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Publikation Validation of UHPLC–MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier and intestinal drug permeability studies(Elsevier, 05.09.2016) Moradi-Afrapoli, Fahimeh; Oufir, Mouhssin; Walter, Fruzsina R.; Deli, Maria A.; Smiesko, Martin; Zabela, Volha; Butterweck, Veronika; Hamburger, Matthias01A - Beitrag in wissenschaftlicher ZeitschriftPublikation GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding(Elsevier, 12/2017) Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic D., Marko; Guillet, Fabrice; Belkacem, Bouaita; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, MouhssinIn 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.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Pharmacokinetics of dietary kaempferol and its metabolite 4-hydroxyphenylacetic acid in rats(Elsevier, 12/2016) Zabela, Volha; Sampath, Chethan; Oufir, Mouhssin; Moradi-Afrapoli, Fahimeh; Butterweck, Veronika; Hamburger, MatthiasSCOPE: Kaempferol is a major flavonoid in the human diet and in medicinal plants. The compound exerts anxiolytic activity when administered orally in mice, while no behavioural changes were observed upon intraperitoneal administration, or upon oral administration in gut sterilized animals. 4-Hydroxyphenylacetic acid (4-HPAA), which possesses anxiolytic effects when administered intraperitoneally, is a major intestinal metabolite of kaempferol. Pharmacokinetic properties of the compounds are currently not clear. METHODS AND RESULTS: UHPLC-MS/MS methods were validated to support pharmacokinetic studies of kaempferol and 4-HPAA in rats. Non-compartmental and compartmental analyses were performed. After intravenous administration, kaempferol followed a one-compartment model, with a rapid clearance (4.40-6.44l/h/kg) and an extremely short half-life of 2.93-3.79min. After oral gavage it was not possible to obtain full plasma concentration-time profiles of kaempferol. Pharmacokinetics of 4-HPAA was characterized by a two-compartment model, consisting of a quick distribution phase (half-life 3.04-6.20min) followed by a fast elimination phase (half-life 19.3-21.1min). CONCLUSION: Plasma exposure of kaempferol is limited by poor oral bioavailability and extensive metabolism. Both compounds are rapidly eliminated, so that effective concentrations at the site of action do not appear to be reached. At present, it is not clear how the anxiolytic-like effects reported for the compounds can be explained.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Pharmacokinetics and in vitro blood-brain barrier screening of the plant-derived alkaloid tryptanthrin(Thieme, 2016) Jähne, Evelyn A.; Eigenmann, Daniela E.; Sampath, Chethan; Butterweck, Veronika; Culot, Maxime; Cecchelli, Roméo; Gosselet, Fabien; Walter, Fruzsina R.; Deli, Maria A.; Smiesko, Martin; Hamburger, Matthias; Oufir, MouhssinThe indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i. v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Development and full validation of an UPLC-MS/MS method for the quantification of the plant-derived alkaloid indirubin in rat plasma(Elsevier, 2016) Jähne, Evelyn A.; Butterweck, Veronika; Hamburger, Matthias; Oufir, Mouhssin; Sampath, ChethanAn UPLC-MS/MS method for the quantification of indirubin in lithium heparinized rat plasma was developed and validated according to current international guidelines. Indirubin was extracted from rat plasma by using Waters Ostro™ pass-through sample preparation plates. The method was validated with a LLOQ of 5.00ng/mL and an ULOQ of 500ng/mL. The calibration curve was fitted by least-square quadratic regression, and a weighting factor of 1/X was applied. Recoveries of indirubin and I.S. were consistent and ≥75.5%. Stability studies demonstrated that indirubin was stable in lithium heparinized rat plasma for at least 3 freeze/thaw cycles, for 3h at RT, for 96h in the autosampler at 10°C, and for 84days when stored below -65°C. Preliminary pharmacokinetic (PK) data were obtained from Sprague Dawley rats after intravenous administration of indirubin (2mg/kg b.w.) and blood sampling up to 12h after injection. PK parameters were determined by non-compartmental analysis. Indirubin had a half-life (t1/2) of 35min, and a relatively high clearance (CL) of 2.71L/h/kg.01A - Beitrag in wissenschaftlicher ZeitschriftPublikation Caco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone(Thieme, 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, MouhssinTryptanthrin 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.01A - Beitrag in wissenschaftlicher Zeitschrift