TY - JOUR A1 - Schmidt, Sebastian A1 - Holzgrabe, Ulrike T1 - Do the enantiomers of ketamine bind enantioselectively to human serum albumin? JF - European Journal of Pharmaceutical Sciences N2 - The binding of drugs to plasma proteins is an important process in the human body and has a significant influence on pharmacokinetic parameter. Human serum albumin (HSA) has the most important function as a transporter protein. The binding of ketamine to HSA has already been described in literature, but only of the racemate. The enantiomerically pure S-ketamine is used as injection solution for induction of anesthesia and has been approved by the Food and Drug Administration for the therapy of severe depression as a nasal spray in 2019. The question arises if there is enantioselective binding to HSA. Hence, the aim of this study was to investigate whether there is enantioselective binding of S-and R-ketamine to HSA or not. Ultrafiltration (UF) followed by chiral capillary electrophoretic analysis was used to determine the extent of protein binding. Bound fraction to HSA was 71.2 % and 64.9 % for enantiomerically pure R- and S-ketamine, respectively, and 66.5 % for the racemate. Detailed binding properties were studied by Saturation Transfer Difference (STD)-, waterLOGSY- and Carr-Purcell-Meiboom-Gill (CPMG)-NMR spectroscopy. With all three methods, the aromatic ring and the N-methyl group could be identified as the structural moieties most strongly involved in binding of ketamine to HSA. pK\(_{aff}\) values determined using UF and NMR indicate that ketamine is a weak affinity ligand to HSA and no significant differences in binding behavior were found between the individual enantiomers and the racemate. KW - protein binding KW - albumin KW - electrophoresis KW - nuclear magnetic resonance spectroscopy KW - ultrafiltration KW - enantioselectivity Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349791 VL - 192 ER - TY - JOUR A1 - Schmidt, Sebastian A1 - Zehe, Markus A1 - Holzgrabe, Ulrike T1 - Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein JF - European Journal of Pharmaceutical Sciences N2 - Most drugs, especially those with acidic or neutral moieties, are bound to the plasma protein albumin, whereas basic drugs are preferentially bound to human alpha-1-acid glycoprotein (AGP). The protein binding of the long-established drugs ephedrine and pseudoephedrine, which are used in the treatment of hypotension and colds, has so far only been studied with albumin. Since in a previous study a stereoselective binding of ephedrine and pseudoephedrine to serum but not to albumin was observed, the aim of this study was to check whether the enantioselective binding behavior of ephedrine and pseudoephedrine, in addition to the derivatives methylephedrine and norephedrine, is due to AGP and to investigate the influence of their different substituents and steric arrangement. Discontinuous ultrafiltration was used for the determination of protein binding. Characterization of ligand-protein interactions of the drugs was obtained by saturation transfer difference nuclear magnetic resonance spectroscopy. Docking experiments were performed to analyze possible ligand-protein interactions. The more basic the ephedrine derivative is, the higher is the affinity to AGP. There was no significant difference in the binding properties between the individual enantiomers and the diastereomers of ephedrine and pseudoephedrine. KW - protein binding KW - AGP KW - ultrafiltration KW - saturation transfer difference NMR KW - epitope mapping KW - ephedrine Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300848 VL - 181 ER - TY - THES A1 - Schmidt, Sebastian T1 - A closer look at long-established drugs: enantioselective protein binding and stability studies T1 - Lang-etablierte Arzneistoffe genauer unter die Lupe genommen: Enantioselektive Proteinbindung und Stabilitätsstudien N2 - The aim of this work was to investigate older, established drugs. The extent of the protein binding of chiral ephedra alkaloids to AGP and of ketamine to albumin was determined. Since enantiomers of these drugs are individual available, the focus was on possible enantioselective binding and structural moieties involved in the binding. Previously