Exploring Microemulsion Systems for the Incorporation of Glucocorticoids into Bacterial Cellulose: A Novel Approach for Anti-Inflammatory Wound Dressings.

The effective pharmacological treatment of inflamed wounds such as pyoderma gangraenosum remains challenging, as the systemic application of suitable drugs such as glucocorticoids is compromised by severe side effects and the inherent difficulties of wounds as drug targets. Furthermore, conventional semi-solid formulations are not suitable for direct application to open wounds. Thus, the treatment of inflamed wounds could considerably benefit from the development of active wound dressings for the topical administration of anti-inflammatory drugs. Although bacterial cellulose appears to be an ideal candidate for this purpose due to its known suitability for advanced wound care and as a drug delivery system, the incorporation of poorly water-soluble compounds into the hydrophilic material still poses a problem. The use of microemulsions could solve that open issue. The present study therefore explores their use as a novel approach to incorporate poorly water-soluble glucocorticoids into bacterial cellulose. Five microemulsion formulations were loaded with hydrocortisone or dexamethasone and characterized in detail, demonstrating their regular microstructure, biocompatibility and shelf-life stability. Bacterial cellulose was successfully loaded with the formulations as confirmed by transmission electron microscopy and surprisingly showed homogenous incorporation, even of w/o type microemulsions. High and controllable drug permeation through Strat-M® membranes was observed, and the anti-inflammatory activity for permeated glucocorticoids was confirmed in vitro. This study presents a novel approach for the development of anti-inflammatory wound dressings using bacterial cellulose in combination with microemulsions.

Glucocorticoids regulate lipid mediator networks by reciprocal modulation of 15-lipoxygenase isoforms affecting inflammation resolution.

Glucocorticoids (GC) are potent anti-inflammatory agents, broadly used to treat acute and chronic inflammatory diseases, e.g., critically ill COVID-19 patients or patients with chronic inflammatory bowel diseases. GC not only limit inflammation but also promote its resolution although the underlying mechanisms are obscure. Here, we reveal reciprocal regulation of 15-lipoxygenase (LOX) isoform expression in human monocyte/macrophage lineages by GC with respective consequences for the biosynthesis of specialized proresolving mediators (SPM) and their 15-LOX-derived monohydroxylated precursors (mono-15-OH). Dexamethasone robustly up-regulated pre-mRNA, mRNA, and protein levels of ALOX15B/15-LOX-2 in blood monocyte-derived macrophage (MDM) phenotypes, causing elevated SPM and mono-15-OH production in inflammatory cell types. In sharp contrast, dexamethasone blocked ALOX15/15-LOX-1 expression and impaired SPM formation in proresolving M2-MDM. These dexamethasone actions were mimicked by prednisolone and hydrocortisone but not by progesterone, and they were counteracted by the GC receptor (GR) antagonist RU486. Chromatin immunoprecipitation (ChIP) assays revealed robust GR recruitment to a putative enhancer region within intron 3 of the ALOX15B gene but not to the transcription start site. Knockdown of 15-LOX-2 in M1-MDM abolished GC-induced SPM formation and mono-15-OH production. Finally, ALOX15B/15-LOX-2 upregulation was evident in human monocytes from patients with GC-treated COVID-19 or patients with IBD. Our findings may explain the proresolving GC actions and offer opportunities for optimizing GC pharmacotherapy and proresolving mediator production.

Structure-Activity Relationships of Flupirtine Analogues for Liver Esterase-Mediated Cleavage of the 4-Fluorobenzylamine Moiety and Its Possible Relevance to Liver Toxicity.

Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv 7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the in vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

Phytochemical profile and anti-inflammatory activity of a commercially available Rhodiola rosea root extract.

