high quinuronium treated animals (Group 3, n = 6) receiving 0, 1.5 or 2.3 mg quinuronium sulphate/kg intraperitoneally, respectively. Each animal was dosed 30 min. before sacrifice. After 30 min. of equilibrium perfusion, BSP was introduced and the experiment continued for 2 hours. Perfusate and bile were collected at designated time intervals and the volume of bile was noted. BSP in perfusate and bile was assayed. Half-life in minutes (t_1/2$pLS.E.) of BSP disappearance from perfusate in groups 1, 2 and 3 were 12.5$pL1.8, 13.7$pL1.0 and 16.8$pL3.3, respectively. Group 2 and 3 rats had a significant decrease of BSP excretion in bile (P<0.01) compared to group 1 rats. Bile volumes decreased in both quinuronium pre-exposed groups of rats compared to control rats. These observations indicate that quinuronium affects liver function.     

Disposition of Bismuth in the Rat. II. Pharmacokinetics and Biliary Excretion

The pharmacokinetics and biliary excretion profile of intravenously administered bismuth ions were investigated in male Sprague Dawley rats. The data indicated that in the dose range studied, the percentage of dose excreted in urine ranged from 58 to 63%. The mean residence time for bismuth ions was 3.93, 4.07, and 5.45 hr for the 0.5, 0.75, and 1.0 mg/kg dose, respectively, while the volume of distribution at steady state was 0.75, 1.24, and 1.38 L/kg for the three doses. Blood clearance values ranged from 0.2 to 0.32 L/hr/kg. Blood bismuth ion concentrations toward the latter part of the sampling schedule indicated significant variability. The bile-to-blood concentration ratio of intravenously administered bismuth exceeded 1.0 for the three doses studied, suggesting that transport of bismuth from blood to bile may be carrier mediated.     

Statistical Moment Analysis of Hepatobiliary Transport of Phenol Red in the Perfused Rat Liver

A new experimental system was applied to study hepatobiliary transport of drugs. Rat livers were perfused using a single-pass technique, and phenol red was momentarily introduced to this system from the portal side. Outflow dilution patterns of phenol red were analyzed using statistical moment theory, and kinetic parameters of hepatic distribution and elimination of phenol red were calculated from moments, namely, the hepatic extraction ratio (E _i) and elimination rate constant (k _el,i). A larger distribution volume (V _i) was obtained for phenol red than for ¹³¹I-human serum albumin (HSA) and ⁵¹Cr-red blood cells (RBC), indicating its extravascular diffusivity. The biliary excretion of conjugated phenol red was delayed relative to that of the free agent. The larger biliary mean transit time (t _bile,conj.) represents the processes of biliary transport and intrahepatic metabolism. Further, the effects of dose and perfusion temperature on the hepatobiliary transport of phenol red were determined. With high doses or low perfusion temperatures (20 and 27°C), E _i, k _el,i, and intrinsic clearance (CL_int,i) of phenol red and biliary recovery of free and conjugated phenol red (F _bile,free, F _bile,conj) significantly decreased. The temperature-dependent and saturable processes in hepatic uptake, metabolism, and biliary excretion of phenol red were assessable to moment analysis.     

Disposition of Glycosidase Inhibitors in the Isolated Perfused Rat Liver: Hepatobiliary and Subcellular Concentration Gradients of 1-Deoxymannojirimycin and N-Methyl-1-Deoxynojirimycin

The hepatic disposition of two glycosidase inhibitors was studied in the isolated perfused rat liver and after subcellular fractionation. The mannosidase inhibitor 1-deoxymannojirimycin (dMM) and the glucosidase inhibitor N-methyl-1-deoxynojirimycin (MedNM) exhibited minimal binding to albumin and reached liver concentrations that approximately equaled their medium concentrations, after 30 min (MedNM) or 90 min (dMM). Within 2 hr 0.5% of the dose of MedNM and 2.9% of dMM were excreted in bile. No metabolites were found for MedNM, whereas minor (bio)degradation was inferred for dMM. After subcellular fractionation, dMM and MedNM were found predominantly in the cytosolic fraction. Compared to the other paniculate fractions, MedNM was elevated in the microsomal fraction, and both compounds were slightly enriched in the lysosomal fraction. We conclude that dMM and MedNM will likely inhibit liver enzymes when sufficiently high plasma levels are reached.     