published work suggested that ephedrine and pseudoephedrine can bind stereoselectively to proteins other than albumin in serum. For the determination of the extent of protein binding, the established ultrafiltration with subsequent chiral CE analysis was used. To determine the influence of basicity on binding, the drugs methylephedrine and norephedrine were also analyzed. Drug binding to AGP increased with increasing basicity as follows: norephedrine < methylephedrine < ephedrine < pseudoephedrine. pKaff was determined both graphically using the Klotz plot and mathematical indicating a low affinity of the ephedra alkaloids to AGP. Using STD-NMR spectroscopy experiments the aromatic protons and the C-CH3 side chain were shown to be most strongly involved in binding, which could be confirmed by molecular docking experiments in more detail. For all drugs, van der Waals-, π π , cationic interactions, hydrogen bonds, and a formation of a salt bridge were observed. The individual enantiomers showed no significant differences and thus the binding of ephedra alkaloids to AGP is not significant. In contrast to the ephedra alkaloids, the possible enantioselective binding to albumin was investigated for R and S ketamine. Again, ultrafiltration followed by CE analysis was performed. The binding of ketamine to one main binding site could be identified. A non-linear fit was used for the determination of pKaff. Using the NMR methods STD-NMR, waterLOGSY-NMR, and CPMG-NMRspectroscopy: the aromatic protons as well as the protons of the NCH3 methyl group showed the largest signal intensity changes, while the cyclohexanone protons showed the smallest changes. pKaff was also determined by the change in the chemical shift at different drug-protein ratios. These obtained values confirm the values obtained from ultrafiltration. Based on this, ketamine is classified as a low-affinity ligand to albumin. There were no significant differences between the individual enantiomers and thus the binding of ketamine to albumin is not a stereoselective process. Using statistical design of experiments an efficient chiral CE method for determining the extent of protein binding of R and S ketamine to albumin was developed and validated according to ICH Q2 (R1) guideline. The stability of ketamine was also investigated because a yellowish discoloration of an aqueous solution of ketamine developed under heat. XRPD investigations showed the same crystal structure for all batches examined. An untargeted screening using LC HRMS as well as LC UV measurements showed no degradation of ketamine or the presence of impurities in stress and non-stressed ketamine solutions, confirming the stability of ketamine under the stress conditions investigated. The lower the quality of the water used in the stress tests, the more intense the yellow discoloration occurred. The impurity or the mechanism that causes the yellow discoloration could not be identified. N2 - Ziel dieser Arbeit war es ältere, etablierte Arzneistoffe zu untersuchen. Das Ausmaß der Proteinbindung von chiralen Ephedra-Alkaloiden an AGP und von Ketamin an Albumin wurde bestimmt. Da Enantiomere dieser Wirkstoffe individuell verfügbar sind, lag der Fokus auf möglichen enantioselektiven Bindungen und strukturellen Funktionalitäten, die an der Bindung beteiligt sind. Zuvor veröffentlichte Arbeiten deuteten darauf hin, dass Ephedrin und Pseudoephedrin stereoselektiv an andere Proteine als Albumin im Serum binden können. Zur Bestimmung des Ausmaßes der Proteinbindung wurde die etablierte Ultrafiltration mit anschließender chiraler CE-Analyse eingesetzt. Um den Einfluss der Basizität auf die Bindung zu bestimmen, wurden auch die Wirkstoffe Methylephedrin und Norephedrin analysiert. Die Bindung des Wirkstoffs an AGP nahm mit zunehmender Basizität wie folgt zu: Norephedrin < Methylephedrin < Ephedrin < Pseudoephedrin. pKaff wurde sowohl grafisch mit Hilfe des Klotz-Plots als auch mathematisch bestimmt, was auf eine geringe Affinität der Ephedra-Alkaloide zu AGP hinweist. Mittels STD-NMR Spektroskopie Experimenten konnte gezeigt werden, dass die aromatischen Protonen und die C-CH3-Seitenkette am stärksten an der Bindung beteiligt sind, was durch molekulare Docking-Experimente detailliert