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Alzheimer's disease (AD) is a multifactorial disease and the most common form of dementia. There are no treatments to cure, prevent or slow down the progression of the disease. Natural products hold considerable interest for the development of preventive neuroprotectants to treat neurodegenerative disorders like AD, due to their low toxicity and general beneficial effects on human health with their anti-inflammatory and antioxidant features. In this work we describe regioselective synthesis of 7-O-ester hybrids of the flavonoid taxifolin with the phenolic acids cinnamic and ferulic acid, namely 7-O-cinnamoyltaxifolin and 7-O-feruloyltaxifolin. The compounds show pronounced overadditive neuroprotective effects against oxytosis, ferroptosis and ATP depletion in the murine hippocampal neuron HT22 cell model. Furthermore, 7-O-cinnamoyltaxifolin and 7-O-feruloyltaxifolin reduced LPS-induced neuroinflammation in BV-2 microglia cells as assessed by effects on the levels of NO, IL6 and TNFα. In all in vitro assays the 7-O-esters of taxifolin and ferulic or cinnamic acid showed strong overadditive activity, significantly exceeding the effects of the individual components and the equimolar mixtures thereof, which were almost inactive in all of the assays at the tested concentrations. In vivo studies confirmed this overadditive effect. Treatment of an AD mouse model based on the injection of oligomerized Aβ\(_{25-35}\) peptide into the brain to cause neurotoxicity and subsequently memory deficits with 7-O-cinnamoyltaxifolin or 7-O-feruloyltaxifolin resulted in improved performance in an assay for short-term memory as compared to vehicle and mice treated with the respective equimolar mixtures. These results highlight the benefits of natural product hybrids as a novel compound class with potential use for drug discovery in neurodegenerative diseases due to their pharmacological profile that is distinct from the individual natural components.
Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 \(\mu\)g/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 \(\mu\)g/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.
Ruxolitinib (RUX) is approved for the treatment of steroid-refractory acute and chronic graft versus host disease (GvHD). It is predominantly metabolized via cytochrome P450 (CYP) 3A4. As patients with GvHD have an increased risk of invasive fungal infections, RUX is frequently combined with posaconazole (POS), a strong CYP3A4 inhibitor. Knowledge of RUX exposure under concomitant POS treatment is scarce and recommendations on dose modifications are inconsistent. A physiologically based pharmacokinetic (PBPK) model was developed to investigate the drug–drug interaction (DDI) between POS and RUX. The predicted RUX exposure was compared to observed concentrations in patients with GvHD in the clinical routine. PBPK models for RUX and POS were independently set up using PK-Sim\(^®\) Version 11. Plasma concentration-time profiles were described successfully and all predicted area under the curve (AUC) values were within 2-fold of the observed values. The increase in RUX exposure was predicted with a DDI ratio of 1.21 (C\(_{max}\)) and 1.59 (AUC). Standard dosing in patients with GvHD led to higher RUX exposure than expected, suggesting further dose reduction if combined with POS. The developed model can serve as a starting point for further simulations of the implemented DDI and can be extended to further perpetrators of CYP-mediated PK-DDIs or disease-specific physiological changes.
Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch
(2023)
The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site‐specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.
Aims: The aim of the current study was to establish a simple and yet as much as possible physiologic approach for a simulation of the pulmonary absorption process to compare different inhaled drugs or drug formulations.
Methodology: We designed a dialysis setting that allowed monitoring the drug release from human lung tissue into a continuous-flow plasma compartment. For proof-of-concept experiments we chose the glucocorticoid fluticasone propionate (FP) as model compound. For subsequent experiments we selected a commercially available metered dose inhaler delivering a fixed combination of the short-acting ß2-agonist fenoterol and the muscarinic antagonist ipratropium bromide.
Results: With the novel dynamic dialysis model we observed high drug transport rates from the lung tissue into plasma including an elimination phase. The concentration profile in the plasma compartment of our model system was similar to the plasma concentration courses after inhalation of FP. Compared to FP significantly higher drug fractions of fenoterol and ipratropium bromide were released into plasma and the transfer of ipratropium was more pronounced compared to fenoterol. Again, concentration profiles in plasma were alike to those described in clinical studies.
