The metabolism of acitretin and isoacitretin in the in situ isolated perfused rat liver

1. The metabolism of acitretin and its 13-cis isomer, isoacitretin, has been investigated in the in situ isolated perfused rat liver in order to differentiate the action of the liver from that of the gut on the metabolism of these isomers.

2. Acitretin undergoes α-oxidation, chain shortening O-demethylation, and glucuronidation in the perfused rat liver.

3. Isoacitretin undergoes glucuronidation as the major, almost exclusive, route of metabolism in the perfused rat liver.

4. The difference in the hepatic metabolism of the cis and trans isomers of this retinoid may explain the differences in their pharmacokinetics, and may help in understanding the pharmacokinetics of related retinoids.     

Uptake and metabolism of diphenylhydantoin in the isolated perfused rat liver

The elimination of diphenylhydantoin (DPH) was studied in isolated perfused rat livers and the dose-dependent kinetics was confirmed. Kinetic analysis using a saturable two-compartmental analogue indicated that, with increasing doses, the V_max remained constant but the elimination halflife (2.8–13 min) for the metabolic component and with it the Michaelis constant (2.9–37 μg/ml) increased progressively. Thus. at first sight, the data appeared to be consistent with the hypothesis of product inhibition. However, the rapid elimination of the metabolic product as well as quantitative results of computer simulations render inhibition unlikely as an explanation for the observed features of DPH elimination. The subcellular distribution of DPH and its metabolites in the liver indicated that DPH and its hydroxylated metabolite were localized mainly in the 600g and microsomal fractions; the glucuronide was mainly in the cytosol. The results were compatible with the possibility of capacity-limiting DPH access to the drug-oxidizing enzyme system.     

The metabolism of 2-allyl-2-isopropylacetamide in vivo and in the isolated perfused rat liver

The metabolism of allylisopropylacetamide (AIA) was studied in normal and phenobarbitone (PB)-pretreated intact male rats and in rats with biliary fistula. Because of the side effects of AIA in the intact rat, the hepatic metabolism of AIA was further investigated in the isolated rat liver perfused with defibrinated rat blood firstly, to confirm the in vivo results and secondly, to further characterize some of the processes involved in the biliary excretion of drugs.At least four days were required to eliminate a single porphyrinogenic dose of 400 mg [2-¹⁴C] AIA per kg body wt from the intact rat, 70 per cent of the administered radioactivity appeared in the urine, as AIA and three metabolites A, B, and C, and 10 per cent in the faeces. AIA, 2-isopropyl-4,5-dihydroxypentanamide (AIA-glycol) and 2-isopropyl-4,5-dihydroxypentanoic acid-γ-lactone (AIA-lactone) were identified in either extracts of glucuronidase-sulphatase-hydrolysed urine. AIA and two other metabolites, D and E, were excreted in bile. The metabolism of AIA by the perfused liver appeared quantitatively similar to that in fistula rats judged by the biliary excretion and by the decline in microsomal cytochrome P-450 after AIA. PB-pretreatment of the rats increased the per cent dose excreted per hour in the bile 2 to 3-fold, enhancing the initial excretion rate of metabolite D from 4 to 5-fold and that of AIA almost 2-fold.The decrease of microsomal P-450 after AIA administration to PB-pretreated rats, previously considered to be biphasic, has been shown to have an additional component with half-life of 4 min. The rapid decline in hepatic P-450 after AIA in PB-pretreated rats correlated with an increased biliary excretion of one particular metabolite of AIA. A pharmacokinetic analysis of the biliary excretion data based on a two-compartment model shows that the rate-limiting step in the biliary excretion of both AIA and metabolite D can be adequately represented as a first-order linear reaction.     

The effects of primaquine stereoisomers and metabolites on drug metabolism in the isolated perfused rat liver and in vitro rat liver microsomes

The effect of the antimalarial drug primaquine, its stereoisomers and its proposed metabolites, on the metabolism of substrates for mixed function oxidase, has been studied in isolated perfused rat livers (IPRL) and/or in vitro microsomal suspension. Following acute administration to an IPRL preparation, racemic primaquine produced a dose related reduction in the hepatic clearance of antipyrine which at the highest dose of primaquine (5.0 mg) represented a decrease to 46% of control values. Antipyrine clearance was reduced to a comparable extent by the (+) and (-) isomers and the racemic mixture (each at a dose of 2.5 mg) with mean reductions of 45, 49 and 47%, respectively. These changes in clearance were reflected by significant increases in half-life relative to control. The apparent volume of distribution of antipyrine was unchanged in all experiments. Racemic primaquine and its (+) and (-) isomers were equipotent in inhibiting aminopyrine N-demethylase activity, producing reductions of 56, 59 and 55%, respectively, relative to control values. These three compounds also produced corresponding reductions of 73, 58 and 73% in ethoxyresorufin O-deethylase activity. The N-acetyl and 5-hydroxy derivative of primaquine produced inhibitory effects comparable to that seen for the parent drug. In contrast the carboxylic acid metabolite of primaquine, 6-desmethylprimaquine and 5-hydroxy-6-desmethyl primaquine did not influence aminopyrine N-demethylase activity. These results indicate that the propensity to inhibit drug metabolism by these primaquine related substances, is influenced by functional group substitution rather than the optical activity of the parent drug.     

