Hepatocyte CYP2B6 Can Be Expressed in Cell Culture Systems by Exerting Physiological Levels of Shear: Implications for ADME Testing

Abstract

Cytochrome 2B6 (CYP2B6) has substantial clinical effects on morbidity and mortality and its effects on drug metabolism should be part of hepatotoxicity screening. Examples of CYP2B6's impacts include its linkage to mortality during cyclophosphamide therapy and its role in determining hepatotoxicity and CNS toxicity during efavirenz therapy for HIV infection. CYP2B6 is key to metabolism of many common drugs from opioids to antidepressants, anesthetics, and anticonvulsants. But CYP2B6 has been extremely difficult to express in cell culture, and as a result, it has been largely deemphasized in preclinical toxicity studies. It has now been shown that CYP2B6 expression can be supported for extended periods of time using suspension culture techniques that exert physiological levels of shear. New understanding of CYP2B6 has identified five clinically significant genetic polymorphisms that have a high incidence in many populations and that convey a substantial dynamic range of activity. We propose that, with the use of culture devices exerting physiological shear levels, CYP2B6 dependent drug testing, including definition of polymorphisms and application of specific inhibitors, should be a standard part of preclinical absorption, distribution, metabolism, and excretion (ADME) testing.

Cell Spinpods: A Simple Inexpensive Device to Determine Suspension Culture and Toxicity Effects on Renal Proximal Tubular Cells

“…Cubilin is the classic example: the protein is so stable that there is scant RNA signal, to the point that investigators had to resort to embryonic tissue to find enough RNA signal to clone it [52]. Similarly, it is no surprise that expression changes in individual genes observed by other groups are absent in our analysis due to differences in cell type, method, timing, and intensity of shear stress application, and choice of renal proximal tubular cell homolog [2,20]. Importantly for drug sensitivity studies, all the drug transporting channels were preserved, except for a small decrease in ABCG2.…”

“…One area ripe for the application of cell spinpod culture is liver and kidney toxicity-each representing major obstacles for safe drug development [2,20]. There are well-established, FDA-approved, high throughput screens for hepatotoxicity, but none for nephrotoxicity [64,65].…”

“…A standard laboratory bottle roller can hold at least 40 cell spinpods. As it rotates, the cell spinpod provides continuous sedimentation of particles through culture medium while rotating as a solid mass in laminar flow with regulatable, physiologic levels of induced cellular shear and with no turbulence, as detailed below [5,20,[37][38][39] (see video 1 in supplementary data).…”

“…Exposure to fluid shear stress in vitro increases PTC transport of proteins [7][8][9][10][11][12], expression of microvilli [10], and formation of tight junctions, with increased transepithelial electrical resistance [13][14][15][16][17][18][19]. Accordingly, in order to serve as meaningful and representative models of living kidneys, cultured PTC in vitro must be exposed to fluid shear stress in a quantifiable manner [2,20].…”

Comparison of the formation, adipogenic maturation, and retention of human adipose‐derived stem cell spheroids in scaffold‐free culture techniques

“…Their work indicated variation in spheroid size over the one‐week culture period, indicating spheroid loss and merging (Baraniak & McDevitt, 2012). Hammond and Birdsall (2017) and Roy, Washizu, Tilles, Yarmush, and Toner (2001) have suggested that the shear force experienced by the cells in suspension culture may introduce phenotypic and functional changes and can hinder cell differentiation and cause cell death. We found that the suspension culture method formed fewer, larger hASC spheroids over the culture period through merging the spheroids together (Figures 6 and 7), but did not result in any significant loss of spheroids or detriment to their adipogenic differentiation (Figures 4 and 5) likely due to the low rotational speed used in our work.…”