Rhodiola rosea roots and rhizomes hold an important place in the folk medicines of Russia, Scandinavia, Mongolia, and China as a health supplement for stimulating the nervous system, enhancing physical and mental performances, and nowadays they constitute the active ingredient in many popular commercial preparations sold worldwide as food additives, pharmaceutical remedies, and drinks. This study was aimed at providing a detailed phytochemical characterization of the Rhodiola 5%, a commercially available extract of R. rosea roots, and resulted in the characterization of 18 secondary metabolites, including 13 polyphenols and 6 terpenoids, and in the discovery of the new rhodiosidin (5), the first R. rosea metabolite to show both terpenoid and cinnamoyl moieties. The 5-lipoxygenase inhibiting activity of the main components was characterized and disclosed that rosiridin (6), kenposide A and rosavins are mainly responsible for this activity of the extract.

Modulation of Inflammation-Related Lipid Mediator Pathways by Celastrol During Human Macrophage Polarization.

Background and Purpose: Celastrol (CS) is a major active ingredient of the Chinese/Asian herb Tripterygium wilfordii that is frequently used as phytomedicine to treat inflammation and autoimmune diseases. We showed before that short-term exposure to CS (1 µM) favorably impacts the biosynthesis of inflammation-related lipid mediators (LM) in human polarized macrophages by modulating the activities of different lipoxygenases (LOXs). However, whether CS regulates the expression of LOXs and other related LM-biosynthetic enzymes during macrophage polarization is unknown. Here, we investigated how CS affects LM-biosynthetic enzyme expression on the protein level and studied concomitant LM signature profiles during polarization of human monocyte-derived macrophages (MDM) towards M1- and M2-like phenotypes. Methods and Results: We used LM metabololipidomics to study the long-term effects of CS on LM profile signatures after manipulation of human monocyte-derived macrophages (MDM) during polarization. Exposure of MDM to low concentrations of CS (ie, 0.2 µM) during polarization to an inflammatory M1 phenotype potently suppressed the formation of pro-inflammatory cyclooxygenase (COX)- and 5-LOX-derived LM, especially prostaglandin (PG)E2. Notably, gene and enzyme expression of COX-2 and microsomal PGE2 synthase (mPGES)-1 as well as M1 markers were strongly decreased by CS during M1-MDM polarization, along with impaired activation of nuclear factor-κB and p38 mitogen-activated protein kinase. During IL-4-induced M2 polarization, CS decreased the capacity of the resulting M2-MDM to generate pro-inflammatory COX and 5-LOX products as well but it also reduced the formation of 12/15-LOX products and specialized pro-resolving mediators, without affecting the levels of liberated fatty acid substrates. Conclusion: Depending on the timing and concentration, CS not only favorably affects LOX activities in macrophages but also the expression of LM-biosynthetic enzymes during macrophage polarization connected to changes of inflammation-related LM which might be of relevance for potential application of CS to treat inflammatory disorders.

Supercritical fluid extraction-supercritical fluid chromatography of saliva: Single-quadrupole mass spectrometry monitoring of caffeine for gastric emptying studies†.

Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2  = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

Beneficial Modulation of Lipid Mediator Biosynthesis in Innate Immune Cells by Antirheumatic Tripterygium wilfordii Glycosides.

Tripterygium wilfordii glycosides (TWG) is a traditional Chinese medicine with effectiveness against rheumatoid arthritis (RA), supported by numerous clinical trials. Lipid mediators (LM) are biomolecules produced from polyunsaturated fatty acids mainly by cyclooxygenases (COX) and lipoxygenases (LOX) in complex networks which regulate inflammation and immune responses and are strongly linked to RA. The mechanism by which TWG affects LM networks in RA treatment remains elusive. Employing LM metabololipidomics using ultra-performance liquid chromatography-tandem mass spectrometry revealed striking modulation of LM pathways by TWG in human monocyte-derived macrophage (MDM) phenotypes. In inflammatory M1-MDM, TWG (30 µg/mL) potently suppressed agonist-induced formation of 5-LOX products which was confirmed in human PMNL and traced back to direct inhibition of 5-LOX (IC50 = 2.9 µg/mL). TWG also efficiently blocked thromboxane formation in M1-MDM without inhibiting other prostanoids and COX enzymes. Importantly, in anti-inflammatory M2-MDM, TWG (30 µg/mL) induced pronounced formation of specialized pro-resolving mediators (SPM) and related 12/15-LOX-derived SPM precursors, without COX and 5-LOX activation. During MDM polarization, TWG (1 µg/mL) decreased the capacity to generate pro-inflammatory 5-LOX and COX products, cytokines and markers for M1 phenotypes. Together, suppression of pro-inflammatory LM but SPM induction may contribute to the antirheumatic properties of TWG.