Conclusion: We suggest that this model is appropriate for rapid assessment of comparative diffusion behaviour of drugs or drug formulations from lung tissue into plasma.
In 1998, the aminoglycoside antibiotic gentamicin sulfate caused several cases of deaths in the United States, after the switch from twice- to once-daily application. Endotoxins were discussed as the cause for the adverse effects and sisomicin was identified as the lead impurity; batches containing sisomicin were contaminated with more impurities and were responsible for the fatalities. In 2016, anaphylactic reactions in horses, and later in humans with one fatality, were observed after application of gentamicin sulfate contaminated with histamine. To determine whether histamine was responsible for the 1990s death cases as well, histamine was quantified by means of liquid chromatography–tandem mass spectrometry (LC-MS/MS) in 30 samples of gentamicin sulfate analyzed in previous studies. Furthermore, a relative quantification of sisomicin was performed to check for a correlation between histamine and the lead impurity. A maximum amount of 11.52 ppm histamine was detected, which is below the limit for anaphylactic reactions of 16 ppm, and no correlation of the two impurities was observed. However, the European Medicines Agency recommends a stricter limit with regard to the maximum single dose of gentamicin sulfate to reach a greater gap between the maximum histamine exposition of 4.3 µg and the quantity known to cause hypotension of 7 µg. The low amounts of histamine and the fact that there is no connection with the contamination with sisomicin showed that histamine was not the cause for the death cases in the United States in 1998, and endotoxins remain the most probable explanation.
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.
Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1–30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy.
The chloroform extract of Valeriana wallichii (V. wallichii) rhizomes was investigated to elucidate the structures responsible for reported antileishmanial activity. Besides bornyl caffeate (1, already been reported by us previously), bioassay-guided fractionation resulted in two additional cinnamic acid derivatives 2–3 with moderate leishmanicidal activity. The structure of a novel nepetolactone derivative 4 having a cinnamic acid moiety was elucidated by means of spectral analysis. To the best of our knowledge villoside aglycone (5) was isolated from this plant for the first time. The bioassay-guided fractionation yielded two new (compounds 6–7) and two known valtrates (compounds 8–9) with leishmanicidal potential against Leishmania major (L. major) promastigotes. In addition, β-bisabolol (10), α-kessyl alcohol (11), valeranone (12), bornyl isovalerate (13) and linarin-2-O-methylbutyrate (14) were identified. This is the first report on the isolation of 4'-demethylpodophyllotoxin (15), podophyllotoxin (16) and pinoresinol (17) in V. wallichii. In total thirteen known and four new compounds were identified from the extract and their cytotoxic and antileishmanial properties were evaluated.
Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization
Azobenzene derivatives with activity against drug‐resistant Candida albicans and Candida auris
(2023)
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.
Many (poly‐)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid‐β 42 (Aβ42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aβ42, which adopts different folds, affecting the propensity to populate fibril‐like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aβ42 aggregation inhibition, glutamate‐induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV‐2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 μm (83 % cell survival), whereas curcumin only showed very low protection at 10 μm (21 % cell survival).
Bile solubilization plays a major role in the absorption of poorly water-soluble drugs. Excipients used in oral drug formulations impact bile-colloidal properties and their molecular interactions. Polymer-induced changes of bile colloids, e.g., by Eudragit E, reduced the flux of the bile interacting drug Perphenazine whereas bile non-interacting Metoprolol was not impacted. This study corroborates these in vitro findings in rats. Eudragit E significantly reduced systemic availability of Perphenazine but not Metoprolol compared to the oral administrations without polymer. This study confirms the necessity to carefully select polymers for bile interacting drugs whereas non-bile interacting drugs are more robust in terms of excipient choice for formulation. The perspective of bile interaction may introduce interesting biopharmaceutical leverage for better performing oral formulations of tomorrow.
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.
