Metabolism of α-amplexichromanol (27a) in a human liver-on-chip model

Neukirch, K. et al. Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation. J Med Chem 64, 11496-11526 (2021). https://doi.org/10.1021/acs.jmedchem.1c00806
Biochips were made from polystyrene by injection molding and were equipped with a poly(ethylene terephthalate) (PET) membrane (TRAKETCH, thickness: 12μm; pore diameter: 8μm; pore density: 1×10^5 pores/cm2; Sabeu, Radeberg, Germany) that was integrated in the upper and lower parts of the biochip by heat-sealing with the bulk material. The top and bottom sides of the biochips and channels were sealed with an extruded PS bonding foil (thickness: 125 μm) by a low-temperature bonding method. The upper and lower parts of the biochips were assembled using a double-sided adhesive film, and the chip surface was hydrophilized by oxygen plasma treatment to facilitate cell adhesion and prevent air bubble formation within the chambers and channels. HUVECs were isolated from human umbilical cord veins and cultivated in an endothelial cell growth medium MV (Promocell, Heidelberg, Germany) with penicillin (100 U/mL) / streptomycin (100 μg/mL, GE Healthcare) up to passage 4. On reaching 95% confluence, cells were sub-cultured at a density of 1.5×10^4 cells/cm2. HepaRG cells (Biopredic International, Rennes, France) were grown in William's medium E (Biochrom, Berlin, Germany) with 10% FCS, 5 μg/mL insulin (Sigma-Aldrich), 2 mM glutamine (Thermofisher Scientific), 50 μM hydrocortisone-hemisuccinate (Sigma-Aldrich), and penicillin (100 U/mL) / streptomycin (100μg/mL, GE Healthcare) at 37°C and 5% CO2 for 14 days prior to differentiation with 2% DMSO for another 14 days. The medium was changed every 3−4 days, and differentiated cells were used up to 4weeks. The liver-on-chip was prepared by seeding HUVECs (top: 3×10^5, bottom: 1.5×10^5) in an endothelial cell growth medium MV on top of the membrane in the upper chamber and on the bottom of the membrane in the lower chamber, giving the cells 3−4 h to adhere before flipping the chip upside and seeding the bottom layer. After 2 days, differentiated HepaRG cells (3×10^5) were seeded on top of the lower and on the bottom of the upper membrane in a hepatocyte culture medium with hydrocortisone-hemisuccinate adjusted to 5 μM for 24 h at 37°C and 5% CO2. The medium between the two membranes was renewed, and the vehicle (DMSO), 12a, or 27a were added. After incubation for 48 h at 37°C and 5% CO2, the medium was collected and the system was washed with 500 μL of methanol. 12a, 27a, and their metabolites were extracted from the combined fractions and analyzed by UPLC-MS/MS as described in Neukirch et al., 2021.