Anti-inflammatory celastrol promotes a switch from leukotriene biosynthesis to formation of specialized pro-resolving lipid mediators.

The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases. However, the molecular mechanisms underlying the anti-inflammatory and inflammation-resolving features of CS are incompletely understood. Here we demonstrate that CS potently inhibits the activity of human 5-lipoxygenase (5-LOX), the key enzyme in pro-inflammatory leukotriene (LT) formation, in cell-free assays with IC50 = 0.19-0.49 µM. Employing metabololipidomics using ultra-performance liquid chromatography coupled to tandem mass spectrometry in activated human polymorphonuclear leukocytes or M1 macrophages we found that CS (1 µM) potently suppresses 5-LOX-derived products without impairing the formation of lipid mediators (LM) formed by 12-/15-LOXs as well as fatty acid substrate release. Intriguingly, CS induced the generation of 12-/15-LOX-derived LM including the specialized pro-resolving mediator (SPM) resolvin D5 in human M2 macrophages. Finally, intraperitoneal pre-treatment of mice with 10 mg/kg CS strongly impaired zymosan-induced LT formation and simultaneously elevated the levels of SPM and related 12-/15-LOX-derived LM in peritoneal exudates, spleen and plasma in vivo. Conclusively, CS promotes a switch from LT biosynthesis to formation of SPM which may underlie the anti-inflammatory and inflammation-resolving effects of CS, representing an interesting pharmacological strategy for intervention with inflammatory disorders.

Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: towards the design of photoswitchable sirtuin inhibitors.

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end-to-end distance. The resulting affinity shifts are intended to enable real-time photomodulation of sirtuins in vitro.

Simultaneous quantification of acidic and basic flupirtine metabolites by supercritical fluid chromatography according to European Medicines Agency validation.

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

Subcritical Fluid Chromatography at Sub-Ambient Temperatures for the Chiral Resolution of Ketamine Metabolites with Rapid-Onset Antidepressant Effects.

Chiral metabolites of ketamine exerting rapid-onset yet sustained antidepressant effects may be marketed directly in the future, but require chemo- and enantio-selective chromatographic methods for quality assurance and control. The chromatographic behavior of S-/R-ketamine, S-/R-norketamine, S-/R-dehydronorketamine, and (2R,6R)-/(2S,6S)-hydroxynorketamine in supercritical fluid chromatography (SFC) was investigated computationally and experimentally with the aim of identifying problematic pairs of enantiomers and parameters for chiral resolution. Retention on three different polysaccharide-based chiral stationary phases (Lux Amylose-2, i-Amylose-3, and i-Cellulose-5) provided new information on the significance of halogen atoms as halogen bond donors and hydrogen bond acceptors for enantioselectivity, which could be corroborated in silico by molecular docking studies. Modifiers inversely affected enantioselectivity and retention. Methanol yielded lower run times but superior chiral resolution compared to 2-propanol. Lower temperatures than those conventionally screened did not impair phase homogeneity but improved enantioresolution, at no cost to reproducibility. Thus, sub-ambient temperature subcritical fluid chromatography (SubFC), essentially low-temperature HPLC with subcritical CO2, was applied. The optimization of the SubFC method facilitated the chiral separation of ketamine and its metabolites, which was applied in combination with direct injection and online supercritical fluid extraction to determine the purity of pharmaceutical ketamine formulations for proof of concept.