Bone concentration of ampicillin/sulbactam: a pilot study in patients with osteonecrosis of the jaw
(2022)
Osteonecrosis of the jaw (ONJ) occurs typically after irradiation of the head and neck area or after the intake of antiresorptive agents. Both interventions can lead to compromised bone perfusion and can ultimately result in infection and necrosis. Treatment usually consists of surgical necrosectomy and prolonged antibiotic therapy, usually through beta-lactams such as ampicillin/sulbactam. The poor blood supply in particular raises the question as to whether this form of antibiosis can achieve sufficient concentrations in the bone. Therefore, we investigated the antibiotic concentration in plasma and bone samples in a prospective study. Bone samples were collected from the necrosis core and in the vital surrounding bone. The measured concentrations in plasma for ampicillin and sulbactam were 126.3 ± 77.6 and 60.2 ± 35.0 µg/mL, respectively. In vital bone and necrotic bone samples, the ampicillin/sulbactam concentrations were 6.3 ± 7.8/1.8 ± 2.0 µg/g and 4.9 ± 7.0/1.7 ± 1.7 µg/g, respectively. These concentrations are substantially lower than described in the literature. However, the concentration seems sufficient to kill most bacteria, such as Streptococci and Staphylococci, which are mostly present in the biofilm of ONJ. We, therefore, conclude that intravenous administration of ampicillin/sulbactam remains a valuable treatment in the therapy of ONJ. Nevertheless, increasing resistance of Escherichia coli towards beta-lactam antibiotics have been reported and should be considered.
Background:
The standardized maritime pine bark extract (Pycnogenol\(^{®}\)) has previously shown symptom alleviating effects in patients suffering from moderate forms of knee osteoarthritis (OA). The cellular mechanisms for this positive impact are so far unknown. The purpose of the present randomized pilot controlled study was to span the knowledge gap between the reported clinical effects of Pycnogenol\(^{®}\) and its in vivo mechanism of action in OA patients.
Methods:
Thirty three patients with severe OA scheduled for a knee arthroplasty either received 100 mg of Pycnogenol\(^{®}\) twice daily or no treatment (control group) three weeks before surgery. Cartilage, synovial fluid and serum samples were collected during surgical intervention. Relative gene expression of cartilage homeostasis markers were analyzed in the patients' chondrocytes. Inflammatory and cartilage metabolism mediators were investigated in serum and synovial fluid samples.
Results:
The oral intake of Pycnogenol\(^{®}\) downregulated the gene expression of various cartilage degradation markers in the patients' chondrocytes, the decrease of MMP3, MMP13 and the pro-inflammatory cytokine IL1B were statistically significant (p ≤ 0.05). Additionally, protein concentrations of ADAMTS-5 in serum were reduced significantly (p ≤ 0.05) after three weeks intake of the pine bark extract.
Conclusions:
This is the first report about positive cellular effects of a dietary supplement on key catabolic and inflammatory markers in patients with severe OA. The results provide a rational basis for understanding previously reported clinical effects of Pycnogenol\(^{®}\) on symptom scores of patients suffering from OA.
Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein
(2023)
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.
The charged aerosol detector (CAD) is the latest representative of aerosol-based detectors that generate a response independent of the analytes' chemical structure. This study was aimed at accurately predicting the CAD response of homologous fatty acids under varying experimental conditions. Fatty acids from C12 to C18 were used as model substances due to semivolatile characterics that caused non-uniform CAD behaviour. Considering both experimental conditions and molecular descriptors, a mixed quantitative structure-property relationship (QSPR) modeling was performed using Gradient Boosted Trees (GBT). The ensemble of 10 decisions trees (learning rate set at 0.55, the maximal depth set at 5, and the sample rate set at 1.0) was able to explain approximately 99% (Q\(^2\): 0.987, RMSE: 0.051) of the observed variance in CAD responses. Validation using an external test compound confirmed the high predictive ability of the model established (R-2: 0.990, RMSEP: 0.050). With respect to the intrinsic attribute selection strategy, GBT used almost all independent variables during model building. Finally, it attributed the highest importance to the power function value, the flow rate of the mobile phase, evaporation temperature, the content of the organic solvent in the mobile phase and the molecular descriptors such as molecular weight (MW), Radial Distribution Function-080/weighted by mass (RDF080m) and average coefficient of the last eigenvector from distance/detour matrix (Ve2_D/Dt). The identification of the factors most relevant to the CAD responsiveness has contributed to a better understanding of the underlying mechanisms of signal generation. An increased CAD response that was obtained for acetone as organic modifier demonstrated its potential to replace the more expensive and environmentally harmful acetonitrile.