The disposition of suramin in the isolated perfused rat liver

We have investigated the disposition of suramin in the isolated perfused rat liver preparation (IPRL) after the administration of suramin (18 mg, 8 muCi). At 30 min post drug administration, almost 100% of the [14C]radioactivity and unchanged suramin were located in the perfusate plasma. During the course of the study, the elimination of suramin from the IPRL was barely perceptible. The AUC0-5 hr of suramin (730.6 +/- 86.2 micrograms hr/ml) corresponded to that of [14C] radioactivity (815.1 +/- 105.5 micrograms ml/hr) at 5 hr, indicating a lack of perfusate suramin metabolites. At 5 hr only a small proportion of [14C] radioactivity was recovered from the livers (2.5 +/- 1.1%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged suramin. Sub-cellular fractionation of the homogenised livers revealed suramin to be distributed in the liposomal rich tissue fractions (10,000 g pellet, 1.6 +/- 0.8%; 105,000 g supernatant, 1.1 +/- 0.35%). Biliary excretion of [14C] radioactivity was low (2.1 +/- 0.7%), however, none could be accounted for as unchanged suramin. Previously undetected metabolites of suramin may have accounted for the unidentified biliary radioactivity.     

The disposition of pyrimethamine in the isolated perfused rat liver

We have investigated the disposition of pyrimethamine base in the isolated perfused rat liver (IPRL) preparation after the administration of pyrimethamine (0.5 mg, 5 microCi). In the first half hour of the study, pyrimethamine underwent marked hepatic uptake, thereafter perfusate plasma drug levels declined monoexponentially with a half life (t 1/2) of 3.0 +/- 1.0 hr. Area under the perfusate plasma concentration/time curve (AUC)0----infinity was 6.9 +/- 1.9 microgram/hr/ml. Pyrimethamine was found to be a low clearance compound (78.4 +/- 25.3 ml/hr identical to 8.6% of liver perfusate flow) with a large volume of distribution (267.5 +/- 55.3 ml) in the IPRL. The combined AUCS(0----5hr) for pyrimethamine (AUC 4.8 +/- 0.5 microgram/hr/ml) and pyrimethamine 3-N-oxide (AUC0----5hr 0.9 +/- 0.6 microgram/hr/ml) accounted for 57% of the total AUC0----5hr of [14C] radioactivity (10.0 +/- 2.6 micrograms/hr/ml). This indicates the presence of metabolites of pyrimethamine as yet unidentified in the perfusate. Biliary excretion of [14C] during the course of the IPRL preparations was extensive (29.0 +/- 10.3%) though only a small proportion was due to pyrimethamine and the 3-N-oxide metabolite. The majority of radioactivity in the bile was attributable to highly polar, but unidentified metabolites of pyrimethamine. At the conclusion of each experiment (5 hr), a significant proportion of [14C] radioactivity was recovered from the livers (22.9 +/- 5.3%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged pyrimethamine, with trace levels of the 3-N-oxide metabolite. Sub-cellular fractionation of the homogenized livers revealed the most pronounced localisation of pyrimethamine to be in the lipid rich 10,000 g pellet (13.0 +/- 2.6%), the remainder being distributed equally between the 105,000 g pellet and supernatant. Neither pyrimethamine, [14C] radioactivity, nor pyrimethamine 3-N-oxide were extensively taken up by red cells throughout the study. Therefore, the large volume of distribution (267.5 +/- 55.3 ml) underlines the extent of pyrimethamine localisation in the liver.     

Reductive drug metabolism in isolated perfused rat liver under restricted oxygen supply

1. Hepatic azo and nitro reductase activities were studied in the perfused rat liver under normal and restricted oxygen supply. 2. Formation of sulphanilamide or p-aminobenzoic acid from neoprontosil or p-nitrobenzoic acid under aerobic conditions of liver perfusion was negligible, even at a reduced oxygen saturation of a pO2 of 300 mm Hg in the haemoglobinfree perfusion system. At a pO2 of 200 mm Hg reductase activities were almost maximal. 3. Conjugation of sulphanilamide (0-08 mM) was similar under aerobic and anaerobic conditions. Hepatic elimination of p-aminobenzoic acid (0-08 mM) showed an oxygen-dependent increase for 15 min after addition of substrate. 4. p-Nitroanisole demethylation was inhibited 80% under hypoxic perfusion at 200 mm Hg pO2 and was completely inhibited after gassing with anoxic mixtures. 5. Restitution of aerobic conditions after 30 min anaerobic perfusion restored hepatic respiration, lactate pyruvate ratio, and pH value to levels found under aerobic conditions, but bile flow remained 50% reduced.     

