GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding

J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jan 1:1072:379-389. doi: 10.1016/j.jchromb.2017.11.036. Epub 2017 Dec 2.

Abstract

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.

Keywords: GABA(A); In vitro metabolism; Piperine analogs; Silensomes; UHPLC-Q-TOF-MS.

MeSH terms

  • Alkaloids* / analysis
  • Alkaloids* / chemistry
  • Alkaloids* / metabolism
  • Benzodioxoles* / analysis
  • Benzodioxoles* / chemistry
  • Benzodioxoles* / metabolism
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme Inhibitors* / analysis
  • Cytochrome P-450 Enzyme Inhibitors* / chemistry
  • Cytochrome P-450 Enzyme Inhibitors* / metabolism
  • Cytochrome P-450 Enzyme System* / analysis
  • Cytochrome P-450 Enzyme System* / classification
  • Cytochrome P-450 Enzyme System* / metabolism
  • Humans
  • Microsomes, Liver / metabolism
  • Piperidines* / analysis
  • Piperidines* / chemistry
  • Piperidines* / metabolism
  • Polyunsaturated Alkamides* / analysis
  • Polyunsaturated Alkamides* / chemistry
  • Polyunsaturated Alkamides* / metabolism
  • Receptors, GABA-A / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Alkaloids
  • Benzodioxoles
  • Cytochrome P-450 Enzyme Inhibitors
  • Piperidines
  • Polyunsaturated Alkamides
  • Receptors, GABA-A
  • Cytochrome P-450 Enzyme System
  • piperine