Investigations on the biotransformation of mebendazole using an isolated perfused rat gut system.

1. The intestinal metabolism of the benzimidazole, mebendazole (MEB), has been investigated using isolated perfused jejunal segments of rats. Significant absorption and intestinal metabolism of the substance was observed. 2. The metabolites, the reduced alpha-hydroxy-analogue, its glucuronide, and the decarbamoylated 2-amino-analogue, were transported into the resorbate collected at the serosal side or were resecreted into the gut lumen. 3. The intestinal decarbamoylation of mebendazole increased up to 20-fold after pretreatment with 3-methylcholanthrene (MC), and complete re-secretion of this metabolite into the gut lumen led to a total loss of the absorption of mebendazole and metabolites across the gut wall. 4. The results indicate the ability of the gut to metabolize mebendazole by phase I and II reactions. 5. An almost complete loss of bioavailability after induction of the gut enzyme system by MC was observed.     

Intestinal uptake of genistein and its glycoside in the rat using various isolated perfused gut segments

Genistein receives much attention because of its potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of this compound like, for instance, their intestinal uptake by humans and systematic bioavailability. In this study, the fate of the absorption of genistein and its glycoside has been analysed in various isolated perfused gut segments of the rat. In all perfused gut segments the transport of genistein was higher compared to its glycoside. Furthermore, it appeared that the resorbate (i.e. serosal side) concentration of genistein was the highest in ileac segments, whereas the transport of genistein in the various other segments tested showed no difference between intestinal compartments. Less than 0.2% of genistin appeared in the resorbate fluid of all isolated gut segments. The main site of metabolism of genistein and its glycoside appears to be located in the jejunal compartment of the rat gut. About 38% of genistein and about 29% of genistin metabolised within 2h of perfusion. In the ileac and colonic intestinal segments, genistein metabolised for only 10%. For the first time, this study demonstrated that genistin could be metabolised by epithelial cells present in isolated colonic segments. However, the metabolites of genistin did not occur at the serosal side (the resorbate) of isolated colonic segments. We assume that there is no absorption of genistin and/or its metabolites in or through colonic tissue of the rat.     

The uptake of oestrone from the lumen of the isolated perfused rat gut

1. In the isolated perfused rat gut oestrone was not taken up from the perfusate but was rapidly taken up from the gut lumen when administered in dimethyl sulphoxide or in 10% acetone in water.

2. Uptake from corn oil was much slower and the rate was concentration-dependent.

3. When given in dimethyl sulphoxide or in aqueous acetone, the residual oestrone in the gut lumen was largely unchanged, with only about 10% being present as the glucuronide. Of the oestrone in the gut perfusate, some 40% was glucuronidated.

4. No evidence was obtained for any reduction or hydroxylation, or for any sulphation, during uptake of oestrone by the gut.

5. No secretion of oestrone or its metabolites into lymph could be detected.     

The uptake of oestrone from the lumen of the isolated perfused rat gut

1. In the isolated perfused rat gut oestrone was not taken up from the perfusate but was rapidly taken up from the gut lumen when administered in dimethyl sulphoxide or in 10% acetone in water. 2. Uptake from corn oil was much slower and the rate was concentration-dependent. 3. When given in dimethyl sulphoxide or in aqueous acetone, the residual oestrone in the gut lumen was largely unchanged, with only about 10% being present as the glucuronide. Of the oestrone in the gut perfusate, some 40% was glucuronidated. 4. No evidence was obtained for any reduction or hydroxylation, or for any sulphation, during uptake of oestrone by the gut. 5. No secretion of oestrone or its metabolites into lymph could be detected.     

Uptake and biotransformation of ibuterol and terbutaline in isolated perfused rat and guinea pig lungs

The lung uptake and biotransformation of [³H]terbutaline and [³H]ibuterol (diisobutyrate ester of terbutaline) was studied using isolated perfused and ventilated rat and guinea pig lungs. The lung extraction ratio, as calculated from the concentration of drug in the inflowing and outflowing medium, in single pass studies of ibuterol ranged from 0.32 to 0.41 at inflowing concentrations 1 × 10^?5 and 1 × 10^?7M and that of terbutaline ranged from 0.013 to 0.021. Ibuterol was hydrolyzed to terbutaline but no further biotransformation of terbutaline was found. The lung clearance of ibuterol (1 × 10^?7M) was estimated to 0.092 ml/sec. When ibuterol (1 × 10^?7M) was infused together with eserine, an esterase inhibitor, the clearance of ibuterol decreased to 0.088 ml/sec (eserine 1 × 10^?5M) and 0.045 ml/sec (eserine 1 × 10^?4M). The latter value was significantly lower (P < 0.001) than control.     