Biliary Excretion of Bisacodyl and Picosulphate in Man: Studies in Gallstone Patients after Biliary Tract Surgery

Abstract: Bisacodyl (=BIS) and picosulphate (=PICO) have been given perorally to postoperative gallstone patients, who have undergone biliary tract surgery with the insertion of an indwelling T-tube. The doses corresponded to 7.7 mg of their common free diphenol desacetylbisacodyl (=DES). The bile was sampled in hourly fractions from the external limb of the T-tube; these fractions were analysed by a modification of the HPLC method previously used to study biliary excretion in the rat. In the BIS-patients (n=8), DES in conjugated form occurred in significant concentration already in the first fractions; peak excretion values equivalent to 4–8 μg DES/ml bile were reached in 2–5 hours. Unchanged BIS could not be detected, and the concentration levels of unconjugated DES were insignificant. The PICO-patients (n=8) on the other hand showed low DES concentrations (conjugated+free≤0.5 μg DES/ml) in all fractions, or low initial concentrations followed by a more or less pronounced rise in the later fractions. These results are, qualitatively, as in the rat. However, the dose fractions excreted in bile (assuming a total hepatic output of 50 ml/hour) seem smaller than those to be expected from rat experiments, at least as far as BIS is concerned.     

Metabolism of I4C-Codeine in the Isolated,Perfused Rat Liver

Abstract: The metabolism of ¹⁴C-codeine in the isolated rat liver was studied in single-pass and recirculation perfusion experiments. The perfusate, a semi-synthetic medium with bovine erythrocytes, was delivered at a constant rate (12 ml/min.) and contained codeine in the range of 3–57 nmol/ml. Samples of perfusate were collected and analyzed for codeine and its metabolites after extraction and thin-layer chromatographic separation. In single-pass perfusion, steady state was reached within 20 min. The codeine concentration in the effluent perfusate varied from 17 to 48% of that in the affluent corresponding to extraction ratios of 0.83 to 0.52. There was a significant negative correlation between codeine dose and extraction ratio (r = 0.86, P<0.05, N = 6). The steady state concentration of free and conjugated morphine made a total of 21 to 49% of the molar concentration of codeine at the inflow side. The recovery of radioactivity at the end of the perfusions was on an average 89%. In recirculation perfusion experiments the codeine extraction ratios varied from 0.65 to 0.35. The amount of free morphine in the reservoir increased to a maximum within 25–45 min. Our results suggest a relatively high hepatic first-pass metabolism of codeine in the rat which is apparently dose-dependent. The quantitatively most significant metabolites of codeine are morphine and conjugated morphine. The rate and extent of morphine formation is compatible with the hypothesis that metabolically produced morphine may be responsible for the analgesic effect of codeine.     

Biliary Excretion of Diazepam and its Metabolites in Man

Abstract: Ten mg diazepam was given intravenously to 12 patients with a T-tube in the common bile duct after choledochotomy (Group I) and to 10 patients after cholecystectomy (Group II). The concentrations of diazepam, of its main metabolite, N-demethyldiazepam, and of free oxazepam in the plasma of both groups and the conjugated and free concentrations of diazepam, N-demethyldiazepam, and oxazepam in the bile in the Group I were measured by gas chromatography. In Group I significantly lower plasma diazepam concentrations were obtained as compared with Group II indicating an enterohepatic circulation of diazepam. There was no significant difference in the concentrations of N-demethyldiazepam in the plasma between the two groups. In Group I the patients had frequently more free oxazepam in their plasma than in Group II. The main conjugated metabolite in the bile was N-demethyldiazepam (about 74 %). Traces of diazepam were in the conjugated form, but no conjugated oxazepam was found in the human bile. The enterohepatic circulation of diazepam and its metabolites may be partly responsible for the prolonged effects of diazepam.     

