In vitro models for CYP inhibition and induction studies

These are cDNA CYPs expressed in baculovirus-insect cell or an Escherichia coli (E. coli) based expression system. Only single enzyme is expressed in such systems, hence resulting to higher activity when a specific substrate is applied (Foti et al., 2010). Recombinant enzymes are usually employed in high throughput screening (HTS) of new candidate drugs at early stages of drug development. The absence of contribution of other metabolic enzyme in recombinant CYP assays is a major setback associated with this system (Kramer and Tracy, 2008).

1.4.4.2 Human liver microsomes

It is regarded as the gold standard enzyme source for most in vitro inhibition studies. Human liver microsomes (HLM) are very convenient, easy to use and affordable. HLM contains multiple phase I hepatic drug metabolizing enzymes compared to the recombinant CYPs. Microsomes obtained from liver has vesicles of the hepatocytes endoplasmic reticulum collected from liver preparation such as fresh human liver, liver slices, liver cell lines and primary

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hepatocytes at specific differential centrifugation. HLMs do not represent complete in vivo situation since only CYP and UGT enzymes are present in HLM (Brendon et al., 2003).

1.4.4.2 Human liver S9 fractions

These are subcellular fractions containing drug metabolism enzymes such as CYPs, flavin monooxygenases and UDP-glucuronyltransferases. S9 fractions depict a complete representation of metabolic profile compare to HLM since they contain both phase I and phase II drug- metabolism enzymes. However, the enzyme activity is relatively lower than that of HLM (Brendon et al., 2003).

1.4.4.3 Liver slices

The liver slices have been developed as an in vitro model for assessment of hepatic drug metabolism. The tissue slices maintain the cytoarchitecture of the liver which can be easily examined when needed. Phase II enzymes, albumin production, and gluconeogenesis have been shown to decrease slightly but remain fairly stable for up to 20-96 hours in cultured liver slices (Toutain et al., 1998). Major challenges with liver slices include erratic supply of the liver, maintenance of slice cultures with quality viability over the study period and the high interindividual variability (Thohan and Rosen, 2002).

1.4.4.4 Primary hepatocytes

Cultured human hepatocyte is currently the gold standard for studying CYP induction (Lecluyse, 2001). Availability of tissue is a major concern and restricts the use of human hepatocytes for in vitro applications. Furthermore, there is time-dependent decline in the expression of mRNA for the major CYPs resulting in reduced enzyme activities. To overcome this flaw, dishes and plates for human hepatocytes assays are coated with an extracellular matrix such as Matrigel or collagen which is termed as sandwich culturing to mimic physiological condition. Human hepatocytes in sandwich culture have cytoarchitecture similar to that found in vivo.

1.4.4.5 Cell lines

Fa2N-4, a non-tumorigenic hepatic cell line, is the most utilized cell system by the pharmaceutical industry (Sinz et al., 2008). It shows inducible CYP1A1/2, CYP3A4, CYP2C9, UGT1A and MDR1 mRNA expression, as well as increased enzymatic activity of CYP1A2, CYP2C9 and CYP3A4 when treated with prototypical inducers. Fa2N-4 is however flawed with

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low basal enzyme activity; hence sensitive analytical methods are required to measure enzyme activity. Because of the low basal enzyme activity, mRNA detection is often used as an end point when using this cell line.

Human hepatoma cell lines such as HepG2, HepaRG and BC2 have been employed for induction studies. HepaRG and HepG2 cells have been shown to respond differently to CYP1A1/2 and 3A4 inducers whilst BC2 cells have been reported to respond to CYP1A inducers (Aninat et al., 2006; Guillouzo et al., 2007). These cell lines however do not maintain the complete phenotypic characteristics of human hepatocytes including enzyme, receptor function or expression.

1.4.4.6 Reporter gene

In the reporter gene assay, plasmids containing elements of the target gene promoter of interest fused into the reporter gene expression vector is transiently or stably transfected into cell lines. HepG2 cell line transfected with two plasmids containing 5’-regulatory elements (PCR5) of CYP3A4 linked to a gene expressing luciferase and PXR expression vector is used for the assessment of PXR activation or CYP3A4 induction in most research laboratories (Sinz et al., 2006). The cells are treated with the test samples and PXR activation or CYP3A4 induction measure using luciferase activity as depicted in Figure 1.7 above. Cells used in reporter gene assays are easy to maintain, and the luciferase measurement applicably for high throughput screenings. However, the levels of target genes and receptor expression are not at physiological levels and the cell lines may not contain all the regulatory factors present in hepatocytes. Hence reporter gene assays are used only for screening purposes to flag compounds as potential inducers.

1.4.4.7 Transgenic/Chimeric animals

Humanized nuclear receptor transgenic mice, knock out mice and chimeric mice with human transplanted hepatocytes have emerged as useful models for investigating the regulation of drug metabolizing enzymes by potential drug candidates. These in vivo models provide an advantage over the above-mentioned in vitro models. Transgenic animals have dynamic systems where drug absorption, distribution, metabolism, and elimination occur simultaneously. Animals can be administered with drug candidates at doses equivalent to those expected in human plasma to improve assessment of induction or inhibition potential of such drugs. The major flaw with the transgenic/knock out systems are that only single or double genes are “humanized” and the

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cross-talk between nuclear receptors/enzymes/transporters may be compromised as the human and mouse systems may or may not be working in a similar fashion (Sinz et al., 2008).