Quinolone antibiotics present an attractive oral treatment option in patients with cystic fibrosis (CF). Prior studies have reported comparable clearances and volumes of distribution in patients with CF and healthy volunteers for primarily renally cleared quinolones. We aimed to provide the first pharmacokinetic comparison for pefloxacin as a predominantly nonrenally cleared quinolone and its two metabolites between both subject groups. Eight patients with CF (fat-free mass [FFM]: 36.3 ± 6.9 kg, average ± SD) and ten healthy volunteers (FFM: 51.7 ± 9.9 kg) received 400 mg pefloxacin as a 30 min intravenous infusion and orally in a randomized, two-way crossover study. All plasma and urine data were simultaneously modelled. Bioavailability was complete in both subject groups. Pefloxacin excretion into urine was approximately 74% higher in patients with CF compared to that in healthy volunteers, whereas the urinary excretion of metabolites was only slightly higher in patients with CF. After accounting for body size and composition via allometric scaling by FFM, pharmacokinetic parameter estimates in patients with CF divided by those in healthy volunteers were 0.912 for total clearance, 0.861 for nonrenal clearance, 1.53 for renal clearance, and 0.916 for volume of distribution. Nonrenal clearance accounted for approximately 90% of total pefloxacin clearance. Overall, bioavailability and disposition were comparable between both subject groups.
Extraction is a key step in studying compounds from plants and other natural sources. The common use of high temperatures in pressurized microwave‐assisted extraction (PMAE) makes it unsuitable for the extraction of compounds with low or unknown thermal stability. This study aimed at determining the suitability of low‐temperature, short‐time PMAE in attaining yields comparable to those of prolonged maceration at room temperature. Additionally, we explored the phytochemical differences of the extracts from both techniques. Maceration at room temperature for 24 hr and PMAE at 40–45°C and 10 bar for 30 min were carried out on 18 samples from 14 plant species at a solvent‐to‐feeds ratio of 10. The PMAE yields of 16 out of 18 samples were within the proportions of 91–139.2% as compared with the respective extracts from maceration. Varying numbers of nonmatching peaks were noted in MS chromatograms of five extract pairs, indicating selective extraction of some compounds. Low‐temperature PMAE can attain reasonable extraction efficiency with the added value of sparing compounds of low thermal stability. The method can also enable the recovery of compounds distinct from those obtained by maceration.
Ellagitannins are signature constituents of oak wood and their consumption has been associated with various health benefits. In vivo, they undergo metabolic degradation including gut microbial metabolism yielding urolithins. Only limited data is available about compounds being present in blood after intake of an extract from French oak wood, Robuvit®. In the course of a randomized, double-blind, controlled clinical investigation, 66 patients undergoing hysterectomy received placebo or 300 mg Robuvit® per day before and over 8 weeks after surgery. Serum and blood cell samples were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The number of urolithin producers and the urolithin levels increased after intake of Robuvit®. In serum samples, the median concentration of urolithin A was 14.0 ng/ml [interquartile range (IQR) 57.4] after 8 weeks. Urolithin B was determined at 22.3 ng/ml (IQR 12.6), urolithin C at 2.66 ng/ml (IQR 2.08). In blood cells, lower concentrations and only urolithins A and B were detected. A statistically significant association of lower post-surgical pain scores with metabotype A was detected (p < 0.05). To conclude, supplementation with French oak wood extract raised urolithin generation in patients and suggested health advantages for urolithin-producers.
Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space.
The title compound, C\(_{12}\)H\(_{9}\)N\(_{3}\)O, is an intermediate in the synthesis of the muscarinic M2 receptor antagonist AFDX-384. The seven-membered ring adopts a boat conformation and the dihedral angle between the planes of the aromatic rings is 41.51 (9)°. In the crystal, molecules are linked into [001] chains of alternating inversion dimers formed by pairs of N-H・・・O hydrogen bonds and pairs of N-H・・・N hydrogen bonds. In both cases, R\(_{2}\)\(^{2}\)(8) loops are generated.
Formation of the Aurora-A–MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A–MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A–MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.