Hepatic Distribution and Clearance of Antisense Oligonucleotides in the Isolated Perfused Rat Liver

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. Methods. The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5-TAC-GCC-AAC-AGC-TCC-3), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr; perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. Results. MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [Cl = 1.05 ± 0.21 mL/min; extraction ratio = 0.06 ± 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 ± 2.2%) than for the MP oligonucleotide (53.0 ± 4.7%). Conclusions. Backbone modifications significantly influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.     

不同内酯型比例9-硝基喜树碱对胆汁排泄的影响

目的考察9-硝基喜树碱(9-nitrocamptothecin,9-NC)不同形式(内酯型或羧酸盐型)给药对胆汁排泄的影响。方法建立HPLC测定胆汁中9-NC浓度的方法,比较不同内酯型比例的9-NC溶液以4 mg·kg?1剂量静脉注射后大鼠胆汁中原形药物的排泄量。结果静脉注射100%,75%,50%,25%,0%内酯型比例的9-NC在8 h内的胆汁累积排泄百分数分别为(7.03±2.23)%,(13.36±0.83)%,(22.68±4.83)%,(28.01±6.71)%,(32.65±2.82)%。结论 9-NC羧酸盐型更易经胆汁途径排泄。     

Effect of 3,4-Dihydroxyphenylpyruvic Acid on the Metabolism of L-DOPA in Isolated Perfused Rat Liver

Abstract: The effect of 3,4-dihydroxyphenylpyruvic acid (DHPPA) on the hepatic metabolism of L-DOPA was investigated in isolated perfused rat liver. DHPPA decreased the initial hepatic extraction and prolonged the elimination half-life of L-DOPA when they were added simultaneously at the ratio 1:4 (L-DOPA: DHPPA) to the perfusate. At the ratio 1:1 DHPPA had no effect on the elimination half-life, but decreased the initial hepatic extraction of L-DOPA. When L-DOPA was added alone the initial loss of L-DOPA was 30% at 5 min. At that time the perfusion medium had passed the liver 2.5 times. Between 5 to 60 min. about 5% of the dose was extracted in a single pass through the liver. DHPPA added alone or together with L-DOPA was rapidly converted to L-DOPA, only about half of the DHPPA dose remained unmetabolized in the “plasma” at 2.5 min. The main metabolites in bile of all the compounds tested were conjugates of homovanillinic acid and 3,4-dihydroxyphenylacetic acid.     

Enterohepatic Circulation,Urinary Excretion and Laxative Action of Some Bisacodyl Derivatives after Intragastric Administration in the Rat

Abstract: Bisacodyl (BIS), the parent diphenol (DES) and its sulphuric acid di-ester (picosulphate = PICO) were given by stomach tube to fasted rats at a dose of 3.1 μmol/100 g rat. Bile was sampled in the periods 0–6, 6–12 and 12–18 hrs after drug administration, and assayed for total diphenol (= free + conjugated) by HPLC. Mean fractions (% of dose±S.E.M.) excreted in 5 rats per compound and period were: BIS 74.0±4.7, 51.9±7.9 and 30.8±2.5; DES 41.2±4.3, 46.8±4.7 and 25.1±2.5; PICO 9.0±0.9, 26.0±5.4 and 19.6±3.1. Only minor amounts were excreted as free diphenol. Urine samples taken by bladder puncture and assayed as above furthermore showed that the renal excretion of total diphenol was insignificant compared to the amounts excreted in bile. Practically no diphenol was present in urine 0–6 hrs after the administration of PICO. In experiments with BIS and DES at 0.85 μmol/100 g, total diphenol excreted in bile during 0–6 hrs was: BIS 67.1±2.6 (n = 5); DES: 55.4±3.0 (5). - The latency time for laxative effect was studied in groups of 10 unfasted rats per compound. Cumulative time response curves showed that PICO caused diarrhoea more promptly at 0.85 μmol/100 g than either BIS or DES. In most rats, this delayed action of BIS and DES persisted also at 1.7 μmol/100 g. At 3.1 μmol/100 g, however, the majority of the rats reacted as promptly to these two compounds as to PICO. These results are discussed in relation to the biliary excretion experiments, and interpreted in terms of the relative importance at the different dose levels of: 1. The enterohepatic recirculated fraction, and 2. The non-absorbed fraction, which passes directly to the large intestine. For PICO, the latter fraction is the single determinant of the effect, which is triggered when the di-ester is being hydrolyzed to active diphenol in this part of the GI-tract.     

