M.José Alcaraz

Synthesis and anti-inflammatory activity of chalcone derivatives

Chalcones and their derivatives were synthesized and evaluated for their anti-inflammatory activity. In vitro, chalcones 2, 4, 8, 10 and 13 inhibited degranulation and 5-lipoxygenase in human neutrophils, whereas 11 behaved as scavenger of superoxide. Only four compounds (4-7) inhibited cyclo-oxygenase-2 activity. The majority of these samples showed anti-inflammatory effects in the mouse air pouch model.

Morelloflavone, a novel biflavonoid inhibitor of human secretory phospholipase A2 with anti-inflammatory activity.

The flavanonylflavone morelloflavone inhibited secretory phospholipase A2 (PLA2) in vitro, with a high potency on the human recombinant synovial and bee venom enzymes (IC50 = 0.9 and 0.6 microM, respectively). The inhibition was apparently irreversible. In contrast, the compound was inactive on cytosolic PLA2 activity from human monocytes. Morelloflavone scavenged reactive oxygen species generated by human neutrophils (IC50 = 2.7 and 1.8 microM for luminol and lucigenin, respectively) but did not modify cellular responses such as degranulation or eicosanoid release. This biflavonoid exerted anti-inflammatory effects in animal models, with a potent inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear inflammation in mice after topical administration. In this test, morelloflavone was found to decrease oedema and myeloperoxidase levels in ear homogenates ID50 = 58.5 and 74.3 micrograms/ear, respectively). In contrast, this biflavonoid failed to modify arachidonic acid-induced ear inflammation or eicosanoid levels in ear homogenates. A significant anti-inflammatory effect was also observed in the mouse paw carrageenan edema after oral administration, with the highest inhibition at 3 hr after induction of inflammation. Morelloflavone is an inhibitor of secretory PLA2 with selectivity for groups II and III enzymes and may be a pharmacological tool. In addition, it shows anti-inflammatory activity apparently not related to the synthesis of eicosanoids, but likely dependent on other mechanisms such as scavenging of reactive oxygen species.

Avarol and avarone, two new anti-inflammatory agents of marine origin.

The anti-inflammatory activity of avarol and avarone, sesquiterpenoid derivatives from the Mediterranean sponge Dysidea avara, was investigated. Both compounds potently inhibited paw oedema induced by carrageenan (approximated ED50 = 9.2 and 4.6 mg/kg, p.o., respectively) as well as ear oedema induced by 12-O-tetradecanoylphorbol acetate (TPA; ED50 = 97 and 397 micrograms/ear, respectively) in mice, with effects comparable to those of indomethacin. In A23187-stimulated rat peritoneal leukocytes, avarol showed an IC50 = 0.6 and 1.4 microM for inhibition of leukotriene B4 and thromboxane B2 release, respectively, with avarone showing a slightly lower potency. Both marine metabolites failed to show xanthine oxidase inhibitory activity or superoxide scavenging effects but were potent inhibitors of superoxide generation in rat peritoneal leukocytes activated by different stimuli, with an IC50 below the microM range. Only avarol was able to inhibit human recombinant synovial phospholipase A2 activity with an IC50 = 158 microM, and thus this compound showed a potency higher than that of mepacrine. Avarol and avarone effectively control acute inflammation in experimental models after either oral or topical administration and their anti-inflammatory activity may result from inhibition of eicosanoid release and depression of superoxide generation in leukocytes.

Novel anti-inflammatory chalcone derivatives inhibit the induction of nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages

In a previous work, we tested a series of chalcone derivatives as possible anti‐inflammatory compounds. We now investigate the effects of three of those compounds, CH1, CH8 and CH12, on nitric oxide and prostanoid generation in mouse peritoneal macrophages stimulated with lipopolysaccharide and in the mouse air pouch injected with zymosan, where they showed a dose‐dependent inhibition with inhibitory concentration 50% values in the μM range. This effect was not the consequence of a direct inhibitory action on enzyme activities. Our results demonstrated that chalcone derivatives inhibited de novo inducible nitric oxide synthase and cyclooxygenase‐2 synthesis, being a novel therapeutic approach for inflammatory diseases.

Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2

Cacospongionolide B is a novel marine metabolite isolated from the sponge Fasciospongia cavernosa. In in vitro studies, this compound inhibited phospholipase A2 (PLA2), showing selectivity for secretory PLA2 (sPLA2) versus cytosolic PLA2 (cPLA2), and its potency on the human synovial enzyme (group II) was similar to that of manoalide. This activity was confirmed in vivo in the 8 h zymosan‐injected rat air pouch, on the secretory enzyme accumulating in the pouch exudate. Cacospongionolide B, that is bioavailable when is given orally, reduced the elevated levels of sPLA2 present in paw homogenates of rats with adjuvant arthritis. This marine metabolite showed topical anti‐inflammatory activity on the mouse ear oedema induced by 12‐O‐tetradecanoylphorbol acetate (TPA) and decreased carrageenin paw oedema in mice after oral administration of 5, 10 or 20 mg kg−1. In the mouse air pouch injected with zymosan, cacospongionolide B administered into the pouch, induced a dose‐dependent reduction in the levels of eicosanoids and tumour necrosis factor α (TNFα) in the exudates 4 h after the stimulus. It also had a weak effect on cell migration. The inflammatory response of adjuvant arthritis was reduced by cacospongionolide B, which did not significantly affect eicosanoid levels in serum, paw or stomach homogenates and did not induce toxic effects. Cacospongionolide B is a new inhibitor of sPLA2 in vitro and in vivo, with anti‐inflammatory properties in acute and chronic inflammation. This marine metabolite was active after oral administration and able to modify TNFα levels, and may offer an interesting approach in the search for new anti‐inflammatory agents.

Nitric oxide synthase and cyclo‐oxygenase pathways in the inflammatory response induced by zymosan in the rat air pouch

We have studied the participation of nitric oxide (NO) in an animal model of inflammation, the rat air pouch stimulated with zymosan. Saline or zymosan was injected into 6‐day rat air pouches at different time points and measurements were made of cell migration, levels of nitrite/nitrate (NO2−/NO3−), prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and secretory phospholipase A2 (sPLA2) in exudates. Nitric oxide synthase (NOS) activity was determined in high speed supernatants from cells present in pouch exudates. Western blot analysis was also performed on these samples. Zymosan injection induced a time‐dependent increase in leukocyte infiltration, NO2−/NO3− levels and cellular NOS activity that reached a peak by 8 h. Western blot analysis showed the same time course for induction of NOS protein. Colchicine administration to rats inhibited cellular infiltration and decreased the levels of NO metabolites and cellular NOS activity zymosan‐injected air pouch at 8 h. NOS activity was present in polymorphonuclear leukocytes (PMNs) and monocytes, but not in the lymphocytes present in exudates. This enzyme is calcium‐independent and needs NADPH for activity. PGE2 levels in exudates showed a time course inverse to that of NOS activity and NO metabolites, with maximum levels of PGE2 observed at 4 h after zymosan injection. Administration of NG‐nitro‐l‐arginine methyl ester (l‐NAME) or aminoguanidine to rats significantly reduced cellular NOS activity, NO2−/NO3− levels and chemiluminescence, whereas they were without effect on cell migration and degranulation, eicosanoid levels and sPLA2 activity. Treatment of animals with dexamethasone inhibited cellular NOS activity, NO2−/NO3− levels, chemiluminescence and the increase in the levels of PGE2 and LTB4, with only a weak effect on elastase release. Administration of the selective cyclo‐oxygenase‐2 (COX‐2) inhibitor NS398 to rats strongly reduced PGE2 levels in exudates without affecting NO metabolites or NOS activity at 4 h after zymosan injection. Our data indicate that NOS is induced in the zymosan‐stimulated rat air pouch model of inflammation. This enzyme is expressed in the cells migrating into the air pouch and caused an increased production of NO metabolites in exudates. The results also suggest the presence of an earlier phase in which eicosanoids play the main role, with participation of COX‐2 activity, and a later phase mediated by NO. The endogenous release of NO does not modify prostaglandin biosynthesis in this in vivo model.

