• Home
  • Search Results
  • Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models.

Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models.

Journal of pharmaceutical and biomedical analysis (2014-06-21)
Evelyn A Jähne, Daniela E Eigenmann, Maxime Culot, Roméo Cecchelli, Fruzsina R Walter, Mária A Deli, Robin Tremmel, Gert Fricker, Martin Smiesko, Matthias Hamburger, Mouhssin Oufir
ABSTRACT

The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties [1]. To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hydrocortisone, BioReagent, suitable for cell culture
Sigma-Aldrich
Sodium selenite, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
Sodium selenite, 99%
Sigma-Aldrich
Hydrocortisone, ≥98% (HPLC)
Sigma-Aldrich
Hydrocortisone, γ-irradiated, powder, BioXtra, suitable for cell culture
Sigma-Aldrich
Calcein-AM, BioReagent, suitable for fluorescence, ≥95.0% (HPLC)
Sigma-Aldrich
Sodium selenite, γ-irradiated, lyophilized powder, BioXtra, suitable for cell culture
Sigma-Aldrich
Calcein AM solution, 4 mM in DMSO, ≥90% (HPLC), solution
Supelco
Hydrocortisone, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Calcein-AM, Small Package (20 X 50 μg ), ≥95.0% (HPLC)
Sigma-Aldrich
Sodium selenite, anhydrous, ≥90.0% (RT)
USP
Hydrocortisone, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Hydrocortisone, meets USP testing specifications
Hydrocortisone for peak identification, European Pharmacopoeia (EP) Reference Standard
Hydrocortisone, European Pharmacopoeia (EP) Reference Standard
Hydrocortisone, British Pharmacopoeia (BP) Assay Standard
Supelco
Hydrocortisone, VETRANAL®, analytical standard