Metabolism of Roxithromycin in the Isolated Perfused Rat Liver

Roxithromycin is a macrolide antibiotic with high clinical potency. N-Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver. After addition of roxithromycin (30 μM) to the perfusion medium the parent compound and one major metabolite were detected in bile by high-performance liquid chromatography. The metabolite was identified as monodesmethylated roxithromycin by mass spectrometric analysis. Onset of biliary excretion of native roxithromycin was fast, reaching a maximum (130.52 ±43.88 pmol g^?1 min^?1) after only 10 min, whereas excretion of the metabolite was delayed (maximum 75.83 ± 11.92 pmol g^?1 min^?1 at 30 min). The cumulative excretion of roxithromycin and its metabolite into bile during the 60 min of application amounted to only 1.09 ± 0.30 and 0.64 ± 0.22% of the roxithromycin cleared from the perfusate during the same time. The liver content was 0.48 μmol (g liver)^?1, indicating high retention within the organ. No release of the metabolite into the perfusate was detected. In conclusion, this study has demonstrated the importance of phase-I metabolism for the biliary excretion of roxithromycin in rat liver. These findings might be predictive of roxithromycin biotransformation and biliary excretion in man.     

The Mechanism of Biliary Excretion of Methyl Mercury: Studies with Methylthiols

Abstract: The S-methylated derivatives of N-acetylpenicillamine, thiola and cysteine as well as methyl iodide decreased biliary excretion of methyl mercury markedly. Excretion of sulfhydryl in bile was not influenced by S-methyl-cysteine, S-methylthiola, S-methyl-N-acetylpenicillamine or a low dose of methyliodide (0.5 mmol/kg body weight). This seems to indicate that coupling of methyl mercury to glutathione in the liver before biliary excretion is a glutathione S-transferase dependent reaction. It also indicates that the methylthiols tested, or metabolites of these compounds are likely to be inhibitors of S-transferase. The effect of S-methylcysteine and low doses of methyl iodide probably reflects glutathione S-transferase inhibition as both compounds are metabolized to the S-transferase inhibitor S-methylglutathione in the liver. A higher dose of methyl iodide (1 mmol/kg body weight) seems to deplete the liver of reduced glutathione through S-methylation as illustrated by decreased biliary excretion of sulfhydryl. S-methylthiola and S-methyl-N-acetylpenicillamine are metabolized in the liver to unknown components which are excreted in bile. Whether S-methylthiola and S-methyl-N-acetylpenicillamine are inhibitors of S-transferase themselves or cause inhibition through metabolites cannot be stated from the present investigation.     

