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

Dvorak, Z.; Vyhlidalov, B.; Pecinkov, P.; Li, H.; Anzenbacher, P.; spicakova, a.; anzenbacherova, e.; Chow, V.; LIU, J.; Krause, H.; Wilson, D. J.; Beres, T.; Tarkowski, P.; Chen, D.; Mani, S.

doi:10.1101/2023.10.21.563410

bioRxiv · 10.1101/2023.10.21.563410

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

Dvorak, Z.·Vyhlidalov, B.·Pecinkov, P.·Li, H.·Anzenbacher, P.·spicakova, a.·anzenbacherova, e.·Chow, V.·LIU, J.·Krause, H.·Wilson, D. J.·Beres, T.·Tarkowski, P.·Chen, D.·Mani, S.

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Abstract

Background and purposeBased 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 approachA complex series of cell-free and cell-based assays were employed. The organic synthesis, and advanced analytical chemistry methods were used. Key resultsFKK6-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 x 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 x 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 implicationsThe PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development. BULLET POINT SUMMARYWhat is already known: O_LIMicrobial metabolite mimic FKK6 is a hPXR agonist with anti-inflammatory properties in mice and human. C_LIO_LIThe in vitro PXR binding, absorption, and metabolism have not been completely characterized. C_LI What this study adds: O_LIPXR selectivity with unique binding mode, high intestinal cell permeability, rapid and complex microsomal metabolism. C_LIO_LIInitial testing for predicted metabolites shows reduced potency as PXR agonists. C_LI Clinical significance: O_LIPXR effects of FKK6 are predicted to be predominantly in the intestines. C_LIO_LIFKK6 has a suitable pharmacological profile supporting its potential preclinical development. C_LI

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Dvorak, Z., Vyhlidalov, B., Pecinkov, P., Li, H., Anzenbacher, P., spicakova, a., anzenbacherova, e., Chow, V., LIU, J., Krause, H., Wilson, D. J., Beres, T., Tarkowski, P., Chen, D., Mani, S.. 2023-10-21. In Vitro Safety Signals for Potential Clinical Development of the Anti-Inflammatory Pregnane X Receptor Agonist FKK6. https://doi.org/10.1101/2023.10.21.563410

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