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Session A6: Organ Studies I (Liver and Drug Metabolism)Chairs: Vera Rogiers (Belgium) and Jose Castell (Spain) A6: Major Phase I Biotransformation Pathways of Trichostatin A in Rat Hepatocytes and Rat and Human Liver Microsomes Phase I biotransformation of Trichostatin A (TSA), a histone deacetylase inhibitor with promising antifibrotic and antitumoral properties, was investigated in rat and human liver microsomes and in suspensions of rat heptocytes. 50 µM TSA was completely metabolized by rat hepatocytes in suspension (2.106/ml) within 30 minutes. Its phase I metabolites were separated by high-performance liquid chromatography (HPLC) and detected with simultaneous UV and electrospray ionization mass spectrometry (ESI-MS). ESI tandem mass spectrometry (ESI-MS/MS) was used to identify the metabolites. The two major phase I biotransformation pathways in rat hepatocytes were N-demethylation and reduction of the hydroxamic acid function to its corresponding amide. Trichostatic acid, N-monodemethylated Trichostatic acid, and N-didemethylated TSA were identified as minor metabolites. Lower concentrations of TSA (5 µM and 25 µM) degraded more rapidly and were subjected to a faster N-demethylation than 50 µM TSA. In contrast, incubations of TSA with rat and human microsomes led to an incomplete biotransformation with the formation of two major metabolites, N-mono- and N-didemethylated TSA and one minor metabolite, Trichostatic acid. Further biotransformation studies of TSA and its structural analogues need to be performed in hepatocytes, since they reflect the in vivo situation better than microsomes. A6: Drug Biotransformation by Human Hepatocytes: In Vitro/In Vivo Metabolism by Cells from the Same Donor Cultured human hepatocytes are considered a close model to human liver. However, the fact that hepatocytes are placed in a microenvironment that differs from that of the cell in the liver raises the question of to what extent drug metabolism in vitro reflects that of the liver in vivo. This issue was examined by investigating the in vitro and in vivo metabolism of aceclofenac, an analgesic/anti-inflammatory drug. Hepatocytes isolated from programmed liver biopsies were incubated with aceclofenac, and the metabolites formed were investigated by HPLC. During the course of clinical recovery, patients were given the drug and the metabolites, largely present in urine, were analyzed. In vitro and in vivo data of the same individual were compared. The relative abundance of oxidized metabolites in vitro (i.e. 4'OH-aceclofenac + 4'OH-diclofenac vs. total hydroxylated metabolites; Spearman's p = 0.855) as well as the hydrolysis of aceclofenac (4'OH-diclofenac vs 4'OH-aceclofenac + 4'OH-diclofenac; p = 0.691) correlated well with in vivo data. The conjugation of the drug in vitro was lower than in vivo. The rate of 4'OH-aceclofenac formation in vitro correlated with the amount of metabolites excreted in urine after 16 hours (p = 0.95). In conclusion, the in vitro/in vivo metabolism of the drug was surprisingly similar in each patient. The variability observed in vitro reflected an existing phenotypic variability among donors. A6: Hepatocytes Cultivated In Vitro as an Alternative for Testing of Substances with Potential Hepatoprotective Effect Studies oriented on testing of hepatoprotective effect are usually performed on experimental animals, causing the suffering of a great number of animals. We decided to replace in vivo study with a primary hepatocyte culture. We tested the effect of S-adenosylmethionine (SAMe), which plays a protective role against glutathione depletion on a hepatocyte culture exposed to a non-specific peroxidating agent t-butylhydroperoxide (BHP). Hepatocytes were isolated from male Wistar rats (230-270 g) by two-step perfusion, using collagenase and cultured in William's E. Medium. The effect of various doses of SAMe was tested under different experimental conditions. The toxicity was analyzed by lactate dehydrogenase activity, concentration of malondialdehyde, and urea production. Incubation of hepatocytes with BHP caused elevation of lactate dehydrogenase and malondialdehyde concentrations. SAMe treatement attenuated the toxic effect of BHP. These findings support our hypothesis that a primary culture of hepatocytes can be used as an alternative to experiments on laboratory animals. Supported by grant MSM 111500003. A6: A New Human Hepatoma Cell Line to Study Repeated Cell Toxicity Early screening of new drugs is performed to select the candidates for development. Many cell models are used to assess basic cytotoxicity showing good correlation with acute toxicity. However their correlation with chronic in vivo exposure is insufficient. The new human cell line (HBGBC2) possesses the capacity of being reversibly differentiated in vitro and of maintaining a relatively higher metabolic rate when in differentiated phase (3 weeks) as compared to HepG2 cells. This allows repeated toxicity testing on cells in culture. In order to evaluate the genetic background of HBGBC2 cells, the expression of selected genes was analyzed in untreated cultures, confirming the MTT reduction was used to evaluate the toxicity of propranolol, perhexilline, aspirin, and paracetamol, after both single and repeated treatment (3 times/week for 2 weeks). Under conditions of repeated treatment, cytotoxicity was observed at lower doses when compared with single administration. Moreover, the first non-toxic doses were in the same range as plasma concentrations measured in humans under therapeutic use. Our results suggest that the new human hepatoma HBGBC2 cell line may be of interest for the evaluation of cell toxicity under repeated treatment conditions. A6: In Vitro Methods Employing hCYP2D6 Polymorphic Forms to Detect Polymorphism-related Problem Compounds Genetically-engineered V79 cells expressing CYP2D6 cDNA polymorphic alleles (CYP2D6*1 [the wild type], CYP2D6*2, CYP2D6*9, CYP2D6*17, and a mock transfected control) were used as a screening tool to investigate and compare the metabolic competences of the different alleles toward selected drugs, namely, bufuralol, imipramine, debrisoquine, and haloperidol, and twelve pharmaceutical development compounds. Following the exposure to several CYP2D6 substrate drugs, the influence of the CYP2D6 genotype was evaluated. The study resulted in the identification of two compounds, haloperidol and compound X, which displayed CYP2D6*17 activity comparable to wt CYP2D6*1 activity, implying that mutation at the DNA level does not always mean a reduction in metabolic capacity. Yeast microsomes over-expressing hCYP2D6*1 and hCYP2D6*17 were used in classical disappearance experiments to calculate Km and Vmax, in order to evaluate whether the affinity for the enzyme could be an explanation for these differences in activity.
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