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Christian Kretzer,1 Paul M Jordan,1 Rossella Bilancia,2 Antonietta Rossi,2 Tuğçe Gür Maz,3 Erden Banoglu,3 Ulrich S Schubert,4,5 Oliver Werz1,5 1Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Jena, 07743, Germany; 2Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Naples, I-80131, Italy; 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Yenimahalle, 06560, Ankara, Turkey; 4Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Jena, 07743, Germany; 5Jena Center for Soft Matter (JCSM) Friedrich Schiller University Jena, Jena, 07743, GermanyCorrespondence: Oliver Werz, Email oliver.werz@uni-jena.deBackground and Purpose: Lipid mediators (LM) play crucial roles in the complex inflammation process with respect to initiation, maintenance, and resolution. Proinflammatory leukotrienes (LTs), generated by 5-lipoxygenase (LOX) and the 5-LOX-activating protein (FLAP), initiate and maintain inflammation while specialized pro-resolving mediators (SPMs) formed by various LOXs as key enzymes promote inflammation resolution and the return to homeostasis. Since 5-LOX also contributes to SPM biosynthesis, smart pharmacological manipulation of the 5-LOX pathway and accompanied activation of 12-/15-LOXs may accomplish suppression of LT formation but maintain or even elevate SPM formation. Here, we demonstrated that the FLAP antagonist BRP-201 possesses such pharmacological profile and causes a switch from LT toward SPM formation.Methods and Results: Comprehensive LM metabololipidomics with activated human monocyte-derived macrophages (MDM) of M1 or M2 phenotype showed that BRP-201 strongly inhibits LT formation induced by bacterial exotoxins. In parallel, SPM levels and 12/15-LOX-derived products were markedly elevated, in particular in M2-MDM. Intriguingly, in unstimulated MDM, BRP-201 induced formation of 12/15-LOX products including SPM and caused 15-LOX-1 subcellular redistribution without affecting 5-LOX. Experiments with HEK293 cells stably expressing either 5-LOX with or without FLAP, 15-LOX-1 or 15-LOX-2 confirmed suppression of 5-LOX product formation due to FLAP antagonism by BRP-201 but activated 15-LOX-1 in the absence of FLAP. Finally, in zymosan-induced murine peritonitis, BRP-201 (2 mg/kg, ip) lowered LT levels but elevated 12/15-LOX products including SPMs.Conclusion: BRP-201 acts as FLAP antagonist but also as 12/15-LOX activator switching formation of pro-inflammatory LTs toward inflammation-resolving SPM, which reflects a beneficial pharmacological profile for intervention in inflammation.Keywords: lipoxygenase, specialized pro-resolving mediators, leukotrienes, lipid mediators

