Elaboración de videos didácticos como alternativa a los ensayos in vivo en las prácticas de farmacología / Didactic video as an alternative to in vivo assays in practical lessons of pharmacology

La Farmacología es una ciencia eminentemente práctica, en la que tiene gran relevancia la investigación "in vivo" con animales de experimentación. Los conocimientos impartidos en las clases teóricas, seminarios y tutorías, se completan con la enseñanza en las sesiones prácticas. Sin embargo, algunos aspectos importantes de esta enseñanza práctica, que incluyen la realización de ensayos “in vivo”, resulta problemático impartirlos correctamente con la normativa actual sobre la utilización de animales de laboratorio. Por ello, nos planteamos la realización de unos vídeos demostrativos de las técnicas experimentales utilizadas en algunas de las sesiones prácticas de las asignaturasde Farmacologia I y II del Grado en Farmacia. En cada sesión el profesor realiza una breve introducción del modelo experimental, indicando los objetivos que se plantea el investigador asi como las posibilidades de dicha técnica. A continuación en los vídeos, los estudiantes ven el desarrollo completo del experimento, los materiales necesarios y las condiciones experimentales adecuadas para su realización, asi como los diferentes parámetros y variables que se pueden medir. Al finalizar la proyección del vídeo se plantean dos tipos de tareas a los estudiantes:- diseño de un protocolo de evaluación de un fármaco con la metodología descrita- análisis, presentación y discusión de resultados, tras proporcionarles ejemplos de datos obtenidos en el ensayo.El procedimiento seguido para la elaboración de los vídeos es: 1) Diseño del ensayo de laboratorio. 2) Preparación del material necesario y las condiciones para una correcta grabación. 3) Grabación. Montaje de las imágenes (incluye fotografías, esquemas) y del sonido. 4) Edición del material filmado (una versión para Video- DVD y otra para incluirla en el Servidor Multimedia de la plataforma de la Universitat de València)(AU)

Pharmacology is a primarily practical science, in which “in vivo” research using experimental animal models plays a relevant role. The topics covered in the theoretical classes, seminars and tutorials are complemented with learning in practical sessions. However, certain important aspects of the practical learning, which include performing “in vivo” assays, represents a challenge given the actual regulations regarding laboratory animal utilization. Therefore, we proposed to produce didactic videos for various practical sessions. The teacher gives a brief introduction of the selected experimental animal model, indicating the intended objectives to be achieved. The students can see in the video the complete experiment progression, necessary materials and the proper experimental conditions toperform the assay, as well as the different parameters and variables to be measured.- a protocol design to evaluate a drug with the described methodology- analysis, result presentation and discussion of given example data obtained with the assay.The procedure used for the video elaboration was: 1) Design of the laboratory assay. 2) Preparation of the necessary materials and conditions for a correct recording. 3) Recording. Image (including photographs, schemes, figures) and sound download. 4) Editing of the filmed material (one version for Video- DVD and another one to be included in the virtual platform Multimedia Server of the University of Valencia)(AU)

Treatment with a CO-releasing molecule (CORM-3) reduces joint inflammation and erosion in murine collagen-induced arthritis.

OBJECTIVE: CO-releasing molecules (CO-RMs) are a novel class of anti-inflammatory agents. We have examined the possible therapeutic effects of CORM-3 in collagen-induced arthritis (CIA). METHODS: Arthritis was induced in DBA-1/J mice by type II collagen. Animals were treated with CORM-3 (5 and 10 mg/kg/day, intraperitoneally) or the inactive compound iCORM-3 (10 mg/kg/day, intraperitoneally) unable to release CO, from days 22 to 31. Production of anti-type II collagen antibodies, cytokines and cartilage olimeric matrix protein (COMP) was evaluated by enzyme-linked immunosorbent assay, and prostaglandin E(2) (PGE(2)) by radioimmunoassay. Localisation of cyclooxygenase-2 (COX-2), haem oxygenase-1 (HO-1), intercellular adhesion molecule-1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) was examined by immunohistochemistry. RESULTS: Therapeutic administration of CORM-3 suppressed clinical and histopathological manifestations of disease. The levels of PGE(2), interleukin (IL)1beta, IL2, IL6, IL10 and tumour necrosis factor (TNF)alpha in joint tissues were inhibited by CORM-3. By contrast, CORM-3 augmented IL4. Anti-type II collagen antibodies and COMP levels in serum were reduced by CORM-3. Treatment with CORM-3 decreased cellular infiltration, joint inflammation and destruction, as well as the expression of COX-2, ICAM-1 and RANKL, whereas HO-1 increased. These beneficial effects were due to CO release, as iCORM-3 was ineffective. CONCLUSION: This study reveals the antiarthritic properties of CORM-3 in the CIA model and supports the notion that CO-RMs could be developed as a novel strategy for the treatment of inflammatory and arthritic conditions.

Antipsoriatic effects of avarol-3'-thiosalicylate are mediated by inhibition of TNF-alpha generation and NF-kappaB activation in mouse skin.

