In Vitro Safety Signals for Potential Clinical Development of the Anti-Inflammatory Pregnane X Receptor Agonist FKK6

Abstract

Background and purpose: Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane xenobiotic receptor. The lead indole, FKK6, displayed pregnane xenobiotic receptor-dependent protective effects in DSS induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we performed the initial in vitro pharmacological profiling of FKK6. Experimental approach: A complex series of cell-free and cell-based assays were employed. The organic synthesis, and advanced analytical chemistry methods were used. Key results: FKK6-pregnane xenobiotic receptor interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets revealed high pregnane xenobiotic receptor selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 times 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. FKK6 was rapidly metabolized by cytochromes P450, notably by CYP3A4 in human liver microsomes. Two oxidized FKK6 derivatives, including N6 oxide and C19-phenol, were detected, and these metabolites had 5-7 times lower potency as pregnane xenobiotic receptor agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its pregnane xenobiotic receptor effects are predicted to be predominantly in the intestines. Conclusion and implications: The pregnane xenobiotic receptor ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.