@article{GuentzelSchillingHanioetal.2020, author = {G{\"u}ntzel, Paul and Schilling, Klaus and Hanio, Simon and Schlauersbach, Jonas and Schollmayer, Curd and Meinel, Lorenz and Holzgrabe, Ulrike}, title = {Bioinspired Ion Pairs Transforming Papaverine into a Protic Ionic Liquid and Salts}, series = {ACS Omega}, volume = {5}, journal = {ACS Omega}, number = {30}, doi = {10.1021/acsomega.0c02630}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230265}, pages = {19202-19209}, year = {2020}, abstract = {Microbial, mammalian, and plant cells produce and contain secondary metabolites, which typically are soluble in water to prevent cell damage by crystallization. The formation of ion pairs, for example, with carboxylic acids or mineral acids, is a natural blueprint to maintain basic metabolites in solution. Here, we aim at showing whether the mostly large carboxylates form soluble protic ionic liquids (PILs) with the basic natural product papaverine resulting in enhanced aqueous solubility. The obtained PILs were characterized by H-1-N-15 HMBC nuclear magnetic resonance (NMR) and in the solid state using X-ray powder diffraction, differential scanning calorimetry, and dissolution measurements. Furthermore, their supramolecular pattern in aqueous solution was studied by means of potentiometric and photometrical solubility, NMR aggregation assay, dynamic light scattering, zeta potential, and viscosity measurements. Thereby, we identified the naturally occurring carboxylic acids, citric acid, malic acid, and tartaric acid, as being appropriate counterions for papaverine and which will facilitate the formation of PILs with their beneficial characteristics, like the improved dissolution rate and enhanced apparent solubility.}, language = {en} } @article{MarkensteinAppeltMenzelMetzgeretal.2014, author = {Markenstein, Lisa and Appelt-Menzel, Antje and Metzger, Marco and Wenz, Gerhard}, title = {Conjugates of methylated cyclodextrin derivatives and hydroxyethyl starch (HES): Synthesis, cytotoxicity and inclusion of anaesthetic actives}, series = {Beilstein Journal of Organic Chemistry}, volume = {10}, journal = {Beilstein Journal of Organic Chemistry}, issn = {1860-5397}, doi = {10.3762/bjoc.10.325}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114280}, pages = {3087 - 3096}, year = {2014}, abstract = {The mono-6-deoxy-6-azides of 2,6-di-O-methyl-beta-cyclodextrin (DIMEB) and randomly methylated-beta-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu+-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 degrees C (DIMEB-HES) and 84.5 degrees C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.}, language = {en} } @article{OrtizSotoSeibel2016, author = {Ortiz-Soto, Maria Elena and Seibel, J{\"u}rgen}, title = {Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {5}, doi = {10.1371/journal.pone.0155410}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179807}, year = {2016}, abstract = {Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20\%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside.}, language = {en} } @phdthesis{Reggane2019, author = {Reggane, Maude}, title = {Lowering lattice forces of crystalline bases}, doi = {10.25972/OPUS-16380}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163803}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The number of active pharmaceutical ingredients (APIs) exhibiting a low solubility in aqueous media or a slow dissolution rate kept rising over the past years urging formulation scientists to explore new ways to tackle poor solubility and to enable oral absorption from such compounds. Bioavailability of poorly water-soluble compounds can be improved by increasing the dissolution rate and/or by increasing the gastro intestinal concentration through transient supersaturation. The dissolution rate of the API can be typically modified by the choice of the physical form, the polymorphic form, the powder surface area, and the local pH, while a transient supersaturation can be extended mainly by nucleation or crystallization inhibiting effects. In the present thesis, three strategies were explored to tailor the dissolution rate, the supersaturation and the hydrotropic solubilization of APIs, weak bases, respectively. The first part of this thesis followed a bioinspired approach to extend the kinetic solubility of salts and co-crystals. API salts and co-crystals are