Establishment of Rat Precision‐cut Fibrotic Liver Slice Technique and Its Application in Verapamil Metabolism

Abstract

1. Liver fibrosis is the compensatory state of cirrhosis. In the long asymptomatic period, it is imperative to select a proper dosing regimen for drugs that are applicable to hepatic fibrosis. Otherwise, progressive deterioration to uncompensated cirrhosis may occur. The present study explored the characteristics of drug metabolism in fibrotic liver. 2. A rat precision-cut fibrotic liver slice (PCFLS) technique was established and the metabolism of verapamil was studied employing this technique. A rat hepatic fibrosis model was successfully induced integrating complex factors that included a high-fat diet, alcohol and CCl4. The PCFLS were incubated under different conditions and lactate dehydrogenase leakage, glutathione S-transferase activity and 3[4,5-dimethythiazole-2-yl]-2,5-diphenyltetrazolium bromide reduction were used as indices to assess PCFLS viability. Activities of phase I and phase II metabolizing enzymes were monitored following treatment with cytochrome P450 (CYP) inducers. Normal and fibrotic liver slices were incubated individually with 10 micromol/L verapamil. The concentration of verapamil in the medium was determined by high-performance liquid chromatography and intrinsic clearance (Cl(int)) was calculated on the basis of the concentration-time curve. 3. The results showed that the PCFLS viability remained steady throughout the 6 h of culture when the thickness of slices was 300 microm and pH of the medium was 7.0; CYP inducers (phenobarbital and ethanol) enhanced CYP2E1, CYP3A1/2 and uridine diphosphate-glucuronate transferase (UDPGT) activities, respectively, in a time-dependent manner. The Cl(int) (microL/min per mg) values differed significantly between normal (9.7 +/- 1.8) and fibrotic (5.6 +/- 1.4) liver slices (P < 0.01). 4. These results suggested that the PCFLS could remain viable for 2-6 h under appropriate conditions. The stability and inducibility of drug-metabolizing enzymes of PCFLS were also demonstrated. Furthermore, the metabolic rate of verapamil in PCFLS was decreased. These findings add further support to the use of PCFLS as a tool to study drug metabolism and to guide clinical medication.

PBPK Modeling to Unravel Nonlinear Pharmacokinetics of Verapamil to Estimate the Fractional Clearance for Verapamil N-Demethylation in the Recirculating Rat Liver Preparation

“…Since the K m value for metabolism ranges from 60 to 140 mM (Hanada et al, 2008), we employed initial concentrations of 1-100 mM verapamil to revisit the problem of metabolite kinetics in the recirculating perfused rat liver preparation. Mechanism-based auto-inhibition for verapamil that exists in man (Wang et al, 2004(Wang et al, , 2013 was not observed in rat liver microsomes, hepatocytes, and precision-cut liver slices (Obach, 1999;Shibata et al, 2002;Axelsson et al, 2003;Guo et al, 2007), except with gel entrapment after prolonged exposure with verapamil (Yin et al, 2011). Hence, auto-inhibition is not expected to occur within the short time frame for liver perfusion studies.…”

“…In addition, inhibitory effects of verapamil on its own P-gpmediated biliary secretion and metabolite formation, events observed in humans, should be negligible during the short 90 minute perfusion, inasmuch as lack of precedence in rat liver for auto-inhibition (Obach, 1999;Shibata et al, 2002;Axelsson et al, 2003;Guo et al, 2007;Yin et al, 2011). Moreover, incorporation of time-dependent inhibition components (e 2kIt , where k I is the in vivo inhibition rate constant, which equals the in vitro inactivation rate constant, k obs ) (Wang et al, 2004) to the various verapamil eliminatory processes (or CL int ) failed to significantly improve the goodness-of-fit of the models (see Supplemental Table 4 ) and biliary secretions (CL VER int;sec ) were low, ranging from 0.002 to 0.14 min 21 (data not shown).…”

Preventative effect of Astragalus flavescens on hepatic fibrosis in rats and its mechanism of action

“…In the normal control group, a normal diet and water were freely available, 0.9% NaCl was administered to the rats by gavage daily and peanut oil was administered by subcutaneous injection at a dose of 0.5 ml/100 g on the first day and 0.3 ml/100 g once every 4 days thereafter. In the model control group, according to a modification of composite factor modeling methods (12,13), rats were subcutaneously injected with a mixture of 40% CCl 4 and peanut oil at a dose of 0.5 ml/100 g on the first day and 0.3 ml/100 g once every 4 days thereafter. The rats were fed with freely available compound feed containing 79.5% pure flour, 20% lard and 0.5% cholesterol, and water was the only drink.…”

Translational Research in Pharmacology and Toxicology Using Precision-Cut Tissue Slices

“…As it is currently not possible to culture liver slices for weeks or months, induction of end-stage brosis ex vivo has not been achieved yet. Gou et al 104 studied the effects of established alcoholic liver brosis on drug metabolism, utilising brotic slices of livers from ethanol-fed animals; these slices remain viable for at least 6 hours. Different studies have been performed with brotic liver slices derived from animal models of chemicallyinduced brosis.…”