Cacospongionolide B suppresses the expression of inflammatory enzymes and tumour necrosis factor-α by inhibiting nuclear factor-κB activation

The marine product cacospongionolide B, a sesterterpene isolated from the Mediterranean sponge Fasciospongia cavernosa, is an inhibitor of secretory phospholipase A2 with anti‐inflammatory properties. In this work, we have studied the mechanism of action of this compound in the inflammatory response induced by zymosan in primary cells and in the mouse air pouch. In mouse peritoneal macrophages, cacospongionolide B was able to downregulate the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), resulting in decreased production of NO and prostaglandin E2 (PGE2). This compound also reduced tumour necrosis factor‐α (TNF‐α) mRNA expression and TNF‐α levels. Cacospongionolide B inhibited nuclear factor‐κB (NF‐κB)‐DNA binding activity and the nuclear translocation of this transcription factor. Treatment of cells with cacospongionolide B impaired NF‐κB inhibitory protein (IκB‐α) phosphorylation and enhanced IκB‐α expression. Inhibition of iNOS, COX‐2 and inflammatory mediators was confirmed in the mouse air pouch. These results show that cacospongionolide B is able to control NO, PGE2 and TNF‐α production in vitro and in vivo, effects likely dependent on NF‐κB inhibition.

Suppression of leukotriene B4 and tumour necrosis factor α release in acute inflammatory responses by novel prenylated hydroquinone derivatives

A series of prenyl hydroquinone derivatives synthesized as structural analogs of marine products were tested for their effects on inflammatory responses in vitro and in vivo. 2-Prenyl-1,4-hydroquinone (H1), 2-diprenyl-1,4-hydroquinone (H2), 2-triprenyl-1,4-hydroquinone (H3) and 2-tetraprenyl-1,4-hydroquinone (H4) scavenged reactive oxygen species and inhibited 5-lipoxygenase (5-LO) activity in human neutrophils. The inhibition of 5-LO activity was demonstrated in vivo in the mouse air pouch injected with zymosan and arachidonic acid-induced ear inflammation. The four compounds suppressed the production of tumour necrosis factor α (TNFα) in J774 cells stimulated with lipopolysaccharide (LPS) and also in vivo in the mouse air pouch injected with zymosan. In addition, all prenyl-hydroquinones inhibited the release of nitrite and PGE2 in LPS-stimulated J774 cells, without direct effects on cyclo-oxygenase-1 (COX-1), cyclo-oxygenase-2 (COX-2) or inducible nitric oxide synthase (iNOS) activities in several cell-free systems. The reduction in the length of the lateral chain in prenyl-hydroquinones (1-4 isoprene units) with respect to their marine analogs (7-8 isoprene units) has improved the anti-inflammatory activity of this class of compounds. Marine natural products may be a model to design new anti-inflammatory agents.

Effects of marine 2-polyprenyl-1,4-hydroquinones on phospholipase A2 activity and some inflammatory responses

Three 2-polyprenyl-1,4-hydroquinone derivatives (2-heptaprenyl-1,4-hydroquinone: IS1, 2-octaprenyl-1,4-hydroquinone: IS2 and 2-[24-hydroxy]-octaprenyl-1,4-hydroquinone: IS3) isolated from the Mediterranean sponge Ircinia spinosula, were evaluated for effects on phospholipase A2 activity of different origin (Naja naja venom, human recombinant synovial fluid and bee venom), as well as on human neutrophil function and mouse ear oedema induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). IS1 interacted minimally with these responses. In contrast, IS2 and IS3 inhibited human recombinant synovial phospholipase A2 in a concentration-dependent manner, with minor effects on the rest of the enzymes. Both compounds slightly affected superoxide generation and degranulation in human neutrophils, whereas they decreased thromboxane B2 and leukotriene B4 synthesis and release in a mixed suspension of human platelets and neutrophils stimulated by ionophore A23187, with IC50 values in the microM range. IS3 was the most effective inhibitor of the synthesis of thromboxane B2 by human platelet microsomes and of leukotriene B4 by high speed supernatants from human neutrophils. IS2 and IS3 showed topical anti-inflammatory activity against the TPA-induced ear inflammation in mice, with similar effects on oedema and a higher inhibition of IS3 on leukocyte migration, estimated as myeloperoxidase activity in supernatants of ear homogenates. Some structure-activity relationships were established since differences in the prenylated chain attached to the hydroquinone moiety result in important modifications of these inflammatory responses.

A Boron Dipyrromethene (BODIPY)-Based Cu(II) -Bipyridine Complex for Highly Selective NO Detection.

A BODIPY-containing Cu(II) -bipyridine complex for the simple selective fluorogenic detection of NO in air and in live cells is reported. The detection mechanism is based on NO-promoted Cu(II) to Cu(I) reduction, followed by demetallation of the complex, which results in the clearly enhanced emission of the boron dipyrromethene (BODIPY) unit.