@article{SchmidtHolzgrabe2024,
  author    = {Schmidt, Sebastian and Holzgrabe, Ulrike},
  title     = {Do the enantiomers of ketamine bind enantioselectively to human serum albumin?},
  series = {European Journal of Pharmaceutical Sciences},
  volume    = {192},
  journal   = {European Journal of Pharmaceutical Sciences},
  doi       = {10.1016/j.ejps.2023.106640},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349791},
  year      = {2024},
  abstract  = {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.},
  language  = {en}
}
@article{ScheupleinLohrVivoliVegaetal.2023,
  author    = {Scheuplein, Nicolas Julian and Lohr, Theresa and Vivoli Vega, Mirella and Ankrett, Dyan and Seufert, Florian and Kirchner, Lukas and Harmer, Nicholas J. and Holzgrabe, Ulrike},
  title     = {Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei},
  series = {SLAS Discovery},
  volume    = {28},
  journal   = {SLAS Discovery},
  number    = {5},
  doi       = {10.1016/j.slasd.2023.03.004},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349784},
  pages     = {211-222},
  year      = {2023},
  abstract  = {Highlights • Synthesis of a new tracer molecule. • Robust and easy screening method for a broad range of compound activities. • FP assay validation considering limited use of starting material, DMSO tolerance, variation in incubation time and temperature. • Possibility of extension to HTP assay. Abstract The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.},
  language  = {en}
}
@article{TriyasmonoSchollmayerSchmitzetal.2023,
  author    = {Triyasmono, Liling and Schollmayer, Curd and Schmitz, Jens and Hovah, Emilie and Lombo, Cristian and Schmidt, Sebastian and Holzgrabe, Ulrike},
  title     = {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},
  series = {Food Analytical Methods},
  volume    = {16},
  journal   = {Food Analytical Methods},
  number    = {1},
  issn      = {1936-9751},
  doi       = {10.1007/s12161-022-02401-4},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324728},
  pages     = {155-167},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@article{SchmidtHolzgrabe2023,
  author    = {Schmidt, Sebastian and Holzgrabe, Ulrike},
  title     = {Method development, optimization, and validation of the separation of ketamine enantiomers by capillary electrophoresis using design of experiments},
  series = {Chromatographia},
  volume    = {86},
  journal   = {Chromatographia},
  number    = {1},
  issn      = {0009-5893},
  doi       = {10.1007/s10337-022-04229-w},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-324713},
  pages     = {87-95},
  year      = {2023},
  abstract  = {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\%.},
  language  = {en}
}
@article{WaltherKrmarLeistneretal.2023,
  author    = {Walther, Rasmus and Krmar, Jovana and Leistner, Adrian and Svrkota, Bojana and Otašević, Biljana and Malenović, Andjelija and Holzgrabe, Ulrike and Protić, Ana},
  title     = {Analytical Quality by Design: achieving robustness of an LC-CAD method for the analysis of non-volatile fatty acids},
  series = {Pharmaceuticals},
  volume    = {16},
  journal   = {Pharmaceuticals},
  number    = {4},
  issn      = {1424-8247},
  doi       = {10.3390/ph16040478},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311265},
  year      = {2023},
  abstract  = {An alternative to the time-consuming and error-prone pharmacopoeial gas chromatography method for the analysis of fatty acids (FAs) is urgently needed. The objective was therefore to propose a robust liquid chromatography method with charged aerosol detection for the analysis of polysorbate 80 (PS80) and magnesium stearate. FAs with different numbers of carbon atoms in the chain necessitated the use of a gradient method with a Hypersil Gold C\(_{18}\) column and acetonitrile as organic modifier. The risk-based Analytical Quality by Design approach was applied to define the Method Operable Design Region (MODR). Formic acid concentration, initial and final percentages of acetonitrile, gradient elution time, column temperature, and mobile phase flow rate were identified as critical method parameters (CMPs). The initial and final percentages of acetonitrile were fixed while the remaining CMPs were fine-tuned using response surface methodology. Critical method attributes included the baseline separation of adjacent peaks (α-linolenic and myristic acid, and oleic and petroselinic acid) and the retention factor of the last compound eluted, stearic acid. The MODR was calculated by Monte Carlo simulations with a probability equal or greater than 90\%. Finally, the column temperature was set at 33 °C, the flow rate was 0.575 mL/min, and acetonitrile linearly increased from 70 to 80\% (v/v) within 14.2 min.},
  language  = {en}
}
@article{MateraKaukCirilloetal.2023,