bestätigt werden konnte. Für alle Wirkstoffe wurden van-der-Waals-, π π , kationische Wechselwirkungen, Wasserstoffbrückenbindungen und die Bildung einer Salzbrücke beobachtet. Die einzelnen Enantiomere zeigten keine signifikanten Unterschiede, so dass die Bindung von Ephedra-Alkaloiden an AGP nicht signifikant ist. Im Gegensatz zu den Ephedra-Alkaloiden wurde die mögliche enantioselektive Bindung an Albumin für R und S Ketamin untersucht. Auch hier wurde eine Ultrafiltration mit anschließender CE-Analyse durchgeführt. Die Bindung von Ketamin an eine Hauptbindungsstelle konnte identifiziert werden. Für die Bestimmung von pKaff wurde eine nichtlineare Anpassung verwendet. Mit den NMR-Methoden STD-NMR, waterLOGSY NMR und CPMG-NMR Spektroskopie zeigten sowohl die aromatischen Protonen als auch die Protonen der NCH3-Methylgruppe die größten Änderungen der Signalintensität, während die Cyclohexanon-Protonen die geringsten Änderungen afuwiesen. pKaff wurde auch durch die Änderung der chemischen Verschiebung bei verschiedenen Wirkstoff-Protein-Verhältnissen bestimmt. Die Werte bestätigen die durch die Ultrafiltration erhaltenen Werte. Auf dieser Grundlage wird Ketamin als Ligand mit niedriger Affinität zu Albumin eingestuft. Es zeigten sich keine signifikanten Unterschiede zwischen den einzelnen Enantiomeren und somit ist die Bindung von Ketamin an Albumin kein stereoselektiver Prozess. Mit Hilfe der statistischen Versuchsplanung wurde eine effiziente chirale CE-Methode zur Bestimmung des Ausmaßes der Proteinbindung von R und S Ketamin an Albumin entwickelt und gemäß der ICH Q2 (R1) Richtlinie validiert. Die Stabilität von Ketamin wurde ebenfalls untersucht, da sich unter Hitze eine gelbliche Verfärbung einer wässrigen Ketaminlösung entwickelte. XRPD-Untersuchungen zeigten für alle untersuchten Chargen die gleiche Kristallstruktur. Ein nicht zielgerichtetes Screening mittels LC HRMS sowie LC UV-Messungen zeigte keinen Abbau von Ketamin oder das Vorhandensein von Verunreinigungen in Stress- und nicht gestressten Ketaminlösungen, was die Stabilität von Ketamin unter den untersuchten Bedingungen bestätigt. Je schlechter die Qualität des in den Stresstests verwendeten Wassers war, desto intensiver trat die Gelbverfärbung auf. Die Verunreinigung oder der Mechanismus, der die gelbe Verfärbung verursacht, konnte nicht identifiziert werden. KW - Proteinbindung KW - Ephedrin KW - Ketamin KW - Stabilitätsstudien KW - Arzneistoffanalytik KW - Enantioselektivität Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-345945 ER - TY - JOUR A1 - Schmidt, Sebastian A1 - Holzgrabe, Ulrike T1 - Method development, optimization, and validation of the separation of ketamine enantiomers by capillary electrophoresis using design of experiments JF - Chromatographia N2 - Capillary electrophoresis was chosen as cost-effective and robust method to separate ketamine enantiomers. For the method development, first different native and modified cyclodextrins were tested. The most promising chiral selector was α-cyclodextrin. A design of experiments (DoE) was carried out, which started with the screening of relevant factors. Based on these results, the method was optimized according to the significant factors (buffer, cyclodextrin concentration, pH value, voltage, temperature) of the screening based on the response resolution and migration time of the later migrating enantiomer. The optimized conditions consisted of a background electrolyte with 275 mM TRIS, adjusted with 85% phosphoric acid to a pH of 2.50, and 50 mM α-cyclodextrin, at a temperature of 15 °C, an applied voltage of 30 kV and an injection pressure of 1.0 psi for 10 s. A fused-silica capillary with a total length of 70 cm and an effective length to the detector of 60 cm was used. The method was validated according to ICH guideline Q2 R(1). The limit of quantification was 3.51 µg mL\(^{−1}\) for S-ketamine and 3.98 µg mL\(^{−1}\)for R-ketamine. The method showed good linearity for racemic ketamine with R\(^2\) of 0.9995 for S-ketamine and 0.9994 for R-ketamine. The lowest quantifiable content of S-ketamine found in R-ketamine was 0.45%. KW - ketamine KW - capillary electrophoresis KW - design of experiments KW - cyclodextrins KW - enantiomers Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324713 SN - 0009-5893 VL - 86 IS - 1 ER - TY - JOUR A1 - Triyasmono, Liling A1 - Schollmayer, Curd A1 - Schmitz, Jens A1 - Hovah, Emilie A1 - Lombo, Cristian A1 - Schmidt, Sebastian A1 - Holzgrabe, Ulrike T1 - Simultaneous determination of the saponification value, acid value, ester value, and iodine value in commercially available red fruit oil (Pandanus conoideus, Lam.) using \(^1\)H qNMR spectroscopy JF - Food Analytical Methods N2 - Red fruit oil (RFO) can be extracted from fruits of Pandanus conoideus, Lam., an endogenous plant of Papua, Indonesia. It is a commonly used essential original traditional medicine. By applying a newly developed quantitative \(^1\)H NMR (qNMR) spectroscopy method for quality assessment, a simultaneous determination of the saponification value (SV), acid value (AV), ester value (EV), and iodine value (IV) in RFO was possible. Dimethyl sulfone (DMSO\(_2\)) was used as an internal standard. Optimization of NMR parameters, such as NMR pulse sequence, relaxation delay time, and receiver gain, finally established the \(^1\)H NMR-based quantification approach. Diagnostic signals of the internal standard at δ = 2.98 ppm, SV at δ = 2.37–2.20 ppm, AV at δ = 2.27–2.20 ppm, EV at δ = 2.37–2.27 ppm, and IV at δ = 5.37–5.27 ppm, respectively, were used for quantitative analysis. The method was validated concerning linearity (R\(^2\) = 0.999), precision (less than 0.83%), and repeatability in the range 99.17–101.17%. Furthermore, this method was successfully applied to crude RFO, crude RFO with palmitic and oleic acid addition, and nine commercial products. The qNMR results for the respective fat values are in accordance with the results of standard methods, as can be seen from the F- and t-test (< 1.65 and < 1.66, respectively). The fundamental advantages of qNMR, such as its rapidity and simplicity, make it a feasible and existing alternative to titration for the quality control of RFO. KW - quantitative 1H NMR KW - saponification Value KW - acid value KW - ester value KW - iodine value KW - red fruit oil Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324728 SN - 1936-9751 VL - 16 IS - 1 ER - TY - JOUR A1 - Schmidt, Sebastian A1 - Liu, Guoxing A1 - Liu, Guilai A1 - Yang, Wenting A1 - Honisch, Sabina A1 - Pantelakos, Stavros A1 - Stournaras, Christos A1 - Hönig, Arnd A1 - Lang, Florian T1 - Enhanced Orai1 and STIM1 expression as well as store operated \(Ca^{2+}\) entry in therapy resistant ovary carcinoma cells JF - Oncotarget N2 - Mechanisms underlying therapy resistance of tumor cells include protein kinase Akt. Putative Akt targets include store-operated \(Ca^{2+}\)-entry (SOCE) accomplished by pore forming ion channel unit Orai1 and its regulator STIM1. We explored whether therapy resistant (A2780cis) differ from therapy sensitive (A2780) ovary carcinoma cells in Akt, Orai1, and STIM1 expression, \(Ca^{2+}\)-signaling and cell survival following cisplatin (100µM) treatment. Transcript levels were quantified with RT-PCR, protein abundance with Western blotting, cytosolic \(Ca^{2+}\)-activity ([\(Ca^{2+}\)]i) with Fura-2-fluorescence, SOCE from increase of [\(Ca^{2+}\)]i following \(Ca^{2+}\)-readdition after Ca2+-store depletion, and apoptosis utilizing flow cytometry. Transcript levels of Orai1 and STIM1, protein expression of Orai1, STIM1, and phosphorylated Akt, as well as SOCE were significantly higher in A2780cis than A2780 cells. SOCE was decreased by Akt inhibitor III (SH-6, 10µM) in A2780cis but not A2780 cells and decreased in both cell lines by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-ABP, 50µM). Phosphatidylserine exposure and late apoptosis following cisplatin treatment were significantly lower in A2780cis than A2780 cells, a difference virtually abolished by SH-6 or 2-ABP. In conclusion, Orai1/STIM1 expression and function are increased in therapy resistant ovary carcinoma cells, a property at least in part due to enhanced Akt activity and contributing to therapy resistance in those cells. KW - Ca2+ release activated Ca2+ channel KW - SOCE KW - Akt KW - SH-6 KW - 2-APB KW - apoptosis Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121423 UR - www.impactjournals.com/oncotarget VL - 5 IS - 13 ER -