Biliary Excretion of Chromium in the Rat: A Role of Glutathione

Abstract: The relative amount of chromium excreted in rat bile after injection of Cr-III is much less than after injection of Cr-VI, about 0.1% and from 6–8% during 5 hours respectively, for corresponding dose levels. The liver to bile ratio was 50–100 for Cr-III injection, for Cr-VI the ratio was 2–3. With doses up to 18 μmol Cr/kg, only Cr-III was found in bile even after injection of Cr-VI. Glutathione depletion of the liver with cyclohexene oxide decreased chromium excretion in bile. Such treatment also decreased the reduction of Cr-VI to Cr-III in the liver cell as only Cr-VI was found in bile. A different distribution of Cr-III in the liver dependent on whether derived from Cr-VI or taken up by the liver as such must be assumed. Taking into account the usual low penetration of biological membranes by Cr-III, a possible active transport mechanism or a specific diffusable Cr-III compound must be postulated.     

Extensive Biliary Excretion of the Model Opioid Peptide [D-PEN2,5] Enkephalin in Rats

Purpose. This study was designed to test the hypothesis that the enzymatically stable opioid peptide, [D-pen^2,5] enkephalin (DPDPE), is excreted extensively into bile. Methods. Following an i.v. bolus dose of DPDPE (10 mg/kg) to rats, concentrations of DPDPE in serum, bile, liver homogenate and urine were measured by a novel capillary zone electrophoresis method. Data were analyzed to recover the fundamental pharmacokinetic parameters (volumes of distribution; distribution and elimination rate constants governing DPDPE systemic and biliary disposition). Parallel in vitro experiments were performed to evaluate the partitioning of DPDPE between erythrocytes and plasma, as well as to assess the degree of binding of DPDPE to serum proteins. Results. The majority of the administered dose (~80%) was recovered from bile as intact peptide. DPDPE disposition was best described by a two-compartment model with Michaelis-Menten elimination (K_m: 37.5 ± 11 g/ml; V_max: 1143 ± 368 g/min/kg) from the central compartment into bile, suggestive of an active hepatic transport system. DPDPE was associated with a distributional space of 486 ± 62 ml/kg. In vitro incubation of DPDPE with whole blood showed that ~65% of the peptide was associated with erythrocytes. The difference between concentrations of DPDPE in erythrocytes and plasma was statistically significant (29.2 ± 4.9 vs. 18.1 ± 3.1 g/ml, p < 0.05), but not between whole blood and plasma (21.3 ± 2.8 vs. 18.1 ± 3.1 g/ml, p > 0.05). Concentration-independent binding of DPDPE to serum proteins was evidenced between 10 and 100 (g/ml, with an unbound fraction of 0.517 ± 0.182. Conclusions. DPDPE undergoes extensive biliary excretion after i.v administration in rats. The apparent nonlinearity in the biliary excretion of DPDPE revealed by the pharmacokinetic modeling strongly suggests the existence of an active transport system(s) in hepatocytes which may mediate the rapid disappearance of DPDPE from the systemic circulation.     

Microsomal Cytochrome P450 Levels and Activities of Isolated Rat Livers Perfused with Albumin

Purpose. We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-. Here, we hypothesize that BSA-induced increase in the release of TNF-, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. Methods. Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. Results. In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2C11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. Conclusion. Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.     

Induction of Liver Microsomal Cytochrome P-450 and Associated Monooxygenases by Octachlorostyrene in the Rat

Abstract: The inducing effects of octachlorostyrene (OCS) and hexachlorobenzene (HCB), chlorinated hydrocarbon contaminants found in fish, on the microsomal cytochrome P-450 system were studied in rats. Administration of single doses of OCS and HCB at 50mg/kg intraperitoneally and higher led to increases in microsomal protein and cytochrome P-450 content, cytochrome P-450 reductase, ethylmorphine N-demethylase, 4-nitroanisole O-demethylase, acetanilide 4-hydroxylase, but not in benzo(a)pyrene hydroxylase activities. Similar patterns of induction were seen after 14 days repeated intraperitoneal or oral dosing. Sodiumdodecylsulfate polyacrylamide gel electrophoresis of microsomes from OCS and HCB treated rats showed increases in proteins similar to those found after phenobarbital (PB) induction. Under the present experimental conditions OCS and HCB were found to be PB-type inducers.