  author    = {Matera, Carlo and Kauk, Michael and Cirillo, Davide and Maspero, Marco and Papotto, Claudio and Volpato, Daniela and Holzgrabe, Ulrike and De Amici, Marco and Hoffmann, Carsten and Dallanoce, Clelia},
  title     = {Novel Xanomeline-containing bitopic ligands of muscarinic acetylcholine receptors: design, synthesis and FRET investigation},
  series = {Molecules},
  volume    = {28},
  journal   = {Molecules},
  number    = {5},
  issn      = {1420-3049},
  doi       = {10.3390/molecules28052407},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311249},
  year      = {2023},
  abstract  = {In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M\(_1\), M\(_2\), M\(_4\), and M\(_5\) FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M\(_1\)/M\(_4\)-preferring orthosteric agonist Xanomeline 10 and the M\(_1\)-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M\(_1\) mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M\(_1\) and M\(_4\) mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M\(_1\) subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.},
  language  = {en}
}
@article{MasotaOhlsenSchollmayeretal.2022,
  author    = {Masota, Nelson E. and Ohlsen, Knut and Schollmayer, Curd and Meinel, Lorenz and Holzgrabe, Ulrike},
  title     = {Isolation and characterization of galloylglucoses effective against multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae},
  series = {Molecules},
  volume    = {27},
  journal   = {Molecules},
  number    = {15},
  issn      = {1420-3049},
  doi       = {10.3390/molecules27155045},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286179},
  year      = {2022},
  abstract  = {The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid-liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-β-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2-256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.},
  language  = {en}
}
@article{RaschigRamirez‐ZavalaWiestetal.2023,
  author    = {Raschig, Martina and Ram{\´i}rez-Zavala, Bernardo and Wiest, Johannes and Saedtler, Marco and Gutmann, Marcus and Holzgrabe, Ulrike and Morschh{\"a}user, Joachim and Meinel, Lorenz},
  title     = {Azobenzene derivatives with activity against drug-resistant Candida albicans and Candida auris},
  series = {Archiv der Pharmazie},
  volume    = {356},
  journal   = {Archiv der Pharmazie},
  number    = {2},
  doi       = {10.1002/ardp.202200463},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312295},
  year      = {2023},
  abstract  = {Increasing resistance against antimycotic drugs challenges anti-infective therapies today and contributes to the mortality of infections by drug-resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'-dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole-susceptible and fluconazole-resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.},
  language  = {en}
}
@article{SchmidtZeheHolzgrabe2023,
  author    = {Schmidt, Sebastian and Zehe, Markus and Holzgrabe, Ulrike},
  title     = {Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein},
  series = {European Journal of Pharmaceutical Sciences},
  volume    = {181},
  journal   = {European Journal of Pharmaceutical Sciences},
  doi       = {10.1016/j.ejps.2022.106333},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300848},
  year      = {2023},
  abstract  = {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.},
  language  = {en}
}
@article{LeistnerHolzgrabe2021,
  author    = {Leistner, Adrian and Holzgrabe, Ulrike},
  title     = {Impurity Profiling of Baclofen Using Gradient HPLC-UV Method},
  series = {Chromatographia},
  volume    = {84},
  journal   = {Chromatographia},
  number    = {10},
  issn      = {1612-1112},
  doi       = {10.1007/s10337-021-04079-y},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268921},
  pages     = {927-935},
  year      = {2021},
  abstract  = {The GABA\(_{B}\) receptor agonist baclofen is a medication commonly used for the treatment of muscle spasticity. It is an amino acid and related to the neurotransmitter GABA. In this study, we developed a new, gradient high-performance liquid chromatography (HPLC) method for the impurity assessment of baclofen, which is appropriate for pharmacopoeial purposes. Since the impurities related to the synthesis pathway are acids, zwitterionic, or neutral, the method development is challenging. However, the separation of all components was achieved on a C18 stationary phase using a water-acetonitrile-trifluoroacetic acid gradient. A limit of detection (LOD) of at least 0.02\% was registered for all specified impurities. Additionally, CAD detection was performed to detect potential impurities lacking off a chromophore. The baclofen batches analyzed are far more pure than expected. All impurities were found below the specification limit, and thus, they can be regarded as unspecified. Moreover, the required runtime could be significantly reduced compared to the current USP or Ph. Eur. method.},
  language  = {en}
}