Early ADMET profiling of anti-inflammatory alkaloids using validated LC-MS/MS methods

Natural products provide an important and unique source of new lead compounds for drug discovery. Approximately 50% of all new chemical entities are inspired by nature. In the search of novel anti-inflammatory compounds in the ancient medicinal plant Isatis tinctoria, tryptanthrin, indirubin, and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) were identified as pharmacologically active constituents. They inhibit, at low µM to nM concentrations, cyclo-oxygenase-2  (COX-2), 5-lipoxygenase (5-LOX) catalyzed leukotriene synthesis, cyclin-dependent kinase (CDK), glycogensynthase kinase-3b (GSK), and mast cell degranulation. While the molecular modes of action of these alkaloids are not yet fully understood, their unique pharmacological profiles, structural drug-like properties, and low cytotoxicity render these molecules promising anti-inflammatory leads. To further evaluate the potential of these alkaloids as novel anti-inflammatory and anti-allergic leads, an assessment of their ADMET properties was warranted. For exact quantification of the compounds, LC-MS/MS methods were developed and validated according to current regulatory guidelines.
To get a first prognostic picture for the in vivo performance of our compounds, a pilot PK study was performed in male Sprague Dawley rats after intravenous administration at a concentration of 2 mg/kg b.w.. Tryptanthrin and indirubin showed a half-life (t1/2) of around 40 min, while indolinone was quickly eliminated (t1/2 = 4 min).
As most of the drugs are preferentially administered orally, the gastrointestinal tract (GIT) represents the major site of drug absorption. Human colon carcinoma cells (Caco-2 cells) serve as the method of choice to predict human drug absorption across the intestinal wall in vitro. To study the permeability of the three compounds across the epithelial monolayer, the alkaloids were screened at concentrations of 5-10 µM in the Caco-2 assay. As efflux transporters can greatly impact the in vivo absorption and, thus, the bioavailability of a drug candidate, the compounds were tested for possible P-glycoprotein (P-gp) interaction. Therefore, the alkaloids were co-incubated with the P-gp inhibitor verapamil (50 µM). Active efflux was assessed by calculating the efflux ratio (ER) from bidirectional assays. Due to high lipophilicity of indirubin, the compound precipitated in the transporter buffer and was thus excluded for further investigations in aqueous solutions. Tryptanthrin displayed a high permeability (Papp > 32.0 x 10-6 cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and the unchanged Papp values in presence of the P-glycoprotein (P-gp) inhibitor verapamil indicated that tryptanthrin was not involved in P-gp mediated efflux. In the Caco-2 assay, the recovery of indolinone was low, pointing to possibly extensive phase II metabolism. Further investigation by a high-resolution mass spectrometry (HR-MS) system revealed the formation of two sulfate and two glucuronide conjugates for indolinone.
Another well-known biological barrier in the human body is the blood-brain barrier (BBB). To evaluate the BBB permeation potential of tryptanthrin and indolinone, the compounds were tested in three cell-based human and animal BBB models. Data obtained with the human and animal BBB models showed good correlation and were indicative of a high BBB permeation potential of tryptanthrin and indolinone. Furthermore, active-mediated efflux was evaluated by calculating the ER from bidirectional assays. The ERs below 2 suggested that both compounds were not involved in active-mediated efflux.
Besides P-gp, another critical anti-target in drug development is the human ether-a-go-go (hERG) potassium channel. In the late 1990s, an increasing number of non-cardiovascular drugs have been withdrawn from the market due to cardiotoxic side-effects linked to hERG blocking. Since then, regulatory agencies insist on acquiring experimental hERG data of drug candidates before moving into clinical trials. Possible cardiotoxic liability of the compounds was assessed in vitro, by measurement of an inhibitory effect on hERG tail currents in stably transfected HEK 293 cells using the patch-clamp technique. Slight hERG inhibition was found for tryptanthrin (IC50 of 22 µM) and indolinone (IC50 of 25 µM).
Data obtained from the in vitro assays were corroborated by in silico predictions. For tryptanthrin and indolinone, all criteria for high human oral absorption and passive BBB penetration were met. In addition, the slight hERG inhibition found for tryptanthrin and indolinone in vitro could be confirmed by in silico predictions.