Heart failure (HF) is a systemic, multifactorial disease that affects most of the world's population. It causes a large increase in hospitalizations so affecting the health economics. About 50% of patients with HF symptoms have a normal left ventricular ejection fraction (HFpEF) and its prevalence continues to increase compared to heart failure with a reduced ejection fraction (HFrEF). Current research suggests that HFpEF is characterized by cardiac fibrosis and remodeling, and occurs when chronic medical conditions (e.g. obesity, hypertension, diabetes mellitus, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, thrombosis) damage the heart and other organ systems. These diseases are thought to gradually change the structure and function of the heart over time. There is evidence that an imbalance of extracellular purine levels may be associated with increased cardiovascular risk. Adenosine is an endogenous nucleoside with autocrine/paracrine functions, acting as signal molecule to preserve host defense and tissue integrity during inflammation and trauma. Adenosine is produced primarily from sequential dephosphorylation of extracellular adenosine triphosphate (ATP) to AMP by activity of CD39 (ectonucleoside triphosphate diphosphohydrolase), followed by ecto-5'-nucleotidase CD73. There is much evidence for a role of adenosine in cardiac fibrosis associated with the progression of heart failure. Endogenous adenosine appears to play a significant role in reshaping the microenvironment during inflammatory processes through the interaction with four subtypes of cell surface G-protein-coupled adenosine receptors. These receptors are widely expressed on cardiac cells including fibroblast, endothelial cells, smooth muscle cells and leukocytes, all with a cardioprotective role. Scientific evidence has shown that the expression of CD39 and CD73 are involved in the extracellular adenosine accumulation, were upregulated in human circulating leukocytes of heart failure patients, suggesting that HF could benefit from adenosine-based drug therapy. It is well recognized that patients with HF have an increased risk of venous thromboembolism, stroke, and sudden death. The increased cardiovascular risk may be associated with an imbalance of extracellular purine levels. Platelets are an important source of purine nucleotides and nucleosides. In case of stress, adenosine is released in large quantities by the CD39 enzyme. It is expressed on the endothelium, circulating blood cells, and smooth muscle cells. Changes in CD39 expression and activity affect the thrombogenic potential of a tissue. Gender difference in the cardiovascular risk has been extensively observed; however, while the age-dependent difference in the prevalence of cardiovascular events between men and women has been attributed to the loss of the protective effect of estrogens in the postmenopausal period, the physiological mechanism behind gender disparity is still unclear. In the light of these considerations, the aims of this study were: 1) to investigate comparatively platelet functionality in male and female rats and the possible link to CD39 enzyme 2) to evaluate the changes in the adenosine pathway in Dahl salt-sensitive hypertensive rat, a model of heart failure with preserved ejection fraction. We found a reduced in vitro response to ADP of female compared to male platelets, associated to increased platelet CD39 expression and activity. Platelet response to ADP was strongly increased by incubation (10 min) with the CD39 inhibitor, ARL67156, while male platelet response was unaffected. Rat treatment with clopidogrel (30 mg/kg, per os) inhibited ex vivo platelet aggregation. Bleeding time was prolonged in female compared to male. Dahl rats, feeding with a 8% NaCl diet, progressively developed hypertension, compared to LS animals that remained normotensive. While systolic parameters were not altered, diastolic parameters were changed in high salt animals. Indeed, hemodynamic analysis showed a decreased dP/dt min, increased end-diastolic pressure, longer time constant Tau and steeper slope of the end-diastolic pressure-volume relationship. In addition, our data showed an increase of LV systolic and diastolic posterior wall thickness in the HS group of animals at both 5 and 13 weeks, respectively. In our animal model of HFpEF we observed, in the myocardium of 13 HS animals, an increase in perivascular fibrosis due to collagen accumulation and an increase in reactive oxygen species and nitrogen. Western blot analysis showed a reduced production of adenosine in rats with HFpEF, an increased production of inosine, which has pro�inflammatory action, and a greater expression of the A2B receptors which, with their pro�fibrotic, cause cardiac rigidity in the hearts of Dahl rats at 13 weeks and thus worsening the inflammatory state in the latter group. Our results suggest that the sex variability in platelet ATPase and ADPase activity should be taken into account for understanding more in depth the molecular mechanisms behind gender difference in cardiovascular risk. These preliminary results suggest, also, that adenosine pathway may represent an attractive target to test potential therapeutic strategies to prevent and/or delay the progression of HFpEF syndrome.

The design of multitarget drugs represents a promising strategy in medicinal chemistry and seems particularly suitable for the discovery of anti-inflammatory drugs. Here, we describe the identification of an indoline-based compound inhibiting both 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH). In silico analysis of an in-house library identified nine compounds as potential 5-LOX inhibitors. Enzymatic and cellular assays revealed the indoline derivative 43 as a notable 5-LOX inhibitor, guiding the design of new analogues. These compounds underwent extensive in vitro investigation revealing dual 5-LOX/sEH inhibitors, with 73 showing the most promising activity (IC50s of 0.41 ± 0.01 and 0.43 ± 0.10 μM for 5-LOX and sEH, respectively). When challenged in vivo in zymosan-induced peritonitis and experimental asthma in mice, compound 73 showed remarkable anti-inflammatory efficacy. These results pave the way for the rational design of 5-LOX/sEH dual inhibitors and for further investigation of their potential use as anti-inflammatory agents.

Leukotrienes (LTs) and prostaglandin (PG)E2, produced by 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1), respectively, are key players in inflammation, and pharmacological suppression of these lipid mediators (LM) represents a strategy to intervene with inflammatory disorders. Previous studies revealed that the benzenesulfonamide scaffold displays efficient 5-LO-inhibitory properties. Here, we structurally optimized benzenesulfonamides which led to an N-phenylbenzenesulfonamide derivative (compound 47) with potent inhibitory activities (IC50 = 2.3 and 0.4 μM for isolated 5-LO and 5-LO in intact cells, respectively). Compound 47 prevented the interaction of 5-LO with its activating protein (FLAP) at the nuclear envelope in transfected HEK293 cells as shown by in situ proximity ligation assay. Comprehensive assessment of the LM profile produced by human macrophages revealed the ability of 47 to selectively down-regulate pro-inflammatory LMs (i.e. LTs and PGE2) in M1 but to enhance the formation of pro-resolving LMs (i.e. resolvins and maresins) in M2 macrophages. Moreover, 47 strongly inhibited LT formation and cell infiltration in two in vivo models of acute inflammation (i.e., peritonitis and air pouch sterile inflammation in mice). Together, 47 represents a novel LT biosynthesis inhibitor with an attractive pharmacological profile as anti-inflammatory drug that also promotes the biosynthesis of pro-resolving LM.