地尔硫对大鼠离体肝脏灌流中安替比林代谢的影响

以安替比林（AP）为模型药,采用大鼠离体肝脏灌流模型（IPRL）,研究钙离子拮抗剂地尔硫本（DZ）对AP代谢的影响。方法：15只雄性Sprague-Dawley大鼠随机分为3组,A、B组灌胃（iP）生理盐水3d,第4天分别用5mgAP及加用2mgDZ循环灌流3h,C组ipDZ（100·kg-1）3d,第4天同B组灌流。HPLC法测定灌流液中AP及其代谢物的浓度。结果：B、C组中,AP的T1/2从（127．9±9.4）min分别延长至（2742±33.6）和（303.8±80．2）min（P＜0．01）;Cls从（0．67±0.08）ml·min－1分别减少至（0．36±0.06）和（0．34±0．09）ml·min－1（P＜0．01）;4-羟基AP的AUC0-3h分别降低73．95％和68.1％（P＜0．01）;3-羟甲基AP和N-去甲基AP和没有明显变化（P＞0．05）。结论：DZ明显抑制TAP在IPRL中的消除,且选择性地抑制AP的4-羟基化代谢途径。     

Carbaryl metabolism is inhibited by cimetidine in the isolated perfused rat liver and in man

The potential for cimetidine to alter the metabolism of carbaryl has been investigated in the isolated perfused rat liver and in man. In the isolated perfused rat liver, carbaryl was removed by a high intrinsic clearance process, which was inhibited in a dose dependent fashion by cimetidine in the concentration range of 60-240 micrograms/ml. The pharmacokinetic profile in one subject for orally administered carbaryl was altered following administration of cimetidine (200 mg every eight hours for three days). The peak carbaryl level occurred 25 minutes later and was increased by twofold, due to a 46% reduction in apparent oral clearance. The observations indicate that cimetidine can inhibit the metabolism of carbaryl in both rat and man. These data suggest that administration of cimetidine to individuals exposed to carbaryl could result in enhanced toxicity.     

Disopyramide elimination in the isolated perfused rat liver

The elimination kinetics of disopyramide, [14C]disopyramide and [2H]disopyramide have been studied in the isolated perfused rat liver. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0.3-7.5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity. At doses of 0.3 and 7.5 mg the kinetics of [2H]disopyramide showed a small isotope effect probably of negligible importance.     

Disopyramide elimination in the isolated perfused rat liver

1. The elimination kinetics of disopyramide, [¹⁴C]disopyramideand [²H]disopyramide have been studied in the isolated perfused rat liver.

2. Disappearance of disopyramide from perfusate was dose- and time-dependent over the dose range 0-3-7-5 mg. Although the mechanism underlying these observations is unclear, the data are consistent with the presence of enzyme saturation and product inhibition.

3. Biliary secretion of conjugated metabolites appeared to be the rate-limiting step in the perfusate clearance of total radioactivity.

4. At doses of 0-3 and 7-5 mg the kinetics of [²H]disopyramide showed a small isotope effect probably of negligible importance.     

Metabolism of garlic constituents in the isolated perfused rat liver.

The metabolic and kinetic behaviour of different garlic (Allium sativum L., Alliaceae) constituents were investigated in the isolated perfused rat liver, using aqueous extracts of garlic powder as well as isolated allicin, the main product of the enzymatic degradation of alliin. Allicin (allyl thiosulfinate) showed a remarkable first pass effect and passed the liver unmetabolized only at high concentrations which caused considerable cell injuries. Diallyl disulfide and allyl mercaptan were identified as metabolites of allicin, whereby diallyl disulfide probably is the metabolic precursor of allyl mercaptan as shown by perfusion with diallyl disulfide alone. The metabolites diallyl disulfide and allyl mercaptan could be determined in the perfusion medium as well as in the bile and the liver tissue. Other degradation products of garlic were also investigated in this model. Ajoenes and vinyldithiins were detected in perfusion medium after liver passage but no metabolites of them could be identified up to now.     

Cadmium toxicity in the isolated perfused rat liver

Isolated perfused livers from male and female Sprague-Dawley rats were exposed to cadmium chloride (50 and 200 microM). Acute hepatotoxicity was investigated by measuring cadmium-induced changes in bile flow, urea synthesis and alanine aminotransferase (ALT) leakage. Cadmium-induced lipid peroxidation was estimated by formation of conjugated dieners and thiobarbituric acid (TBA) reactants. Cadmium, at both concentrations, caused a rapid decrease in bile flow (within 40 min) and complete cholestasis within 70 min exposure in livers perfused from both male and female rats. Cadmium exposure (50 and 200 microM) also resulted in the leakage of ALT into the perfusate within 60 min. In contrast, exposure of isolated rat hepatocytes to as high as 500 microM cadmium did not result in enzyme leakage until 180 min exposure. Sex differences in cadmium-induced cholestasis and ALT leakage were not observed at these concentrations. Malondialdehyde was not detected in the perfusate nor were conjugated dienes detected in liver tissue following 90 min cadmium exposure. These data demonstrate that the isolated perfused rat liver (IPRL) is a sensitive system in which to study chemically induced hepatotoxicity. Cadmium rapidly causes functional alterations and cellular damage in perfused livers from both male and female rats. Cadmium-induced liver injury was apparently not related to lipid peroxidation.