ADME/Tox
liver is composed of 80% parenchymal cells (hepa- tocytes) and 20% non-parenchymal cells (stellate cells of the connective tissue, endothelial cells of the sinusoids, Kupffer cells functioning as immune cells and macrophages). The hepatocytes are responsible for approximately 80% of all drug metabolism
and biotransformation of small
molecules in the body. In recent years, advances in the understanding of hepatocyte function, cell sig- nalling, liver toxicity mechanisms and cell culture techniques have led to numerous advances in com- pound testing for both preclinical and clinical trial purposes. In this regard, the use of primary human hepatocytes in cell culture has become an integral part of preclinical drug development. In general, the use of primary hepatocytes in cell culture is an established technology, but it still has limitations. When the liver tissue is dis- rupted as part of the hepatocyte isolation and purification procedure, the hepatocytes begin to rapidly lose their identity. The metabolic activity and hepatocyte-specific markers and functions of the cells immediately begin to decline. As a result, hepatocytes tend to under-predict clinical phar- macokinetics. Furthermore, because primary hep- atocytes are removed from their native environ- ment of support cells, they have been of limited use for the prediction of drug-induced liver injury (DILI). DILI remains a leading cause of clinical failure and market withdrawal and is poorly pre- dicted in animal models. The mechanisms of DILI do not often point to the hepatocytes alone as the locus of disease, but include paracrine and endocrine relationships with other cell types throughout the system.
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Current common hepatocyte formats Hepatocytes in suspension taken directly after iso- lation have the highest metabolic activity and are used in this format to determine close to in vivo measurements of intrinsic clearance of a drug. However, these hepatocytes are limited to 2-4 hours of metabolic activity, so as drugs in develop- ment become more and more stable, assays in sus- pension hepatocytes can sometimes not provide any meaningful metabolic data.
Hepatocytes are used in plated formats when drugs are poorly metabolised in short-term suspen- sion assays, or when mechanistic data regarding drug-drug interactions and mechanistic toxicity is needed. Hepatocytes plated in a monolayer on a collagen matrix have an extended life-span com- pared to cells in suspension and will maintain metabolic capacity longer. In vivo, hepatocytes exhibit a polar morphology whereby cell surface proteins are different on the different 3D surfaces. This polar morphology can only be replicated in vitro by also providing an overlay of collagen or other basement membrane extracts such as Matrigel® (Corning) to the top of the hepatocyte monolayer. By ‘sandwiching’ hepa- tocytes between these basement membrane protein mixtures, hepatocytes form the correct basal sur- face structures and a small apical membrane pock- et between cells that is known as bile-canuliculi which enables excretion of bile acids from the hep- atocytes similarly to the in vivo processes. It is gen- erally accepted that the sandwich culture most reflects 3D in vivo cell shape, and functionality in a 2D format, but nonetheless sandwich culture does not solve the issue of metabolic decline.
Drug Discovery World Summer 2017
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