Lipid mediators (LM) encompass pro-inflammatory prostaglandins (PG) and leukotrienes (LT) but also specialized pro-resolving mediators (SPM) which display pivotal bioactivities in health and disease. Pharmacological intervention with inflammatory disorders such as osteoarthritis and rheumatoid arthritis commonly employs anti-inflammatory drugs that can suppress PG and LT formation, which however, possess limited effectiveness and side effects. Here, we report on the discovery and characterization of the two novel benzoxanthene lignans 1 and 2 that modulate select LM biosynthetic enzymes enabling the switch from pro-inflammatory LT to SPM biosynthesis as potential pharmacological strategy to intervene with inflammation. In cell-free assays, compound 1 and 2 inhibit microsomal prostaglandin E2 synthase-1 and leukotriene C4 synthase (IC50 ∼ 0.6–3.4 µM) and potently interfere with 5-lipoxygenase (5-LOX), the key enzyme in LT biosynthesis (IC50 = 0.04 and 0.09 µM). In human neutrophils, monocytes and M1 and M2 macrophages, compound 1 and 2 efficiently suppress LT biosynthesis (IC50

The incidence and severity of asthma preponderate in women versus men. Leukotrienes (LTs) are lipid mediators involved in asthma pathogenesis, and sex disparities in LT biosynthesis and anti-LT pharmacology in inflammation have recently emerged. Here, we report on sex dimorphism in LT production during allergen sensitization and its correlation to lung function. While high plasma levels of IgE, as sensitization index, were elevated in both sexes, LT levels increased only in lungs of female ovalbumin-sensitized BALB/c mice. Sex-dependent elevated LT levels strictly correlated to an enhanced airway hyperreactivity, pulmonary inflammation and mast cell infiltration/activation in female mice. Importantly, this sex bias was coupled to superior therapeutic efficacy of different types of clinically used LT modifiers like zileuton, MK886 and montelukast in female animals. Our findings reveal sex-dependent LT production as a basic mechanism of sex dimorphism in allergic asthma, and suggest that women might benefit more from anti-LT asthma therapy.

The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4–8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE2 than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.

Salvinorin A, a neoclerodane diterpene isolated from Salvia divinorum, exerts a number of pharmacological actions which are not solely limited to the central nervous system. Recently it has been demonstrated that Salvinorin A inhibits acute inflammatory response affecting leukotriene (LT) production. Since LTs are potent lipid mediators implicated in allergic diseases, we evaluated the effect of Salvinorin A on allergic inflammation and on airways following sensitization in the mouse. Mice were sensitized with s.c. injection of ovalbumin (OVA) on days 1 and 8. Sensitized mice received on days 9 and 12 on the shaved dorsal surface air administration to induce the development of the air-pouches. On day 15 animals were challenged by injection of OVA into the air-pouch. Salvinorin A, administered (10 mg/kg) before each allergen exposure, significantly reduced OVA-induced LT increase in the air pouch. This effect was coupled to a reduction in cell recruitment and Th2 cytokine production. In another set of experiments, mice were sensitized with OVA and both bronchial reactivity and pulmonary inflammation were assessed. Salvinorin A abrogated bronchial hyperreactivity and interleukin (IL)-13 production, without effect on pulmonary inflammation. Indeed cell infiltration and peribronchial edema were still present following diterpenoid treatment. Similarly, pulmonary IL-4 and plasmatic IgE levels were not modulated. Conversely, Salvinorin A significantly reduced LTC4 production in the lung of sensitized mice. Finally mast cell activity was evaluated by means of toluidine blue staining. Data obtained evidenced that Salvinorin A significantly inhibited mast cell degranulation in the lung. Our study demonstrates that Salvinorin A inhibits airway hyperreactivity induced by sensitization by inhibition of LT production and mast cell degranulation. In conclusion Salvinorin A could represent a promising candidate for drug development in allergic diseases such as asthma.