high energy forms that can generate supersaturated solutions with respect to any more stable form, typically the most stable API form in physiological environment. The transient kinetic stabilization of supersaturated states, also termed "parachute effect", is considered to improve bioavailability and is one aspect of the formulation that can be tailored. Inspiration from plants, which store high concentrations of aromatic bases in their vacuoles via complexation with polyphenols, sparked the evaluation to use hydroxybenzoic acid derivatives for salt or co-crystal engineering. Imatinib was chosen as the model compound for this investigation as its aromaticity and flat molecular architecture could favor interactions with hydroxybenzoic acid derivatives. One 1:1 Imatinib syringate co-crystal (I-SYA (1:1)) and one 1:2 Imatinib syringate co-crystal salt (I-SYA (1:2)) were obtained. Their dissolution assays in simulated intestinal fluid (SIF; a 50 mM phosphate buffer of pH 6.8) revealed that they formed stable solutions for several hours and days, respectively, in contrast to the marketed Imatinib mesylate salt (approx. 1h). This kinetic stability in solution was linked to the nucleation inhibition of the less soluble Imatinib hydrate by syringic acid (SYA). In solution 1H-NMR studies evidenced the aggregation of Imatinib and SYA. The amphiphilic nature of both Imatinib and SYA is considered to drive their association in solution, additionally, multiple intermolecular interactions such as hydrogen bonds and π-π stacking are likely to contribute. The association in solution enabled a phase of extended supersaturation, i.e., a parachute against desupersaturation, while no negative impact of aggregation on the permeability of both Imatinib and SYA was observed. A prerequisite to reach supersaturation is a rapid dissolution and release of the API from the formulation. Accordingly, the second and third part of this thesis is focused on the so-called "spring effect" of amorphous solid dispersions (ASDs). The addition of a hydrotropic agent, meaning a molecule that can solubilize poorly water-soluble APIs in aqueous solutions (well-known examples of hydrotropes are benzoic acid and nicotinamide) into an amorphous Ciprofloxacin-polymer matrix led to ternary systems with a significantly faster release and higher concentration of the API in SIF as compared to binary ASDs consisting of Ciprofloxacin (CPX) and polymer only. The stronger spring could be rationalized by an improved wetting of the ASD, or/and by a hydrotropic solubilization effect, although these hypotheses need further investigation. Marked differences in the dissolution profiles of binary ASDs were observed in biorelevant fasted simulated intestinal fluid (FaSSIF; a medium containing Na taurocholate (3 mM) and lecithin (0.75 mM) at pH 6.5) as compared to SIF. In FaSSIF, API release from binary polymeric ASDs was largely improved, and the duration of supersaturation was extended. This suggests that the bile salt Na taurocholate and lecithin present in FaSSIF do improve both dissolution rate and supersaturation of ASDs, the two pillars of ASDs as oral enabling formulations. Indeed, bile salts are endogenous surfactants which, together with phospholipids, play an important role in the wetting, solubilization, and absorption of lipophilic compounds. The aim of the third part of the present thesis was to study ASDs as formulation principles reducing the strong positive food effect of Compound A. By inclusion of Na taurocholate (NaTC) within the matrix of polymeric ASDs a significant improvement of the dissolution rate and the kinetic solubility in SIF were achieved. Transient supersaturated states of up to four orders of magnitude over the equilibrium solubility were obtained. Two ASDs were selected for further in vivo evaluation in dog. The first was a NaTC/Eudragit E based ASD meant to dissolve and release Compound A in the acidic environment of the stomach, where its solubility is the highest. The second relied on the release of Compound A in the neutral environment of the duodenum and jejunum by using an enterically dissolving polymer, HPMC-P. Releasing the API at the site of its putative absorption was an attempt to control supersaturation levels in the duodenum and to prevent portioning and thus dilution effects during transfer from the stomach. In fasted dogs, exposure