14CH3-N-pethidine biotransformation in the rat and rabbit isolated perfused liver during the pathological process.

The demonstration of decreased hydrolysis and demethylation of pethidine in the liver subcellular fractions in the course of acute radiation sickness was not verified under the conditions of perfusion of the isolated liver. An attempt was made to interpret the discrepancy in the findings by a change in the intensity of uptake of pethidine and its metabolites in liver tissue after irradiation.     

An investigation of the relationship between the liver and brain using an isolated perfused rat brain preparation

The pathogenesis of portal-systemic encephalopathy (PSE) and hepatic encephalopathy (HE), disorders of the brain attributed to abnormal liver function, are poorly understood. This study was conducted to examine if a fundamental, and possibly exclusive, homeostatic interrelationship exists between the liver and brain that might deteriorate during liver failure to result in the syndrome of PSE and HE. An isolated organ perfusion circuit was devised to accommodate an isolated rat brain and an isolated rat liver preparation perfused concomitantly. The survival time of the brain preparation was measured by the maintenance of the spontaneous electroencephalocorticogram and was extended from a median survival time of 35 min (range 22 to 53 min), when perfused alone, to 210 min (range 172 to 480 min), when perfused simultaneously with a liver. Also, concomitant perfusion with an isolated rat liver reduced perfusate glucose concentrations from 200 mg% to a range between 45 to 60 mg%. These data support our hypothesis that a homeostatic interrelationship exists between the liver and brain that is independent of other metabolic influences; disturbance of this relationship may contribute towards the syndrome of PSE and HE.     

Pharmacokinetic consequences of time-dependent inhibition using the isolated perfused rat liver model

Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibition (TDI) and these have been reported to predict accurately for several marketed drugs. However, their robustness using in-depth, carefully controlled pre-clinical studies has yet to be established. In the current study, the isolated perfused rat liver was employed to investigate the effects of TDI under carefully controlled conditions. A five-compartmental model was used to describe the observed data and bring context to in vitro TDI data (kinact and KI). Co-administration of midazolam with troleandomycin, mifepristone, erythromycin and the discovery compound, AZ-X, increased midazolam area under the curve (AUC) 3.2-, 2.5-, 1.6- and 1.0-fold, respectively, compared with AUC increases of 1.8-, 1.4-, 1.2- and 1.1-fold predicted by the model. These experimental findings, whilst modest in overall effect, support the use of this model in the rat and it is proposed that projections can be made for the likely clinical impact of novel time-dependent inhibitors in man based on predicted human pharmacokinetics and TDI potency determined in vitro.     

Pharmacokinetic consequences of time-dependent inhibition using the isolated perfused rat liver model

Mathematical models exist to describe the pharmacokinetic changes caused by time-dependent inhibition (TDI) and these have been reported to predict accurately for several marketed drugs. However, their robustness using in-depth, carefully controlled pre-clinical studies has yet to be established. In the current study, the isolated perfused rat liver was employed to investigate the effects of TDI under carefully controlled conditions. A five-compartmental model was used to describe the observed data and bring context to in vitro TDI data (k_inact and K_I). Co-administration of midazolam with troleandomycin, mifepristone, erythromycin and the discovery compound, AZ-X, increased midazolam area under the curve (AUC) 3.2-, 2.5-, 1.6- and 1.0-fold, respectively, compared with AUC increases of 1.8-, 1.4-, 1.2- and 1.1-fold predicted by the model. These experimental findings, whilst modest in overall effect, support the use of this model in the rat and it is proposed that projections can be made for the likely clinical impact of novel time-dependent inhibitors in man based on predicted human pharmacokinetics and TDI potency determined in vitro.     

Effect of cadmium on bromosulfophthalein kinetics in the isolated perfused rat liver system.

