Model For Drug Metabolism Studies Research Articles

Extending the viability of human precision-cut intestinal slice model for drug metabolism studies

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Hepatocyte Cocultures with Endothelial Cells and Fibroblasts on Micropatterned Fibrous Mats to Promote Liver-Specific Functions and Capillary Formation Capabilities

The maintenance of hepatocyte phenotype and functions remains as a great challenge in the generation of functional liver tissue and in vitro model for drug metabolism studies. The use of hepatocyte coculture systems plays essential roles in the establishment of cell-cell and cell-extracellular matrix communications similar to native liver tissues. In the current study, micropatterned electrospun fibrous mats were created to load hepatocytes, fibroblasts, and endothelial cells (ECs), which were precisely assembled to establish their spatially controlled coculture for mimicking the in vivo structure of hepatic lobules. Hepatocytes formed compact polyhedral spheroids with an average diameter of 80-100 μm, reorganized actin filaments in the cell-cell contact regions, and well-developed bile canaliculi. Compared with hepatocytes cultured alone, the coculture of hepatocytes with either fibroblasts or ECs led to significantly higher albumin secretion, urea synthesis and cytochrome P-450 expression, which were dramatically improved by the coculture of hepatocytes with both fibroblasts and ECs. The cocultured ECs well spread on patterned regions with little organized filamentous actin, and significantly higher densities and deeper penetration into patterned scaffolds were determined for ECs after coculture with fibroblasts and hepatocytes compared with those after cultured alone or coculture with either fibroblasts or hepatocytes. A Matrigel overlay assay showed that the capabilities of ECs to form capillary tubes were significantly enhanced by micropatterned coculture with fibroblasts and hepatocytes. Thus, the coculture of hepatocytes, fibroblasts, and ECs on micropatterned fibrous mats helps both hepatocytes in the maintenance of hepatic functions and ECs in the formation of capillary-like structures. It is suggested that the micropatterned coculture model described here not only provides functional hepatic tissues for predictions of drug metabolism profiles, but also will enable investigations on more complex and physiological cell-cell communications.

Hepatic Cell Lines for Drug Hepatotoxicity Testing: Limitations and Strategies to Upgrade their Metabolic Competence by Gene Engineering

One key issue in the pharmaceutical development of new compounds is knowledge on metabolism, the enzymes involved and the potential hepatotoxicity of a drug. Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, human hepatocytes show phenotypic instability and have restricted accessibility and high batch-to-batch functional variability, which seriously complicates their use in routine testing. Therefore, several liver-derived cell models have been developed for drug metabolism and hepatotoxicity screening to circumvent these drawbacks. Hepatoma cell lines offer important advantages, availability, an unlimited life span and a stable phenotype, thus rendering them suitable models for such studies. However, currently available human hepatoma cell lines are not a good alternative to cultured hepatocytes as they show very limited expression for most drug-metabolising enzymes. Other approaches have been developed to generate immortalised hepatic cells with metabolic competence (use of plasmids encoding immortalising genes to transform human hepatocytes, cell lines obtained from transgenic animals, hepatocytomes or hydrid cells). Recombinant models heterologously expressing cytochrome P450 enzymes in hepatoma cells have also been generated, and are widely used in drug metabolism and toxicity evaluations. In recent years, new approaches to up-regulate the expression of drug-biotransformation enzymes in human cell lines (i.e., transfection with the expression vectors encoding key hepatic transcription factors) have also been investigated. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism and hepatotoxicity studies, and the state-of-the-art strategies pursued to generate metabolically competent hepatic cell lines.

Cell Lines: A Tool for In Vitro Drug Metabolism Studies

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alternative to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes (transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential enzyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with favourable pharmacokinetic and metabolic properties.

Human Hepatocytes in Primary Culture: The Choice to Investigate Drug Metabolism in Man

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy/pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates/inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction/inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

Application of Chemical Cytochrome P450 Model Systems to Studies on Drug Metabolism .VI: N, N-Coupling Reaction of N-Methylaniline Catalyzed by Polypeptide-Bound Porphyrinatoiron(III) and Cytochrome P450

Polypeptide-bound porphyrinatoiron(III) effectively catalyzed the N, N coupling reaction of N-methylaniline in the presence of NaBH 4/O 2/tetramethyl-ammonium hydroxide or cumenehydroperoxide, whereas the corresponding non-bound monomer porphyrinatoiron(III) had little catalytic activity. Rat liver microsomes also catalyzed the N, N-coupling which was inhibited by cytochrome P-450 specific inhibitors, SKF-525A and metyrapone. The polypeptide-bound porphinatoiron(III) appears to be an excellent cytochrome P450 model for drug metabolism studies.

Cultured human adult hepatocytes: a new model for drug metabolism studies

Cultured human adult hepatocytes: a new model for drug metabolism studies