from the NaTC/HPMC-P ASD was close to that of the reference Compound A formulation under fed conditions, which suggests an improved dissolution rate and kinetic solubility under fasted conditions (historical data). The exposure from the NaTC/Eudragit E ASD was twice as low as from the NaTC/HPMC-P ASD, and also lower compared to Compound A reference formulation, whereas in vitro the parachute effect of the NaTC/Eudragit E ASD was largely superior to that of the NaTC/HPMC-P ASD. A difference in the extend of the parachute could be related to differences in the thermodynamic activity of dissolved molecules from the two ASDs. Indeed, the high instability of the NaTC/HPMC-P ASD could stem from a high thermodynamic activity driving diffusion through membranes, whereas less instable solutions of NaTC/Eudragit E could indicate solubilization effects which often translate into a lower flux through the biological membrane. Additionally, the pH of the environment where dissolution takes place might be an important factor for absorption, and could also account for the difference in exposure from the two ASDs. The aim of this thesis was to explore how the intimate environment of weak, poorly soluble bases could be functionalized to improve dissolution rate and kinetic solubility. The investigations highlighted that the performance of enabling oral delivery formulations of weak bases in aqueous media can be enhanced at different levels. At one end initial dissolution rate of ASDs can be tailored by introducing hydrotropes or/and bile salts within the polymeric matrix of ASDs. Bile salts, when combined with appropriate polymers, had also a precipitation inhibition effect enabling the maintenance of supersaturation for a bio-relevant period of time. These results set the ground for further investigations to comprehend specific interactions between bile salts and APIs, and potentially polymers at the molecular level. It will be interesting to explore how such complex systems can be exploited in the formulation design of poorly water-soluble APIs. In addition, it was observed that the duration of supersaturation generated by salts/co-crystals can be extended by the pertinent selection of counterions or coformers. The in vivo relevance of these tunings remains to be evaluated, as translation from closed, in vitro systems to the highly dynamic gastrointestinal environment is not straightforward. A better understanding of the contribution of each kinetic stage (dissolution, supersaturation, and precipitation) and their interplay with physiological factors impacting absorption is essential to facilitate the design of formulations with improved pharmacokinetics.}, subject = {Kokristallisation}, language = {en} } @article{MasotaVoggOhlsenetal.2021, author = {Masota, Nelson E. and Vogg, Gerd and Ohlsen, Knut and Holzgrabe, Ulrike}, title = {Reproducibility challenges in the search for antibacterial compounds from nature}, series = {PLoS One}, volume = {16}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0255437}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260239}, year = {2021}, abstract = {Background Reproducibility of reported antibacterial activities of plant extracts has long remained questionable. Although plant-related factors should be well considered in serious pharmacognostic research, they are often not addressed in many research papers. Here we highlight the challenges in reproducing antibacterial activities of plant extracts. Methods Plants with reported antibacterial activities of interest were obtained from a literature review. Antibacterial activities against Escherichia coli and Klebsiella pneumoniae were tested using extracts' solutions in 10\% DMSO and acetone. Compositions of working solutions from both solvents were established using LC-MS analysis. Moreover, the availability of details likely to affect reproducibility was evaluated in articles which reported antibacterial activities of studied plants. Results Inhibition of bacterial growth at MIC of 256-1024 μg/mL was observed in only 15.4\% of identical plant species. These values were 4-16-fold higher than those reported earlier. Further, 18.2\% of related plant species had MICs of 128-256 μg/mL. Besides, 29.2\% and 95.8\% of the extracts were soluble to sparingly soluble in 10\% DMSO and acetone, respectively. Extracts' solutions in both solvents showed similar qualitative compositions, with differing quantities of corresponding phytochemicals. Details regarding seasons and growth state at collection were missing in 65\% and 95\% of evaluated articles, respectively. Likewise, solvents used to dissolve the extracts were lacking in 30\% of the articles, whereas 40\% of them used unidentified bacterial isolates. Conclusion Reproducibility of previously reported activities from plants' extracts is a multi-factorial aspect. Thus, collective approaches are necessary in addressing the highlighted challenges.