BACKGROUND AND PURPOSE: Avarol is a marine sesquiterpenoid hydroquinone with anti-inflammatory and antipsoriatic properties. The aim of this study was to evaluate the in vitro and in vivo pharmacological behaviour of the derivative avarol-3'-thiosalicylate (TA) on some inflammatory parameters related to the pathogenesis of psoriasis. EXPERIMENTAL APPROACH: Human neutrophils and monocytes as well as the human keratinocyte cell line HaCaT were used to study the effect of TA on oxidative stress, the arachidonic acid pathway, tumour necrosis factor-alpha (TNF-alpha) release and nuclear factor-kappaB (NF-kappaB) activation. All these parameters were also determined in vivo using the zymosan induced mouse air pouch model and the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced mouse epidermal hyperplasia model. KEY RESULTS: TA showed antioxidant properties in human neutrophils and in the hypoxanthine/xanthine oxidase assay. This compound reduced, in a concentration-dependent manner, leukotriene B(4), prostaglandin E(2) and TNF-alpha production in activated leukocytes. Oral and intrapouch administration of TA in the mouse air pouch model produced a dose-dependent reduction of all these inflammatory mediators. TA also inhibited secretory phospholipase A(2) activity and NF-kappaB DNA-binding in HaCaT keratinocytes. In TPA-induced mouse epidermal hyperplasia, topical administration of TA reduced oedema, leukocyte infiltration, eicosanoid levels and TNF-alpha in skin. In addition, interleukin (IL)-1beta and IL-2 production were also inhibited. Finally, TA was also capable of suppressing NF-kappaB nuclear translocation in vivo. CONCLUSIONS AND IMPLICATIONS: TA inhibited several key biomarkers up-regulated in the inflammatory response of psoriatic skin and this compound could be a promising antipsoriatic agent.

Evaluation of the anti-inflammatory and analgesic activity of Me-UCH9, a dual cyclooxygenase-2/5-lipoxygenase inhibitor.

Recently, we reported the dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) activity by some phenylsulphonyl urenyl chalcone derivatives. 2,4-dichloro-4'N[N'(4''methylphenylsulphonyl)urenyl] chalcone (Me-UCH9), was selected in the present study to determine its potential anti-inflammatory and analgesic effect after oral administration in several animal models related to the activation of COX-2 and 5-LO pathways. In the zymosan stimulated mouse air pouch model, Me-UCH9, reduced in a dose-dependent manner leukotriene B(4) (LTB(4)) levels in pouch exudates obtained at 4 h, as well as prostaglandin E(2) (PGE(2)) generated through COX-2 activation at 24 h. Tumor necrosis factor alpha (TNF-alpha) and myeloperoxidase activity were also strongly inhibited in this model. Me-UCH9 significantly reduced granuloma size and vascular index determined in the murine air pouch granuloma model of angiogenesis. In the carrageenan-induced paw edema, this compound inhibited inflammatory response and pain, as well as PGE(2) and LTB(4) content in paw edematous fluid. Analgesic properties were corroborated in the murine phenyl-p-benzoquinone-induced writhing test. Finally, Me-UCH9 exerted anti-inflammatory effects in the chronic model of rat adjuvant-induced arthritis, both inhibiting paw swelling and reducing PGE(2) content. Our findings confirm that Me-UCH9 can modulate inflammatory and nociceptive responses in relation to the dual inhibition of COX-2 and 5-LO activities presented by this compound.

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase.

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E2 (PGE2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 microM but most of them inhibited the generation of PGE2 with IC50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC50 0.06 microM). Selected compounds also reduced leukotriene B4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC50 values from 0.5 to 0.8 microM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.

A new chloroquinolinyl chalcone derivative as inhibitor of inflammatory and immune response in mice and rats.

The synthetic chalcone derivative 1-(2,4-dichlorophenyl)-3-(3-(6,7-dimethoxy-2-chloroquinolinyl))-2-propen-1-one (ClDQ) was evaluated for its anti-inflammatory, analgesic and immunomodulatory efficacy in-vitro and in-vivo. ClDQ concentration-dependently inhibited the production of nitric oxide (NO) (IC50 4.3 microM) and prostaglandin E(2) (PGE(2)) (IC50 1.8 microM) in RAW 264.7 macrophages stimulated with lipopolysaccharide. Human mononuclear cell proliferation was significantly inhibited by 10 microM ClDQ. Oral administration of ClDQ (10-30 mg kg(-1)) in the 24-h zymosan-stimulated mouse air-pouch model produced a dose-dependent reduction of cell migration as well as NO and PGE(2) levels in exudates. ClDQ (20 mg kg(-1), p.o.) inhibited ear swelling and leucocyte infiltration in the delayed-type hypersensitivity response to 2,4-dinitrofluorobenzene in mice. In the rat adjuvant-arthritis model, this compound reduced joint inflammation as well as PGE(2) and cytokine levels. In addition, ClDQ displayed analgesic effects in the phenylbenzoquinone-induced abdominal constriction model in mice and in the late phase of the nociceptive response to formalin. Our findings indicated the potential interest of ClDQ in the modulation of some immune and inflammatory conditions.

