The invention relates to the production of hepatocyte cell lines useful in toxicology screens

Medicilon's pharmacokinetics department offers the clients a broad spectrum of high quality of services in the areas of in vitro ADME, in vivo pharmacokinetics and bioanalysis services, ranging from small molecules to large molecules, such as protein and antibody. The animal species involved in our services are non-human primate, canine, mice, rat, rabbit and hamster. Meanwhile, non-human primate experimental platform and isotope platform for protein/antibody are certified by the Shanghai Government. Email:marketing@medicilon.com.cn Web:www.medicilon.com

The invention relates to the production of hepatocyte cell lines useful in toxicology screens or therapy. A mammalian hepatocyte comprises, as a single polypeptide, a fusion protein comprising a c-myc protein and an oestrogen receptor, or functional fragments thereof.

This invention relates to conditionally-immortalised hepatocyte cells that can be scaled up for clinical and commercial application. Background to the Invention

The liver is responsible for the detoxification of drugs and poisons from the body. The most common cell type in the liver are hepatocytes; each hepatocyte has the capacity to perform each task required of the whole organ. Due to the metabolism of drugs in the liver, Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/Tox) testing of drug candidates is performed on liver tissue to assess potential toxicity in vivo.

Traditionally ADME/Tox tests have been performed late in the process of developing new drugs, but the number of new drugs that fail because of toxicity is about 30%. There is therefore great interest in the development of high-throughput ADME/Tox screening that can be applied early in the drug development process, thereby saving time and money.

Many in vitro ADME/Tox studies focus on the liver's main metabolic enzymes, the cytochrome P450 enzyme family, although studies on P-glycoprotein and absorption studies with the CaCo-2 cell line are also commonly used.

Several biological assay systems are used to evaluate cytochrome P450 enzyme isoforms, but they all have some major disadvantages in their ability to predict in vivo toxicity. The main disadvantage of current assays is that only a limited number of liver enzymes can be studied at once, limiting the similarity between the assay and the situation in the liver itself1'2.

Human hepatocytes are the closest in vitro model of the human liver. Primary hepatocyte cultures are often used in ADME/Tox studies, as these express a "normal" hepatocyte phenotype. However, the availability of these cells is low and the expression of markers varies between batches. Foetal hepatocytes proliferate readily when dissociated and plated in cell culture under the right culture conditions, but the expression levels of many mature enzymes differ in foetal hepatocytes. For example, the level of cyp3A4, one of the main metabolic enzymes, is much lower in foetal than mature hepatocytes3. Adult hepatocytes also prove problematic in culture. With a low proliferation capacity, it is difficult to induce them to proliferate more then once in vitro, even with stimulation by growth factors4.

The problems associated with primary hepatocytes led to the development of hepatocyte cell lines. Several human hepatocyte cell lines are currently available, some derived from adult liver and some from foetal liver, either immortalized with virus or simply cultured without immortalisation5'6. However, no cell line expresses a "normal" human hepatocyte phenotype, mainly because the expression of many hepatocyte markers, including cytochrome P450, decreases rapidly or completely disappears in cell culture7. There are seven classes of hepatocyte mRNA markers, as indicated in table 1 , together with examples in each class. Table 1 Liver mRNA markers for phenotype assessment.

It is possible to induce and upregulate the expression of some liver markers by adding substances such as 3-methylcholantrene and oncostatin M8'9 to the media.

However.it is not currently possible to upregulate the levels of all or most hepatocyte enzymes to a "normal" level by adding substrates to the media or altering culture conditions10. Several hepatocyte lines are available from other species, including rats and primates11'12, but these also have the problem of low expression of markers and the additional problem of interspecies differences.

There is therefore the need for a human hepatocyte cell line expressing normal liver functions and markers. Such a cell line, that could be used to study most phase I and Il drug metabolism enzymes including the study of cell-cell contact, would be an unlimited cell source, could be used in a high throughput system, give highly reproducible data, have no inter-species differences, and would be cost effective1.