Delayed and limited administration of the JAKinib tofacitinib mitigates chronic DSS-induced colitis
(2023)
In inflammatory bowel disease, dysregulated T cells express pro-inflammatory cytokines. Using a chronic azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis model resembling ulcerative colitis, we evaluated whether and when treatment with the Janus kinase (JAK) inhibitor tofacitinib could be curative. Comparing the treatment with two and three cycles of tofacitinib medication in drinking water – intermittently with DSS induction – revealed that two cycles were not only sufficient but also superior over the 3-x regimen. The two cycles of the 2-x protocol paralleled the second and third cycles of the longer protocol. T cells were less able to express interferon gamma (IFN-γ) and the serum levels of IFN-γ, interleukin (IL)-2, IL-6, IL-17, and tumor necrosis factor (TNF) were significantly reduced in sera, while those of IL-10 and IL-22 increased under the 2-x protocol. Likewise, the frequency and effector phenotype of regulatory T cells (Tregs) increased. This was accompanied by normal weight gain, controlled clinical scores, and restored stool consistency. The general and histologic appearance of the colons revealed healing and tissue intactness. Importantly, two phases of tofacitinib medication completely prevented AOM-incited pseudopolyps and the hyper-proliferation of epithelia, which was in contrast to the 3-x regimen. This implies that the initial IBD-induced cytokine expression is not necessarily harmful as long as inflammatory signaling can later be suppressed and that time-restricted treatment allows for anti-inflammatory and tissue-healing cytokine activities.
The cholinergic hypothesis has been reported first being the cause of memory dysfunction in the Alzheimer’s disease. Researchers around the globe have focused their attention on understanding the mechanisms of how this complicated system contributes to processes such as learning, memory, disorientation, linguistic problems, and behavioral issues in the indicated chronic neurodegenerative disease. The present review reports recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer’s disease (AD) and the study of their therapeutic relevance. The rationale and the design of the bifunctional compounds will be discussed in order to understand their potential as tools to investigate the role of the cholinergic system in AD.
Exposure-efficacy and/or exposure-toxicity relationships have been identified for up to 80% of oral anticancer drugs (OADs). Usually, OADs are administered at fixed doses despite their high interindividual pharmacokinetic variability resulting in large differences in drug exposure. Consequently, a substantial proportion of patients receive a suboptimal dose. Therapeutic Drug Monitoring (TDM), i.e., dosing based on measured drug concentrations, may be used to improve treatment outcomes. The prospective, multicenter, non-interventional ON-TARGET study (DRKS00025325) aims to investigate the potential of routine TDM to reduce adverse drug reactions in renal cell carcinoma patients receiving axitinib or cabozantinib. Furthermore, the feasibility of using volumetric absorptive microsampling (VAMS), a minimally invasive and easy to handle blood sampling technique, for sample collection is examined. During routine visits, blood samples are collected and sent to bioanalytical laboratories. Venous and VAMS blood samples are collected in the first study phase to facilitate home-based capillary blood sampling in the second study phase. Within one week, the drug plasma concentrations are measured, interpreted, and reported back to the physician. Patients report their drug intake and toxicity using PRO-CTCAE-based questionnaires in dedicated diaries. Ultimately, the ON-TARGET study aims to develop a nationwide infrastructure for TDM for oral anticancer drugs.
A variety of in vitro dissolution and gastrointestinal transfer models have been developed aiming to predict drug supersaturation and precipitation. Further, biphasic, one-vessel in vitro systems are increasingly applied to simulate drug absorption in vitro. However, to date, there is a lack of combining the two approaches. Therefore, the first aim of this study was to develop a dissolution-transfer-partitioning system (DTPS) and, secondly, to assess its biopredictive power. In the DTPS, simulated gastric and intestinal dissolution vessels are connected via a peristaltic pump. An organic layer is added on top of the intestinal phase, serving as an absorptive compartment. The predictive power of the novel DTPS was assessed to a classical USP II transfer model using a BCS class II weak base with poor aqueous solubility, MSC-A. The classical USP II transfer model overestimated simulated intestinal drug precipitation, especially at higher doses. By applying the DTPS, a clearly improved estimation of drug supersaturation and precipitation and an accurate prediction of the in vivo dose linearity of MSC-A were observed. The DTPS provides a useful tool taking both dissolution and absorption into account. This advanced in vitro tool offers the advantage of streamlining the development process of challenging compounds.
