Clinically relevant CYP3A4 induction mechanisms and drug screening in 3D spheroid cultures of primary human hepatocytes.
CYP3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with the obligations CYP3A4 induction in drug development prerequisites. Here, we present a 3D spheroid cultures of primary human hepatocytes (PHH) as CYP3A4 induction of new screening models.
Screening of 25 drugs (12 known inducers of CYP3A4 in vivo and 13 negative control) at physiologically relevant concentrations revealed 100% sensitivity and 100% specificity system. Three of inducers of CYP3A4 in vivo induction of CYP3A4 displayed capacity is much higher in comparison with the 3D spherical culture in 2D monolayer culture. Among them, we identify AZD1208, PIM kinase inhibitor in Phase I development stopped because of unexpected autoinduction CYP3A4, CYP3A4 inducer as the only active in 3D spheroids but not in 2D monolayer culture.
Gene knockdown experiments revealed that AZD1208 require PXR to induce CYP3A4. Rifampicin require PXR solely to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of CYPs is not indicative of the absolute dependence on both PXR or CAR demonstrated its ability to switch the activation of nuclear receptors. Mechanistic Study of AZD1208 to find involvement of MAPK / ERK pathway in induction of CYP3A4-sensitive culture format used, as revealed by the inhibition of ERK1 / 2 phosphorylation Tyr204 and sensitivity to EGF pressure. Accordingly, we also identified lapatinib, a dual EGFR / HER2 inhibitor, such as CYP3A4 inducer active just another round in 3D culture.
Our findings offer insight into the pathways involved in the induction of CYP3A4 and suggest spheroids PHH for preclinical screening of CYP3A4 induction.
Glucose and lipid metabolism screening models of hepatocyte spheroids after culture by injecting fiber fragments.
With the increase in obesity, diabetes and other metabolic diseases, in vitro liver cell and tissue models play an important role in the identification of active pharmaceutical ingredients. Up to now three-dimensional (3D) models rarely focuses on the culture of hepatic glucose and lipid metabolism.
In addition, primary human liver cells suffer from limited availability and differences among donors to build the model reproduced. Thus, in this study, the most available human liver cancer cells (HepG2), and primary hepatocytes from rat (RPH) proposed to build a 3D spheroids using fiber fragments graft syringe with galactose (GSF) as a substrate. RPH and spheroids showed HepG2 cells and cell fragments strong-fiber interaction to promote cell survival, albumin and urea synthesis.
Compared with HepG2 spheroids, spheroids RPH demonstrate strong abilities in terms of consumption of glucose metabolism of glucose, glycogen intracellular content, the level of glucose in gluconeogenesis and sensitivity to the hormone modulators such as insulin and glucagon. On the other hand, HepG2 spheroids displays a strong ability of lipid metabolism in produce significantly higher levels of total cholesterol and triglycerides.
Compared with those who did not fragment the fiber, the GSF-supported 3D culture establish an effective model for in vitro glucose (RPH spheroids) and lipid metabolism (HepG2 spheroids). The model confirmed screening of each enzyme activity and gene expression and showed significantly higher sensitivity and clinically relevant response to hypoglycemic and lipid-lowering drugs.Connection error.
Thus, the culture configuration shows the predicted in vitro platform for defining the profiles of glucose and lipid metabolism and screening of therapeutic agents for metabolic disorders such as diabetes and obesity.