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 X receptor (PXR). The lead indole, FKK6, displayed PXR-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-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets revealed high PXR 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 × 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 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. Conclusion and implications The PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development. BULLET POINT SUMMARY What is already known: Microbial metabolite mimic FKK6 is a hPXR agonist with anti-inflammatory properties in mice and human. The in vitro PXR binding, absorption, and metabolism have not been completely characterized. What this study adds: PXR selectivity with unique binding mode, high intestinal cell permeability, rapid and complex microsomal metabolism. Initial testing for predicted metabolites shows reduced potency as PXR agonists. Clinical significance: PXR effects of FKK6 are predicted to be predominantly in the intestines. FKK6 has a suitable pharmacological profile supporting its potential preclinical development.