Opioid receptors (ORs) are classified among the oldest and best investigated drug targets due to their fundamental role in the treatment of pain and related disorders. ORs are divided in three conventional subtypes (μ, κ, δ) and the non‐classical nocicepetin receptor. All ORs are family A G protein‐coupled receptors (GPCRs), and are located on the cell surface. Modern biophysical methods use light to investigate physiological processes at organismal, cellular and subcellular level. Many of these methods rely on fluorescent ligands, thus highlighting their importance. This review addresses the advancements in the development of opioid fluorescent ligands and their use in biological, pharmacological and imaging applications.
A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib,cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for theapplication in daily clinical routine has been developed and validated according to the US Food and Drug Administration andEuropean Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples withacetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5μm(2.1×50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A andmethanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stableisotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min perrun. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2–500 ng/mLfor afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6–1500 ng/mL for cabozantinib, dabrafenib, nilotinib,and osimertinib (coefficients of correlation≥0.99). Validation assays for accuracy and precision, matrix effect, recovery,carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughputand was successfully applied to monitor concentrations of kinase inhibitors in patients.
Our ability of screening broad communities for clinically asymptomatic diseases critically drives population health. Sensory chewing gums are presented targeting the tongue as 24/7 detector allowing diagnosis by “anyone, anywhere, anytime”. The chewing gum contains peptide sensors consisting of a protease cleavable linker in between a bitter substance and a microparticle. Matrix metalloproteinases in the oral cavity, as upregulated in peri-implant disease, specifically target the protease cleavable linker while chewing the gum, thereby generating bitterness for detection by the tongue. The peptide sensors prove significant success in discriminating saliva collected from patients with peri-implant disease versus clinically asymptomatic volunteers. Superior outcome is demonstrated over commercially available protease-based tests in saliva. “Anyone, anywhere, anytime” diagnostics are within reach for oral inflammation. Expanding this platform technology to other diseases in the future features this diagnostic as a massive screening tool potentially maximizing impact on population health.
The present randomized controlled study aimed to investigate the in vivo distribution of constituents or metabolites of the standardized maritime pine bark extract Pycnogenol\(^{®}\). Thirty-three patients with severe osteoarthritis scheduled for a knee arthroplasty were randomized to receive either 200 mg per day Pycnogenol\(^{®}\) (P+) or no treatment (Co) over three weeks before surgery. Serum, blood cells, and synovial fluid samples were analyzed using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (LC-ESI/MS/MS). Considerable interindividual differences were observed indicating pronounced variability of the polyphenol pharmacokinetics. Notably, the highest polyphenol concentrations were not detected in serum. Catechin and taxifolin primarily resided within the blood cells while the microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone, ferulic, and caffeic acid were mainly present in synovial fluid samples. Taxifolin was detected in serum and synovial fluid exclusively in the P+ group. Likewise, no ferulic acid was found in serum samples of the Co group. Calculating ratios of analyte distribution in individual patients revealed a simultaneous presence of some polyphenols in serum, blood cells, and/or synovial fluid only in the P+ group. This is the first evidence that polyphenols distribute into the synovial fluid of patients with osteoarthritis which supports rationalizing the results of clinical efficacy studies.
In DNA-encoded library synthesis, amine-substituted building blocks are prevalent. We explored isocyanide multicomponent reactions to diversify DNA-tagged amines and reported the Ugi-azide reaction with high yields and a good substrate scope. In addition, the Ugi-aza-Wittig reaction and the Ugi-4-center-3-component reaction, which used bifunctional carboxylic acids to provide lactams, were explored. Five-, six-, and seven-membered lactams were synthesized from solid support-coupled DNA-tagged amines and bifunctional building blocks, providing access to structurally diverse scaffolds.
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.
Objectives
Mechanisms of wound healing are often impaired in patients with osteonecrosis of the jaw (ONJ). According to the guidelines for the treatment of this disease, early surgical intervention is indicated. However, surgery often faces complications such as wound healing disorders. The application of platelet-rich fibrin (PRF) after necrosectomy between bone and mucosa may constitute a promising approach to improve surgical results. An aspect that was not investigated until now is that PRF acts as a “bio-carrier” for antibiotics previously applied intravenously.
