title: Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models.
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.
authors.affiliation: Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
authors.forename: Evelyn A
authors.initials: EA
authors.lastname: Jähne
authors.affiliation: Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
authors.forename: Daniela E
authors.initials: DE
authors.lastname: Eigenmann
authors.affiliation: Université Lille Nord de France, UArtois, BBB Laboratory EA 2465, IMPRT: IFR114, 62307 Lens Cedex, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Culot
authors.affiliation: Université Lille Nord de France, UArtois, BBB Laboratory EA 2465, IMPRT: IFR114, 62307 Lens Cedex, France.
authors.forename: Roméo
authors.initials: R
authors.lastname: Cecchelli
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary.
authors.forename: Fruzsina R
authors.initials: FR
authors.lastname: Walter
authors.affiliation: Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary.
authors.forename: Mária A
authors.initials: MA
authors.lastname: Deli
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 329, D-69120 Heidelberg, Germany.
authors.forename: Robin
authors.initials: R
authors.lastname: Tremmel
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 329, D-69120 Heidelberg, Germany.
authors.forename: Gert
authors.initials: G
authors.lastname: Fricker
authors.affiliation: Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Smiesko
authors.affiliation: Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hamburger
authors.affiliation: Institute of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. Electronic address: mouhssin.oufir@unibas.ch.
authors.forename: Mouhssin
authors.initials: M
authors.lastname: Oufir
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000893:Anti-Inflammatory Agents; D005452:Fluoresceins; D007211:Indoles; C085925:calcein AM
keywords: (E,Z)-3-(4-Hydroxy-3,5-dimethoxybenzylidene)indolin-2-one; Anti-inflammatory; Blood–brain barrier (BBB); Isatis tinctoria; LC–MS/MS