Bromosulfophthalein (BSP) is a relatively nontoxic organic anion used as an in vivo indicator of liver performance. Elimination of BSP via the biliary system following iv injection requires dissociation from albumin in plasma, translocation across the sinusoidal membrane, conjugation with glutathione within the hepatocyte, translocation across the bile canalicular membrane, and excretion in bile. The effects of cadmium (Cd), anin vivo hepatotoxicant in rats, on BSP kinetics in the isolated perfused rat liver (IPRL) were studied to investigate the interaction between liver toxicity and BSP kinetics. Livers were isolated from male Fisher 344 rats. After a 30-min period for acclimation to the IPRL system, livers were dosed with Cd (as cadmium acetate), in the presence of 0.25% bovine serum albumin, to give initial concentrations of 10 and 100 microM. Sixty min after Cd dosing, the IPRL system was dosed with BSP to give an initial concentration of 150 microM and the elimination kinetics of BSP from the perfusion medium were monitored. Cadmium concentrations in livers at the end of the experiments were 60 +/- 4 and 680 +/- 210 micro mol/kg for the 10 and 100 microM doses, respectively. Exposure to 10 microM Cd for 60 min resulted in a reduction in bile flow, no significant effect on lactate dehydrogenase (LDH) leakage, and slight effects on BSP clearance. Similar studies following exposure to 100 microM Cd showed a dramatic decrease in bile flow with complete cholestasis 60 min after Cd addition. LDH leakage into perfusion medium at the end of the experiment was less than 10%, indicating that Cd affected bile production well before the liver showed significant signs of necrosis. Clearance of BSP from the perfusion medium was dramatically reduced. Taken together, the data indicate that Cd has a significant effect on the kinetics of BSP in the IPRL and the dominant effects were mediated through the cholestatic effect of Cd.     

Factors affecting the biotransformation of the pesticide parathion by the isolated perfused mouse liver

Single-pass perfusion of mouse livers in situ with the phosphorothioate pesticide parathion resulted in formation of paraoxon, p-nitrophenol, p-nitrophenyl sulfate, and p-nitrophenyl-beta-D-glucuronide. At a perfusate bovine serum albumin (BSA) concentration of 4% (fraction of unbound parathion = 0.04), and a flow rate of 3.2 ml/min/liver, the half-life associated with the approach to steady state of parathion was 6.2 min (SD = 0.4), whereas at steady state the extraction ratio of parathion was 0.19 (SD = 0.03). Alterations in perfusate flow rates had no discernable effects on metabolism of parathion. However, lowering the BSA perfusate concentration to 1.0% (fraction of unbound parathion = 0.12) significantly prolonged the half-life for the approach to steady state while increasing the steady state extraction ratio to 0.49 (SD = 0.08). At perfusate BSA concentrations below 1%, steady state conditions with respect to parathion could not be achieved during the 50-min perfusions. These results suggest that binding of parathion to BSA hinders its biotransformation by mouse livers perfused in situ, probably by limiting the availability of free pesticide to metabolic sites. Consequently, its elimination by the liver is insensitive to changes in hepatic blood flow. However, exclusion of BSA from the perfusate resulted in partitioning of all parathion in perfusate into the liver, leading to high concentrations of parathion within liver and preventing biotransformation of this pesticide.     

Mathematical model for cadmium kinetics in the isolated perfused rat liver system

The isolated perfused rat liver (IPRL) preparation has previously been used to investigate cadmium kinetics. A mathematical model which has been developed to simulate cadmium kinetics in the IPRL is described. The model takes into consideration binding of cadmium to both intra- and extracellular proteins and the mechanisms of membrane transport. In addition, the competitive interaction of cadmium with endogenous zinc is incorporated into the model. Model simulations of the behavior of cadmium and zinc in the perfusion medium, liver, and bile are compared to results from IPRL experiments involving cadmium doses ranging from 0.29 to 15.6 mumol. A major contribution of this model is the identification, from a kinetic point of view, of two high-molecular-weight classes of intracellular cadmium-binding species which can be identified by two distinct peaks in Sephadex G-75 profiles of hepatic cytosol. This model can be utilized for the quantitation of kinetics based on specific mechanisms involved in cadmium hepatic kinetics.     

Experimental study of dexrazoxane-anthracycline combinations using the model of isolated perfused rat heart

We have studied the protective effect of dexrazoxane on the cardiac toxicity induced by the anthracyclines currently used in clinics, doxorubicin, epirubicin, daunorubicin and idarubicin, with special emphasis on determining the optimal dose of dexrazoxane. This was performed using the model of isolated perfused rat heart after 12-day combination treatment of anthracyclines used at equi-cardiotoxic doses, and dexrazoxane used at 10-fold or 20-fold the anthracycline dose. We have shown in this study that dexrazoxane by itself was not cardiotoxic, and was able to significantly reduce anthracycline cardiac toxicity without increasing the general toxicity induced by these drugs. Using dexrazoxane at 20 times the anthracycline dose provided a better cardioprotection than using it at 10 times the anthracycline dose; at the higher dexrazoxane dose, the functional cardiac parameters (developed pressure, contractility and relaxation) were not different from those recorded in control animals.     

