cytochrome P450

/Tag:cytochrome P450

Lack of Effect of Medium Supplementation With Pyruvate and Hormones on Cytochrome P450-mediated Activity of Rat Hepatocytes in Primary Culture

Porntip Wirachwong and Jeffrey R. Fry

The loss of cytochrome P450 (CYP)-dependent activity continues to be a problem in the use of cultured hepatocytes in xenobiotic toxicity studies. It has been reported that the inclusion of pyruvate and various hormones in the culture medium improves the maintenance of various hepatic functions, including that of CYP2C11 mRNA expression. We have studied this further, by investigating the effects of the addition of pyruvate and hormones on various CYP-dependent enzyme activities and on the CYP-dependent toxicity of precocene II in rat hepatocyte cultures. No beneficial effects of this medium supplementation could be demonstrated.
You need to register (for free) to download this article. Please log in/register here.

The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status

Sandra Coecke, Vera Rogiers, Martin Bayliss, José Castell, Johannes Doehmer, Gérard Fabre, Jeffrey Fry, Armin Kern and Carl Westmoreland

In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of interest. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sandwich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process.
You need to register (for free) to download this article. Please log in/register here.

Three-dimensional Co-culture of Primary Human Liver Cells in Bioreactors for In Vitro Drug Studies: Effects of the Initial Cell Quality on the Long-term Maintenance of Hepatocyte-specific Functions

Katrin Zeilinger, Igor M. Sauer, Gesine Pless, Catrin Strobel, Jeannette Rudzitis, Aiguo Wang, Andres K. Nüssler, Alexander Grebe, Lei Mao, Stefan H.G. Auth, Juliane Unger, Peter Neuhaus and Jörg C. Gerlach

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (P450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.
You need to register (for free) to download this article. Please log in/register here.

In Vitro Methods in the Prediction of Kinetics of Drugs: Focus on Drug Metabolism

Hannu Raunio, Päivi Taavitsainen, Paavo Honkakoski, Risto Juvonen and Olavi Pelkonen

The absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of a candidate drug influence its final clinical success. These properties have traditionally been evaluated by using various in vivo animal approaches, but recently, a number of in vitro and in silico methods have been introduced to determine key ADMET features. Basic events, such as absorption through the gut wall, binding to plasma proteins, active and passive transfer through the blood–brain barrier, and various metabolic parameters, can now be screened with rapid in vitro and computer modelling methods. The focus in this short review is on the basic in vitro and in silico methods that are used for studying the metabolism properties of new drug molecules.
You need to register (for free) to download this article. Please log in/register here.
2017-01-09T06:33:08+00:00 Tags: , , |

In Silico Toxicology in Drug Discovery — Concepts Based on Three-dimensional Models

Angelo Vedani and Martin Smiesko

Animal testing is still compulsory worldwide, for the approval of drugs and chemicals produced in large quantities. Computer-assisted (in silico) technologies are considered to be efficient alternatives to in vivo experiments, and are therefore endorsed by many regulatory agencies, e.g. for use in the European REACH initiative. Advantages of in silico methods include: the possible study of hypothetical compounds; their low cost; and the fact that such virtual experiments are typically based on human data, thus making the question of interspecies transferability obsolete. Since the mid-1990s, computer-based technologies have become an indispensable tool in drug discovery — used primarily to identify small molecules displaying a stereospecific and selective binding to a regulatory macromolecule. Since toxic effects are still responsible for some 20% of the late-stage failures, there is a continuing need for in silico concepts which can be used to estimate a compound’s ADMET (adsorption, distribution, metabolism, elimination, toxicity) properties — in particular, toxicity. The aim of this paper is to provide an insight into computational technologies that allow for the prediction of toxic effects triggered by pharmaceuticals. As most adverse and toxic effects are mediated by unwanted interactions with macromolecules involved in biological regulatory systems, we have focused on methodologies that are based on three-dimensional models of small molecules binding to such entities, and discuss the results at the molecular level.
You need to register (for free) to download this article. Please log in/register here.

The Use and Value of In Vitro Technologies in Metabolism Studies

Johannes Doehmer

The detailed investigation of the metabolism of drugs is one of the key issues in drug development. Several in vitro metabolism assays have been developed over the last two decades, to replace time-consuming and expensive animal studies. These have the potential to speed up drug development and increase drug safety, as they can be used to improve the prediction of the effects of drugs on humans. The key factors to be identified in metabolism are: a) the enzymes involved, and b) the metabolites produced by these enzymes. Cytochromes P450 (CYP-450s) are the key enzymes in drug metabolism. Cloning the genes encoding the CYP-450s, and the genetic engineering of suitable cells for heterologous expression, have provided new cell lines for studies on drug metabolism in vitro, under highly defined conditions. The V79 cell line, derived from Chinese hamster lung fibroblasts, was found to be suitable for heterologous expression, as these cells themselves do not express CYP-450s, thus providing a clean background for genetically engineering for the stable expression of any cloned CYP-450. In this way, V79 cell lines were created which specifically express CYP-450s from human, mouse, rat, and fish. These recombinant V79 cells have been applied in several drug metabolism and toxicity studies.
You need to register (for free) to download this article. Please log in/register here.