Materials and methods
We investigated the antimicrobial properties of PRF in 24 patients presenting ONJ undergoing systemic antibiosis with ampicillin/sulbactam. We measured the concentration of ampicillin/sulbactam in plasma and PRF and performed agar diffusion tests. Ampicillin/sulbactam was applied intravenously to the patient 10 minutes for blood sampling for PRF. No further incorporation of patients’ blood or PRF product with antibiotic drugs was obtained. Four healthy patients served as controls.
Results
Our results revealed that PRF is highly enriched with ampicillin/sulbactam that is released to the environment. The antibiotic concentration in PRF was comparable to the plasma concentration of ampicillin/sulbactam. The inhibition zone (IZ) of PRF was comparable to the standard ampicillin/sulbactam discs used in sensitivity testing.
Conclusions
The results of our study demonstrated that PRF is a reliable bio-carrier for systemic applied antibiotics and exhibits a large antimicrobial effect.
Clinical relevance
We describe a clinically useful feature of PRF as a bio-carrier for antibiotics. Especially when applied to poorly perfused tissues and bone such as in ONJ, the local release of antibiotics can reduce wound healing disorders like infections.
Background: Cyclic aminals are core features of natural products, drug molecules and important synthetic intermediates. Despite their relevance, systematic investigations into their stability towards hydrolysis depending on the pH value are lacking.
Results: A set of cyclic aminals was synthesized and their stability quantified by kinetic measurements. Steric and electronic effects were investigated by choosing appropriate groups. Both molecular mechanics (MM) and density functional theory (DFT) based studies were applied to support and explain the results obtained. Rapid decomposition is observed in acidic aqueous media for all cyclic aminals which occurs as a reversible reaction. Electronic effects do not seem relevant with regard to stability, but the magnitude of the conformational energy of the ring system and pK a values of the N-3 nitrogen atom.
Conclusion: Cyclic aminals are stable compounds when not exposed to acidic media and their stability is mainly dependent on the conformational energy of the ring system. Therefore, for the preparation and work-up of these valuable synthetic intermediates and natural products, appropriate conditions have to be chosen and for application as drug molecules their sensitivity towards hydrolysis has to be taken into account.
Many plant secondary metabolites exhibit some degree of biological activity in humans. It is a common observation that individual plant-derived compounds in vivo are present in the nanomolar concentration range at which they usually fail to display measurable activity in vitro. While it is debatable that compounds detected in plasma are not the key effectors of bioactivity, an alternative hypothesis may take into consideration that measurable concentrations also reside in compartments other than plasma. We analysed the binding of constituents and the metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1), that had been previously detected in plasma samples of human consumers of pine bark extract Pycnogenol, to human erythrocytes. We found that caffeic acid, taxifolin, and ferulic acid passively bind to red blood cells, but only the bioactive metabolite M1 revealed pronounced accumulation. The partitioning of M1 into erythrocytes was significantly diminished at higher concentrations of M1 and in the presence of glucose, suggesting a facilitated transport of M1 via GLUT-1 transporter. This concept was further supported by structural similarities between the natural substrate α-D-glucose and the S-isomer of M1. After cellular uptake, M1 underwent further metabolism by conjugation with glutathione. We present strong indication for a transporter-mediated accumulation of a flavonoid metabolite in human erythrocytes and subsequent formation of a novel glutathione adduct. The physiologic role of the adduct remains to be elucidated.
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.
Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.
Substandard and falsified (SF) medicines have emerged as a global public health issue within the last two decades especially in low- and middle-income countries (LMICs). Serious consequences of this problem include a loss of trust and increased financial costs due to less disease control and more frequent complications during therapy. Of note, antimicrobial resistance is an additional long-term implication of poor-quality antimicrobials. This review covers information technology tools including medicines authentication tools (MAT) as mobile apps and messaging service, 2D barcoding approaches with drug safety alert systems, web based drug safety alerts, radiofrequency identification tags, databases to support visual inspection, digital aids to enhance the performance of quality evaluation kits, reference libraries for identification of falsified and substandard medicines, and quality evaluation kits based on machine learning for field testing. While being easy to access and simple to use, these initiatives are gaining acceptance in LMICs. Implementing 2D barcoding based on end-to-end verification and "Track and Trace" systems has emerged as a step toward global security in the supply chain. A breakthrough in web-based drug safety alert systems and data bases was the establishment of the Global Surveillance and Monitoring System by the World Health Organization in 2013. Future applications include concepts including "lab on a chip" and "paper analytical devices" and are claimed to be convenient and simple to use as well as affordable. The principles discussed herein are making profound impact in the fight against substandard and falsified medicines, offering cheap and accessible solutions.
