SAR Studies on Curcumin's Pro-inflammatory Targets: Discovery of Prenylated Pyrazolocurcuminoids as Potent and Selective Novel Inhibitors of 5-Lipoxygenase.

The anticarcinogenic and anti-inflammatory properties of curcumin have been extensively investigated, identifying prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), key enzymes linking inflammation with cancer, as high affinity targets. A comparative structure-activity study revealed three modifications dissecting mPGES-1/5-LO inhibition, namely (i) truncation of the acidic, enolized dicarbonyl moiety and/or replacement by pyrazole, (ii) hydrogenation of the interaryl linker, and (iii) (dihydro)prenylation. The prenylated pyrazole analogue 11 selectively inhibited 5-LO, outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects underlie the inhibition of mPGES-1 and 5-LO by curcumin

The novel benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5-lipoxygenase-activating protein (FLAP).

BACKGROUND AND PURPOSE: Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH: We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTS: BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONS: BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development

BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC50=7-10nM), and upon activation by ionophore A23187 (IC50=10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E2 synthase-1 (IC50=0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation

Implementierung einer neuen Separationstechnolgie in die pharmazeutische Herstellung von Erythrozytenkonzentraten aus Vollblut

Erythrozytenkonzentrate (EK) sind Blutzubereitungen aus humane Spender-Erythrozyten, die in einer Additivlösung zur Erhaltung der Zellfunktionen resuspendiert sind. Für das Inverkehrbringen von Blutprodukten zur Anwendung am Menschen bedarf der pharmazeutische Unternehmer in Deutschland einer Zulassung durch das Paul-Ehrlich-Institut. Zur Herstellung von EK stehen verschiedene Methoden zur Verfügung. Eines der fortschrittlichsten ist das automatisierte Vollblutaufbereitungssystem Atreus. Es vereint die individuellen Teilschritte der Zentrifugation und Separation in einem Gerät. Es galt zu klären, ob die mit Atreus gewonnenen EK den Zulassungsprozess bestehen. Die wesentlichen Abweichungen zur konventionellen Vollblutverarbeitung, besonders die erhöhte Zentrifugationstemperatur und deren Einfluss auf die Qualität der Endprodukte, waren zu untersuchen und zu bewerten. Zudem sollte das System in die Routineherstellung zur Patientenversorgung am Institut für Transfusionsmedizin des Universitätsklinikum Jena integriert werden. Insgesamt wurden 49 EK in SAGM-Additivlösung in vier Validierungsläufen am Tag der Herstellung und danach wöchentlich über einen Lagerungszeitraum von 42 Tagen geprüft. Die Parameter Hämolyserate, Konzentration extrazelluärer Kaliumionen und der Anteil Adenosintriphosphat pro Gramm Hämoglobin dienten zur Beurteilung der Produktqualität. Die Validierungsszenarien umfassten eine 2-stündige Vollblutlagerung vor Atreus-Verarbeitung mit Zentrifugationstemperaturen zwischen 24 und 28 °C, eine 15-stündige Vollblutlagerung vor Verarbeitung sowie zwei Validierungsläufe bei erhöhten Vollblut-Zentrifugationstemperaturen von bis zu 34 °C. Die Datenlage zeigte, dass alle EK den aufgestellten Spezifikationen entsprachen. Dennoch ergab die statistische Auswertung signifikante Unterschiede einiger Parameter der EK der Validierungsläufe. Die Zulassung für das Arzneimittels Erythrozytenkonzentrat SAGM Th-J wurde erlangt und das Verfahren in die Routine eingeführt

Malgré nous / Léon Weinigel

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Rigid and high-numerical-aperture two-photon fluorescence endoscope

We present a rigid miniaturized optical system block fiber-optic two-photon endoscope based on a compact two-axis piezo scanner system and a miniature high (0.65) NA GRIN lens objective. The optical system is scanned as a whole by a piezo scanner allowing always an on-axis beam irradiation of the optical system. A photonic crystal fiber is used for excitation and ultrashort laser pulses can be delivered with typical power up to 100 mW at 800 run. Two-photon fluorescence signal is collected by the use of a multimode fiber. Lateral resolution values for the system were experimentally measured to be 0.67 mu m vertically and 1.08 mu m horizontally. Axial resolution was found to be 5.8 mu m. The endoscope is highly flexible and controllable in terms of time acquisition, resolution, and magnification. Fluorescence images were acquired over a 420 mu m x 420 mu m field of view. Results presented here demonstrate the ability of the system to resolve subcellular details and the potential of the technology for in vivo applications

Humudifucol and Bioactive Prenylated Polyphenols from Hops (Humulus lupulus cv. “Cascade”)

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the antiinflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E-2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low mu M concentrations