Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines

Abstract

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

Shell engineering in soft alginate‐based capsules for culturing liver spheroids

“…Metabolic assay was carried out as previously reported. [ 34 ] Briefly, HepG2 spheroids in AL and AL‐CS MCs were prepared in advance in a 24 well‐plate and cultured at 37°C under 5% CO 2 for a week with each well containing around 100 capsules. The metabolic activity of encapsulated HepG2 cells was tested upon incubation with testosterone (20 μM) for 4 h. The concentration of testosterone and androstenedione was measured using liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) in multiple reaction monitoring (MRM) mode (see Supporting Information).…”

“…Metabolic assay was carried out as previously reported. [34] Briefly, HepG2 spheroids in AL and AL-CS MCs were prepared in advance in a 24 well-plate and cultured at 37 The AL and AL-CS capsules show no obvious diameter and morphology differences, when generated under the same flow rates (Figure 1C). the values of 86% (50 µm), 57% (100 µm), and 11% (150 µm).…”

Preclinical evaluation of an 18F-labeled Nε-acryloyllysine piperazide for covalent targeting of transglutaminase 2

“…Microsome experiments with [ 18 F]7b in the presence of NADPH were performed according to the procedure recently described by us with slight modifications (Lemm et al 2022 ). Incubations had a final volume of 250 µL.…”

Impact of Viscosity on Human Hepatoma Spheroids in Soft Core–Shell Microcapsules

“…After verifying the MC permeability, we generated the MCs (with different concentrations of incorporated CMC in the core: 0%, 1%, 2%, and 2.5% w/v) and GBs containing HepG2 cells as models of hepatocellular carcinoma. [38,39] Figure 4 summarizes the findings about the character of assembly, proliferation, and viability of HepG2 cells and spheroids grown within different groups of MCs and GBs (used as control reactors).…”

“…In this study, we focused on spheroids derived from HepG2 cells, which are used both as a model for liver function and as a model for hepatocellular carcinoma, [38,39] to investigate the influence of fluid viscosity on 3D cell assemblies (Figure 1). To perform the investigation, we generated core-shell microcapsules (MCs) with diameters of ≈500 μm as bioreactors for cell culturing using the microfluidic platform that we designed previously.…”