Selenite biotransformation to volatile metabolites in an isolated hepatocyte model system

The biotransformation of selenite to dimethylselenide was studied in an oxygenated hepatocyte model system. The concentrations of selenite used were 20-100 microM. A lag period of one hour or more, during which no net formation of selenide could be detected characterized the system. The maximal rate of volatilization was recorded during the second hour and was 0.13 nmoles/10(6) cells/min with 50 microM selenite. The rate then declined and volatilization eventually ceased. Two-thirds of the added amount of Se was lost within 4 hr. Oxidation of glutathione (GSH) by cumene hydroperoxide delayed volatilization. An inhibitor of gluconeogenesis, p-tert-butylbenzoic acid (3 microM) prevented volatilization. There were indications that GSSG reductase dependent metabolism was the only major metabolic pathway in hepatocytes under the conditions studied. During the lag period Se accumulated in cells, but was subsequently partially released during volatilization. The accumulation of Se was paralleled by an increase in oxygen uptake. The above mentioned inhibitors of volatilization prolonged the phase of accumulation. With 50 microM selenite the rate of accumulation was 0.06 nmoles/10(6) cells/min and maximally 30-35% of the added dose was retained in the cells. The results are compatible with the assumption that Se mainly accumulated as Se-glutathione complexes. The possibility that such complexes autooxidized and entered futile redox cycles during the lag period is discussed.     

Effects of Concanavalin A on the isolated perfused rat liver

Summary In the isolated perfused liver, Concanavalin A provoked a significant decrease of flow rate within 2 to 4 min which was dose-dependent and could be partly inhibited by specific antagonists.Furthermore it was found that the lectin led to a decline of the respiration, an increase of the lactate/pyruvate ratio and a release of the transaminases into the medium. It was suggested that Concanavalin A displaced endothelial cells in the liver capillaries, which occluded the vessels and decreased the flow rate. The decreased respiration was considered to be secondary to this effect.     

Effects of disophenol on the isolated, perfused rat heart

Perfusion of hearts with disophenol caused significant alterations in spontaneous heart rate, coronary flow, isometric systolic tension, metabolite levels and electrical activity. Phosphorylase a levels were normal at 10 and 100 $\text{ng}\text{ml}$ but decreased significantly at 1000 $\text{ng}\text{ml}$. Disophenol markedly depressed spontaneous heart rate at 1000 $\text{ng}\text{ml}$ and similar decreases were noted in coronary flow and isometric systolic tension. Diastolic tension, however, increased greater than threefold after 60 min of perfusion at a level of 1000 $\text{ng}\text{ml}$. At this concentration, serious disturbances in electrical activity were also evident. Disophenol depleted tissue glycogen, adenosine-5'-triphosphate, total adenine nucleotides and creatine phosphate levels while it elevated lactate levels. No disophenol-induced lesions, gross or microscopic, were found.     

Effects of chlorpromazine on the isolated perfused rat heart.

Perfusion of hearts with chlorpromazine · HCl (50–5000 ng/ml) caused significant changes in heart rate, coronary flow, isometric systolic tension, certain metabolite concentrations, and electrical activity. Phosphorylase a activity remained normal. Chlorpromazine (50 ng/ml) decreased isometric systolic tension and prolonged QT interval in the electrocardiogram. Fructose 1,6-diphosphate and pyruvate concentrations were elevated, suggesting aldolase inhibition and decreased pyruvate utilization. Similar results were obtained at 500 ng/ml. At 5000 ng/ml, heart rate and isometric systolic tension were markedly diminished. Coronary flow increased briefly but returned to normal. PR, QT_a, and QT intervals were prolonged. Fructose 1,6-diphosphate, glyceraldehyde-3-phosphate, and pyruvate concentrations in the tissue increased, while the amount of dihydroxyacetone phosphate and l-(?)-glycerol-1-phosphate decreased. No chlorpromazine-induced lesions, gross or microscopic, were found.