Caco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone

Abstract

Tryptanthrin and (E,​Z)​-​3-​(4-​hydroxy-​3,​5-​dimethoxybenzylidene)​indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-​inflammatory and antiallergic alkaloids, and shown to inhibit COX-​2, 5-​LOX catalyzed leukotriene synthesis, and mast cell degranulation at low μM to nM concns. To assess their suitability for oral administration, we screened the compds. in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compds., validated UPLC-​MS​/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coeff. > 32.0 × 10-​6 cm​/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coeff. values in the presence of the P-​glycoprotein inhibitor verapamil (50 μM) indicated that tryptanthrin was not involved in P-​glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-​resoln. mass spectrometry pointed to extensive phase II metab. of indolinone (sulfation and glucuronidation)​. Possible cardiotoxic liability of the compds. was assessed in vitro by measurement of an inhibitory effect on human ether-​a-​go-​go-​related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-​a-​go-​go-​related gene inhibition was found for tryptanthrin (IC50 > 10 μM) and indolinone (IC50 of 24.96 μM)​. The anal. of compds. using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-​a-​go-​go-​related gene potassium channel at micromolar concns.