Baclofen (1) is a potent and selective agonist for bicuculline-insensitive GABAB receptors and is used clinically as an antispastic and muscle relaxant agent. In the search for new bioactive chemical entities that bind specifically to GABAB receptors, we report here the synthesis of certain baclofen homologues, namely (R,S)-5-amino-3-arylpentanoic acid hydrochlorides (R,S)-1a–h as well as (R,S)-5-amino-3-methylpentanoic acid [(RS)-1i] to be evaluated as GABABR agonists. Compound 1a is an agonist to GABAB receptors with an EC50 value of 46 μM on tsA201 cells transfected with GABAB1b/GABAB2/Gqz5, being the most active congener among all the synthesized compounds.
Sacha inchi oil is a premier raw material with highly nutritional and functional features for the foodstuff, pharmaceutical, beauty, and personal care industries. One of the most important facts about this oil is the huge chemical content of unsaturated and polyunsaturated fatty acids. However, the current available information on the characterization of the triglyceride composition and the advance physicochemical parameters relevant to emulsion development is limited. Therefore, this research focused on providing a detailed description of the lipid composition using high-resolution tandem mass spectrometry and thorough physicochemical characterization to find the value of the required hydrophilic–lipophilic balance (HLB). For this, a study in the interfacial tension was evaluated, followed by the assessment of different parameters such as creaming index, droplet size, viscosity, zeta potential, pH, and electrical conductivity for a series emulsified at thermal stress condition. The results show that fatty acids are arranged into glycerolipids and the required HLB to achieve the maximum physical stability is around 8.
Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.
H/D exchange reactions can be observed by NMR spectroscopy of acebutolol (ACE). The results obtained showed deuterium incorporation at α-posi t ion of the carbonyl group of acebutolol, when using deuterium oxide or deuterated methanol as deuterium source and solvent. The spontaneous deuteration is proceeded by the following pathway CH\(_{3}\)→CH\(_{2}\)D→CHD→CD\(_{2}\), through a keto-enol tautomerization reaction. Furthermore, LC-MS / QTOF analyses have confirmed the proposed H/D exchange. In order to reduce the time of total deuteration observed at the acetyl group alkaline catalysts were employed.
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.
Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17β-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by questionnaire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tissue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible.
Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).
Investigation of isomerization of dexibuprofen in a ball mill using chiral capillary electrophoresis
(2021)
Besides the racemate, the S‐enantiomer of ibuprofen (Ibu) is used for the treatment of inflammation and pain. Since the configurational stability of S‐Ibu in solid state is of interest, it was studied by means of ball milling experiments. For the evaluation of the enantiomeric composition, a chiral CE method was developed and validated according to the ICH guideline Q2(R1). The addition of Mg\(^{2+}\), Ca\(^{2+}\), or Zn\(^{2+}\) ions to the background electrolyte (BGE) was found to improve Ibu enantioresolution. Chiral separation of Ibu enantiomers was achieved on a 60.2 cm (50.0 cm effective length) x 75 μm fused‐silica capillary using a background electrolyte (BGE) composed of 50 mM sodium acetate, 10 mM magnesium acetate tetrahydrate, and 35 mM heptakis‐(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin (TM‐β‐CD) as chiral selector. The quantification of R‐Ibu in the mixture was performed using the normalization procedure. Linearity was evaluated in the range of 0.68–5.49% R‐Ibu (R\(^{2}\) = 0.999), recovery was found to range between 97 and 103%, the RSD of intra‐ and interday precision below 2.5%, and the limit of quantification for R‐ in S‐Ibu was calculated to be 0.21% (extrapolated) and 0.15% (dilution of racemic ibuprofen), respectively. Isomerization of S‐Ibu was observed under basic conditions by applying long milling times and high milling frequencies.
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.
It began with citrus
(2013)