Neue antientzündliche Zielstrukturen und Mechanismen von Boswelliasäuren und Celecoxib

Extracts of Boswellia serrata, also known as Indian frankincense, have been used to treat inflammatory diseases in the Indian ayurvedic medicine or Chinese traditional medicine (TCM) for over 3000 years, but the molecular mechanisms of the anti-inflammatory effects are still not well understood. It is obvious that the boswellic acids, the major compounds in the extracts, are responsible for the efficacy. This work employed a protein fishing technique to identify putative targets of boswellic acids at different stages within the inflammatory cascade. For fishing experiments, boswellic acids were immobilized to sepharose and incubated with cell lysates. After washing and boiling, fished proteins were separated by SDS-PAGE and analysed by MALDI-TOF-MS. CatG, DNA-PK and the protein kinase Akt were identified by protein pulldowns with immobilised BAs and characterised as selective and important targets for BAs with an IC50 in the range of physiologically achievable plasma levels up to 5 microM. In addition, the influence on several signal transductions by BAs was tested. Calcium influx, arachidonic acid release, platelet aggregation and TNFalpha-release were assayed to reveal further pharmacological effects of BAs. Celecoxib is a well-known selective COX-2 inhibitor that is in clinical use. In this work, it is demonstrated that celecoxib is also a highly potent direct 5-LO inhibitor. Celecoxib is used in arthritis and its gastro-intestinal side effects are reduced compared to non-selective NSAIDs. In patients with a familiar disposition to polyp forming, celecoxib reduced polyps and the incidence of colon cancer. Because of lowered leukotriene levels in patients under celecoxib therapy it was plausible to test whether celecoxib interferes with 5-LO. Here it is shown that the activity of 5-LO is inhibited in PMNL and cell-free assays with IC50 of 8 microM in intact cells, 20 microM with supplemented arachidonic acid and 30 microM in cell-free systems. Thus, celecoxib is a dual inhibitor of COX-2 and 5-LO. Since 2006, celecoxib has been approved as an orphan drug for the treatment of familial adenomatous polyposis. Aside from this indication, it could be useful for treatment of asthma and other diseases where 5-LO is implicated.Extrakte des indischen Weihrauchs werden seit mehr als 3000 Jahren in der Volksmedizin zur Behandlung chronisch entzündlicher Erkrankungen sowie in der ayurvedischen Medizin in Indien und in der Traditionellen Chinesischen Medizin (TCM) verwendet. Pharmakologisch wirksame Inhaltsstoffe sind vor allem Boswelliasäuren, die den Hauptbestandteil der Säurefraktion des Harzes ausmachen. Bislang sind die molekularen Mechanismen nur unvollständig aufgeklärt. Diese Arbeit befasst sich mit der Auffindung und Charakterisierung von Zielstrukturen, die innerhalb der Entzündungskaskade von Boswelliasäuren moduliert werden. Zur Identifizierung wurden 11-Keto-Boswelliasäure sowie beta-Boswelliasäure an Sepharose-Beads gebunden und damit immobilisiert. Die immobilisierten Boswelliasäuren wurden zusammen mit Zelllysaten aus verschiedenen Zelltypen (PMNL, Thrombozyten, MM6, LNCaP, MCF-7, HL-60 und RBL-1) inkubiert. Bei diesem als „Fischen“ bezeichneten Verfahren wurden selektiv folgende Proteine als mögliche Interaktionspartner von BAs gefunden: CatG, Rap1b, DNA-PK, Akt, Proteinase-3, Prohibitin, UNC-112, cathelicidin antimicrobial peptide (hCAP18), farnesylpyrophosphate synthase (FPPs), VAT-1 und ATP-synthase. Die meisten dieser Proteine sind wichtige Mediatoren in der Zelltransduktion, im Immunsystem oder spielen eine Rolle im Entzündungsgeschehen sowie bei der Apoptose. Der Einfluss der BAs auf PKC und FPPs ist noch unklar, da ex vivo und in vitro Experimente zwar negativ ausfielen, Untersuchungen in klinischen Studien aber auf eine Beeinflussung schließen lassen. Rap1b wird durch BAs in seiner Aktivität gehemmt, hier sind noch weitere Studien zur genaueren Charakterisierung nötig. CatG, DNA-PK und die Proteinkinase Akt