}, language = {en} } @phdthesis{Renner2003, author = {Renner, Jan}, title = {Restmonomergehalt von Kompositen bei Aush{\"a}rtung mit unterschiedlichen Polymerisationsger{\"a}ten bzw. -verfahren in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-6378}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2003}, abstract = {Das Ziel der vorliegenden Arbeit war die Bestimmung der freigesetzten Bestandteile aus Kompositkunststoffen bei unterschiedlichen Bestrahlungsverfahren. Miteinander verglichen wurden die Halogenstandard-, die Halogensoftstarth{\"a}rtung und die Plasmah{\"a}rtung. Zudem sollten die initiale und die mittelfristige Monomerfreisetzung bzw. Wasseraufnahme verglichen werden. Es wurden die Hypothesen {\"u}berpr{\"u}ft, dass Proben, die nach Softstart- oder Schnellh{\"a}rtungsprotokollen belichtet worden waren, eine st{\"a}rkere Monomerfreisetzung aufweisen und somit mehr von der Nachh{\"a}rtung abh{\"a}ngen als konventionell geh{\"a}rtete Komposite. Außerdem sollte festgestellt werden, ob die mittelfristige L{\"o}slichkeit bei verschiedenen H{\"a}rtungsverfahren {\"a}hnlich ist, da die Nachh{\"a}rtung urspr{\"u}ngliche Defizite kompensieren soll. Schließlich sollte {\"u}berpr{\"u}ft werden, ob eine fehlende Abstimmung zwischen den Absorptionseigenschaften der Photoinitiatoren und dem Emissionsspektrum von Lichtpolymerisationsger{\"a}ten die Polymerisation beeintr{\"a}chtigt und folglich zu einer h{\"o}heren initialen und mittelfristigen L{\"o}slichkeit f{\"u}hrt. Insgesamt wurden f{\"u}nf Komposite getestet, drei Feinkorn-Hybridkomposite [Herculite XRV (Kerr), Solitaire 2 (Kulzer) und Z 250 (3M)], ein inhomogenes Mikrof{\"u}llerkomposit [Silux Plus] und ein Ormocer [Keramikkomposit Definite (Degussa)]. Die Bestrahlungsprotokolle umfassten die Halogenstandardh{\"a}rtung mit drei verschiedenen Intensit{\"a}ten (TriLight, ESPE), die Exponentialpolymerisation (Ramp Curing) (dito), die Stufenpolymerisation (Step Curing) (HiLight, ESPE), die Pulspolymerisation (VIP Light, Bisco) und die Plasmah{\"a}rtung (Apollo 95E, DMDS; Lightning Cure, ADT). Die initiale L{\"o}slichkeit wurde bestimmt, indem die Komposite in simulierte Kavit{\"a}ten (Hohlzylinder-Formen mit 6 mm Innendurchmesser und 2 mm H{\"o}he aus gepresster Keramik) gef{\"u}llt und 24 Stunden in demineralisiertem Wasser bei 37°C eluiert wurden. Die mittelfristige L{\"o}slichkeit wurde mittels reiner Komposit-Proben gleicher Gr{\"o}ße bestimmt, die im Dunkeln bei 37°C f{\"u}r 24 Stunden gelagert und in 50\% Methanol- Wasser-Gemisch bei 37°C f{\"u}r 72 Stunden extrahiert wurden. Nachdem die Proben auf ein konstantes Gewicht getrocknet waren, wurden L{\"o}slichkeit und L{\"o}sungsmittelaufnahme gravimetrisch bestimmt. Die mittelfristige L{\"o}slichkeit und L{\"o}sungsmittelaufnahme war in allen Versuchsreihen h{\"o}her als die initiale. Die Bestrahlung mit verminderter Intensit{\"a}t hat die L{\"o}slichkeit und L{\"o}sungsmittelaufnahme im Vergleich zur Standardh{\"a}rtung mit hoher Intensit{\"a}t erh{\"o}ht. Dies war bei der Exponentialpolymerisation, der Stufenpolymerisation und der Pulspolymerisation (bei den meisten Materialien) nicht der Fall. Die Plasmah{\"a}rtung funktionierte gut bei Z250 und Herculite XRV. Bei Silux Plus und Definite erzielte sie {\"a}hnliche Resultate wie die Halogenstandardh{\"a}rtung bei mittlerer oder niedriger Intensit{\"a}t. Bei Solitaire 2 f{\"u}hrte sie zu einer hohen (Lightning Cure) oder sehr hohen (Apollo 95E) L{\"o}slichkeit. Somit kann aus den Ergebnissen verallgemeinernd die Schlussfolgerung gezogen werden, dass eine Verringerung der Bestrahlungsintensit{\"a}t die L{\"o}slichkeit und L{\"o}sungsmittelaufnahme erh{\"o}ht, Softstart-Protokolle jedoch nicht. Die Wirksamkeit der Plasmah{\"a}rtung h{\"a}ngt in starkem Maß von der Art der verwendeten Photoinitiatoren ab.}, language = {de} }