Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism.

BACKGROUND: Compound failures have been emerging in later stages of pharmaceutical drug development and are in many cases not detected until the administration to humans in clinical trials or even after approval. Among the most frequent adverse effects are drug-induced liver injury generated by species-specific susceptibilities (e.g., in xenobiotic metabolism and/or the occurrence of idiosyncratic drug hepatotoxicity). OBJECTIVES: Detecting or predicting unfavorable drug effects on the liver as early as possible in drug development is crucial in making the drug development process more efficient and the application of new drugs to humans in clinical studies and medical use safer. METHODS: To achieve this goal, primary hepatocyte cultures from various species, including humans, are analyzed for morphological, functional and gene expression alterations after compound treatment. RESULTS/CONCLUSION: Primary hepatocyte cultures appear to be a promising tool for the detection of general or liver toxicity and the evaluation of species-specific drug effects.

Understanding the biokinetics of ibuprofen after single and repeated treatments in rat and human in vitro liver cell systems.

Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100µM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions.

Polyethylene glycol-polyvinyl alcohol grafted copolymer: study of the bioavailability after oral administration to rats.

The absorption, urinary excretion, and the biliary excretion of a single oral dose of 10 or 1000 mg/kg bw of (14)C-polyethylene glycol-polyvinyl alcohol (PEG-PVA) grafted copolymer were studied in adult male and female rats. In a balance/excretion experiment, the total excretion of ingested radioactivity was determined over a period of 168 h and residual radioactivity was detected in selected tissues and the carcass. In a biliary excretion experiment, excretion of radioactivity via the bile duct was determined over a period of 48 h after administration of the substance to cannulated rats. Most, if not all, of the radioactivity (>100%) was excreted within 48 h via the feces regardless of sex or dose. Urinary excretion was very limited: 0.45-0.50% of dose at the low dose and 0.22-0.27% of dose at the high dose. At both dose levels, residual radioactivity in the carcass and all organs and tissues after 168 h was ≤ 0.02% of dose. Biliary excretion was 0.01-0.02% of dose. Based on these findings, the bioavailability of PEG-PVA grafted copolymer was determined to be <1% demonstrating that absorption was virtually negligible following a single oral administration to male and female rats.

Polyethylene glycol-g-polyvinyl alcohol grafted copolymer: reproductive toxicity study in Wistar rats.

Polyethylene glycol-g-polyvinyl alcohol (PEG-PVA) grafted copolymer was administered by gavage to groups of 25 male and 25 female young Wistar rats at doses of 0 (vehicle control), 100, 300, or 1000 mg/kg bw/day for one generation (F0). The study followed the treated F0 generation through mating, gestation, lactation, and weaning of the F1 generation. F1 animals were mated and followed to gestation day (GD) 15-17 at which time F2 implants were evaluated. There were no indications from the various clinical and gross pathological examinations that the oral administration of PEG-PVA grafted copolymer to the F0-parental rats produced any signs of general, reproductive, or developmental toxicity in the F0 or F1 animals or F2 implants. Based on the lack of any dose-related or biologically relevant effects on fertility, reproduction, development, and overall health of rats gavaged with PEG-PVA grafted copolymer and their progeny, the no-observed-adverse effect level (NOAEL) was determined to be the highest dose tested of 1000 mg/kg bw/day.

Developmental toxicity of polyethylene glycol-g-polyvinyl alcohol grafted copolymer in rats and rabbits.

Polyethylene glycol-g-polyvinyl alcohol (PEG-PVA) grafted copolymer was evaluated in developmental toxicity studies with Wistar rats and Himalayan rabbits. Pregnant Wistar rats were gavaged with 0 (vehicle control), 100, 300, or 1000 mg PEG-PVA grafted copolymer/kg bw/day from gestation day (GD) 6-15. Pregnant Himalayan rabbits received the same treatment from GD 6 to 19. On GD 20 and 29 for rats and rabbits, respectively, the animals were euthanized and were examined grossly. For each dam, corpora lutea were counted and number and distribution of implantation sites were determined. The fetuses were removed, sexed, weighed, and evaluated for any external, soft tissue, and skeletal findings. No significant findings were found that could be attributed to administration of PEG-PVA grafted copolymer. Under the conditions of these studies, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity in both species was the highest dose tested of 1000 mg/kg bw/day.

Subchronic toxicity of polyethylene glycol-g-polyvinyl alcohol grafted copolymer.

The safety of polyethylene glycol-g-polyvinyl alcohol (PEG-PVA) grafted copolymer was evaluated in a 13-week oral toxicity study in rats and in a 9-month oral toxicity study in dogs. Wistar rats were administered 600, 3000, or 15,000 ppm PEG-PVA grafted copolymer in their drinking water whereas beagle dogs were fed 3000, 10,000, or 30,000 ppm PEG-PVA grafted copolymer in the diet. There were no mortalities, no adverse clinical signs, no toxicologically adverse effects on body weight or body weight gain, feed consumption, hematological, clinical chemistry or urinary parameters, or histopathology in either species. In rats, no treatment-related effects were observed in the functional observational battery (FOB) or related measurements of motor activity. Increased water consumption observed in rats at the highest dose was the only test substance-induced effect noted. The no-observed-adverse-effect level (NOAEL) was the highest concentration tested in both species: 15,000 ppm in rats (corresponding to a daily intake of 1611 mg/kg bw for males and 2191 mg/kg bw for females) and 30,000 ppm in dogs (corresponding to a mean daily intake of 783 mg/kg bw for males and 811 mg/kg bw for females).

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes.

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.