konnten als Zielstrukturen von BAs mit hoher Affinität (IC50-Werte < 5 mikroM) identifiziert werden, wobei die erreichbaren Plasmakonzentrationen mancher BAs pharmakologische Relevanz implizieren. Andere in der Literatur beschriebene Interaktionspartner benötigen für eine Hemmung sehr hohe Konzentrationen an BAs (z.B. 5-LO: 15-30 mikroM), so dass eine pharmakologische Relevanz in vivo bei der 5-LO und der HLE fraglich erscheint. Die proteolytische Aktivität von CatG wird konzentrationsabhängig, kompetitiv und reversibel mit IC50-Werten von 0,6 mikroM (AKBA), 0,8 mikroM (beta-BA), 1,1 mikroM (A beta-BA) und 3,7 mikroM (KBA) gehemmt. Den BAs ähnliche Triterpene wie Amyrin oder Ursolsäure konnten die Aktivität von CatG bis zu einer Konzentration von 30 mikroM nicht signifikant supprimmieren. Auch die Aktivität anderer Serinproteasen wurde zum Teil sehr potent inhibiert, so konnte für Chymotrypsin ein IC50-Wert von 4,8 mikroM (A beta-BA) ermittelt werden, die anderen BAs waren weniger stark wirksam. Insgesamt ist die Effektivität bei CatG aber am höchsten. Nicht nur die proteolytische Aktivität von CatG wird in vitro inhibiert, auch die CatG-induzierte Migration von PMNL durch Matrigel wird mit einem IC50 von 2,9 mikroM (A beta-BA) gehemmt. DNA-PK und Akt sind Kinasen in der Signaltransduktion von Rezeptor-Tyrosinkinasen, deren Liganden Wachstumsfaktoren sind. Durch zellbasierte und zellfreie Experimente wurde gezeigt, dass BAs die Aktivität von DNA-PK und Akt hemmen und somit Wachstumssignale inhibieren. Die Phosphorylierung von Akt wird intrazellulär und im zellfreien System durch BAs stark reduziert, und die Aktivität von Akt wird extrazellulär durch BAs konzentrationsabhängig im nanomolaren Bereich inhibiert. Durch die Identifizierung dieser hochaffinen Targets ist es möglich, die zelluläre Wirkung der BAs genauer aufzuklären und die BA-Grundstruktur zur Entwicklung neuer selektiver Arzneistoffe gegen chronisch entzündliche Erkrankungen und Krebs zu nutzen. Des Weiteren wurde gezeigt, daß Celecoxib, ein gut charakterisierter, selektiver COX-2-Inhibitor, als hoch potenter 5-LO Inhibitor fungiert. Der Wirkstoff wird zur Therapie von Arthrosen und Rheumatoider Arthritis angewendet und kam als erster selektiver COX-2-Hemmer auf dem Markt. Die gastrointestinalen Nebenwirkungen sind gegenüber den unselektiven NSAR deutlich reduziert, zusätzlich konnte durch klinische Studien gezeigt werden, dass die Gefahr für kardiovaskuläre Ereignisse im Vergleich zu den anderen Coxiben – Rofecoxib (Vioxx®) wurde deshalb vom Markt genommen – stark erniedrigt ist und auf dem Niveau der klassischen Antirheumatika wie Ibuprofen oder Diclofenac liegt. Durch Zufall wurde entdeckt, dass neben der bisher bekannten Wirkung Celecoxib bei Patienten mit familiärer Prädisposition die Polypentstehung im Darm stoppen kann. Diese Darmpolypen sind eine Vorstufe von Darmkrebs. Dieser Effekt konnte weder mit klassischen NSAR noch mit den anderen Coxiben erzielt werden, weshalb neben der COX-2 noch weitere Zielstrukturen beeinflusst werden mussten. Bei weiteren Studien konnte mit 800 mg/d Celecoxib bei einigen Patienten eine Remission von Darmtumorvorstufen erreicht werden, unzureichend mit einer COX-Hemmung erklärbar ist. Durch Untersuchungen an Patientenvollblut konnten verminderte Level an Leukotrienen unter Therapie gezeigt werden, die auf eine Beeinflussung des Leukotrienstoffwechsels hindeuten. Die Aktivität der 5-LO wird sowohl in PMNL als auch in zellfreien Systemen inhibiert und zwar mit folgenden IC50-Werten: 8 mikroM in intakten PMNL, 20 mikroM nach Zugabe von 20 mikroM Arachidonsäure und 30 mikroM im zellfreien System

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging

Triterpene acids from frankincense and semi-synthetic derivatives that inhibit 5-Lipoxygenase and Cathepsin G

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging

Prepectoral versus subpectoral implant-based breast reconstruction after skin-sparing mastectomy or nipple-sparing mastectomy (OPBC-02/ PREPEC): a pragmatic, multicentre, randomised, superiority trial.

