Abstract
The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics. Numerous CYP allelic variants have been identified; the functional characterisation of these variants is required for their application in precision medicine. Results from heterologous expression systems using mammalian cells can be integrated in in vivo studies; however, other systems such as E. coli, bacteria, yeast, and baculoviruses are generally used owing to the difficulty in expressing high CYP levels in mammalian cells. Here, by optimising transfection and supplementing conditions, we developed a heterologous expression system using 293FT cells to evaluate the enzymatic activities of three CYP isoforms (CYP1A2, CYP2C9, and CYP3A4). Moreover, we established co-expression with cytochrome P450 oxidoreductase and cytochrome b5. This expression system would be a potential complementary or beneficial alternative approach for the pharmacokinetic evaluation of clinically used and developing drugs in vitro.
Highlights
The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics
carbon monoxide (CO)-difference spectroscopy of microsomal fractions transfected with the CYP3A4 plasmid using different transfection reagents (PEI-Max, TransFectin, and Lipofectamine 3000) revealed that holoprotein levels were relatively higher with the utilisation of polyethyleneimine "Max" (PEI-Max) (30 and 45 μL) and Lipofectamine 3000 (Fig. 1A)
Amongst the several complementary DNA (cDNA) expression systems that have been successfully used for the functional characterisation of CYP allelic variants, mammalian cell expression systems would be preferable since the post-translational processing of proteins may influence enzymatic activity in some C YPs7–9
Summary
The evaluation of Cytochrome P450 (CYP) enzymatic activity is essential to estimate drug pharmacokinetics. Over 80% of all variations in pharmacogenes are newly discovered and observed at low frequencies across their respective populations; their impact on pharmacokinetics has not been evaluated[6] Taken together, these rare variants account for a considerable percentage of the whole population; the determination of their effects on drug response and adverse effects would be essential for the improvement of personalised medicine. It would be beneficial to use a heterologous expression system in mammalian cells to reveal the enzymatic activities of CYP variants in humans, the low CYP levels in mammalian cells would hinder the establishment of an effective expression system. Exogenous DNA delivery into animal cells is a widely used process in biological sciences for the expression of functional recombinant proteins When this same strategy is applied with mammalian cells, costs significantly increase due to low expression levels[10]. Several studies have used the pcDNA3.4 TOPO vector, containing a full-length CMV promoter and other expression elements, as it allows for higher expression than other pcDNA-based expression constructs, making it suitable to achieve higher levels of CYP expression in 293FT c ells[13,14]
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