2017-12, Zabela, Volha, Hettich, Timm, Schlotterbeck, Götz, Wimmer, Laurin, Mihovilovic D., Marko, Guillet, Fabrice, Belkacem, Bouaita, Shevchenko, Bénédicte, Hamburger, Matthias, Oufir, Mouhssin

In a screening of natural products for allosteric modulators of GABAA receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABAA and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds.

2016, Jähne, Evelyn A., Eigenmann, Daniela E., Moradi-Afrapoli, Fahimeh, Verjee, Sheela, Butterweck, Veronika, Hebeisen, Simon, Hettich, Timm, Schlotterbeck, Götz, Smiesko, Martin, Hamburger, Matthias, Oufir, Mouhssin

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.