INTRODUCTION The emphasis on aesthetic outcomes and quality of life (QoL) has motivated surgeons to develop skin-sparing or nipple-sparing mastectomy (SSM/ NSM) for breast cancer treatment or prevention. During the same operation, a so-called immediate breast reconstruction is performed. The breast can be reconstructed by positioning of a breast implant above (prepectoral) or below (subpectoral) the pectoralis major muscle or by using the patients' own tissue (autologous reconstruction). The optimal positioning of the implant prepectoral or subpectoral is currently not clear. Subpectoral implant-based breast reconstruction (IBBR) is still standard care in many countries, but prepectoral IBBR is increasingly performed. This heterogeneity in breast reconstruction practice is calling for randomised clinical trials (RCTs) to guide treatment decisions. METHODS AND ANALYSIS International, pragmatic, multicentre, randomised, superiority trial. The primary objective of this trial is to test whether prepectoral IBBR provides better QoL with respect to long-term (24 months) physical well-being (chest) compared with subpectoral IBBR for patients undergoing SSM or NSM for prevention or treatment of breast cancer. Secondary objectives will compare prepectoral versus subpectoral IBBR in terms of safety, QoL and patient satisfaction, aesthetic outcomes and burden on patients. Total number of patients to be included: 372 (186 per arm). ETHICS AND DISSEMINATION This study will be conducted in compliance with the Declaration of Helsinki. Ethical approval has been obtained for the lead investigator's site by the Ethics Committee 'Ethikkommission Nordwest- und Zentralschweiz' (2020-00256, 26 March 2020). The results of this study will be published in a peer-reviewed medical journal, independent of the results, following the Consolidated Standards of Reporting Trials standards for RCTs and good publication practice. Metadata describing the type, size and content of the datasets will be shared along with the study protocol and case report forms on public repositories adhering to the FAIR (Findability, Accessibility, Interoperability, and Reuse) principles. TRIAL REGISTRATION NUMBER NCT04293146

Prognostic and predictive impact of genetic markers in patients with CLL treated with obinutuzumab and venetoclax

Genetic parameters are established prognostic factors in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy, but are less well studied with novel compounds. We assessed immunoglobulin heavy variable chain (IGHV) mutation status, common genomic aberrations, and gene mutations in 421 untreated patients within the CLL14 trial (NCT02242942), comparing obinutuzumab1chlorambucil (GClb) vs obinutuzumab1venetoclax (VenG). The incidences of genomic aberrations considering the hierarchical modelwere del(17p) 7%, del(11q) 18%, 112 18%, and del(13q) 35%, whereas IGHV was unmutated in 60% of patients. NOTCH1 mutations weremost common (23%), followed by SF3B1 (16%), ATM (13%), and TP53 (10%). Although the overall response rate (ORR) for GClb was lower in patients with del(17p), del(11q), mutated TP53, ATM, andBIRC3, none of these parameters reduced complete remission (CR) rate and ORR with VenG. At a median follow-up of 28 months, del(17p) and mutated TP53 were the only abnormalities with an effect on progression-free survival (PFS) for both treatment groups: GClb (hazard ratio [HR], 4.6 [P <.01]; HR, 2.7 [P <.01], respectively) and VenG (HR, 4.4 [P <.01]; HR, 3.1 [P <.01], respectively). No other factors affected outcome with VenG, whereas for GClb del(11q), BIRC3, NOTCH1, and unmutated IGHV were associated with shorter PFS. Multivariable analysis identified del(17p), del(11q), unmutated IGHV, and mutated TP53, BIRC3, and SF3B1 as independent prognostic factors for PFS with GClb, whereas for VenG, only del(17p) was significant. VenG was superior to GClb across most genetic subgroups. Patients with adverse genetic markers had the strongest benefit from VenG, particularly subjects with unmutated IGHV, which was identified as a predictive factor in a multivariable treatment-interaction analysis