1-(2,3,4-trimethoxyphenyl)-3-(3-(2-chloroquinolinyl))-2-propen-1-one, a chalcone derivative with analgesic, anti-inflammatory and immunomodulatory properties.

OBJECTIVE AND DESIGN: The synthetic chalcone derivative 1-(2,3,4-trimethoxyphenyl)-3-(3-(2-chloroquinolinyl))-2-propen-1-one (TQ) was evaluated for its immunomodulatory and anti-inflammatory efficacy in vitro and in vivo. MATERIAL AND SUBJECTS: Human neutrophils and lymphocytes from healthy volunteers and RAW 264.7 murine macrophages. Swiss mice and Lewis rats were randomly divided into groups of six animals. TREATMENT: TQ was orally administered in all in vivo assays (10-30 mg/kg). METHODS: Elastase, superoxide and LTB(4) release were assayed in human neutrophils, NO/PGE(2) production and NF-kappaB activation in RAW 264.7, and (3)H thymidine incorporation in human lymphocytes. Zymosan-stimulated air pouches, DNFB-DTH, PBQ-induced writhings and formalin-induced pain were assayed in mice. Adjuvant-induced arthritis was tested in rats. Dunnett's t-test was employed for statistical analysis. RESULTS: Human T-cell proliferation, neutrophil functions and NO/PGE(2) production in murine macrophages were inhibited by TQ (IC(50) in the microM range), which showed anti-inflammatory, immunomodulatory and analgesic effects. CONCLUSIONS: Our findings indicate the potential interest of TQ in the modulation of some immune and inflammatory responses probably by NF-kappaB inhibition.

Inhibition of leukocyte functions by the alkaloid isaindigotone from Isatis indigotica and some new synthetic derivatives.

The alkaloid isaindigotone (1a) and seven derivatives have been synthesized to study their influence on several leukocyte functions and the generation of inflammatory mediators. Isaindigotone (1a) was found to be a scavenger of superoxide generated either by the hypoxanthine/xanthine oxidase system or stimulated human neutrophils. Isaindigotone (1a) and its acetylated derivative (1b) also inhibited 5-lipoxygenase activity and leukotriene B(4) production in these cells, whereas none of the compounds affected degranulation. In RAW 264.7 macrophages stimulated with lipopolysaccharide, synthetic derivatives exerted higher inhibitory effects on prostaglandin E(2) (PGE(2)) and nitric oxide (NO) generation when compared with (1a). The presence of an acetoxyl group at C-4' favors the inhibition of NO and PGE(2) production, whereas the fluoro substituent at C-4' or the absence of substituents on the aromatic ring of the benzylidene unit improves the inhibition of PGE(2). Thus, this series of compounds can attenuate the production of mediators relevant to the inflammatory response.

Inhibition of 5-lipoxygenase activity by the natural anti-inflammatory compound aethiopinone.

OBJECTIVE AND DESIGN: We have investigated the mechanisms of action of aethiopinone, an anti-inflammatory compound from Salvia aethiopis L. roots. MATERIAL AND SUBJECTS: Human neutrophils from healthy volunteers and murine peritoneal macrophages. Swiss mice were randomly divided into groups of six animals. TREATMENT: Test compounds were applied topically in the mouse ear oedema test. In the air pouch, mice received aethiopinone (0.001-0.5 pmol/pouch or 12.5-50 mg/kg p.o.). METHODS: LTB4 production was assayed in human neutrophils and COX-2 and iNOS activities in murine macrophages. Air pouches were induced subcutaneously in mice and injected with zymosan on the day six. Mouse ear oedema was induced by arachidonic acid. Dunnett's t-test was employed for statistical analysis. RESULTS: We have observed potent inhibitory effects on human neutrophil LTB4 production without effects on COX or NOS activities. Aethiopinone is an in vitro inhibitor of 5-LO from human neutrophils (IC50 = 0.11 microM). In addition, aethiopinone reduced leukocyte accumulation and showed in vivo inhibitory activity on this enzyme. CONCLUSIONS: Our results indicate that inhibition of 5-LO could participate in the anti-inflammatory properties of this natural product.

Solid-phase synthesis and inhibitory effects of some pyrido[1,2-c]pyrimidine derivatives on leukocyte formations and experimental inflammation.

A number of pyrido[1,2-c]pyrimidines bearing a nitrogen, oxygen, or sulfur functionality at C-1 were synthesized on solid-phase using the iminophosphorane methodology and tested for their effects on leukocyte functions in vitro and antiinflammatory activity. Compound 5c was found to be a strong scavenger of superoxide anion and an inhibitor of chemiluminescence induced by 12-O-tetradecanoylphorbol 13-acetate in human neutrophils. These pyrido[1,2-c]pyrimidines inhibited the generation of PGE(2) by COX-2 in RAW 264.7 macrophages stimulated with lipopolysaccharide. Compounds 7, 5f, 6, and 8 inhibited enzyme activity, whereas the remaining compounds also acted on the induction phase. In addition, 5a-f, 6, and 7 administered p.o. at a dose of 20 mg/kg showed antiinflammatory activity in the carrageenan mouse paw edema model, where they inhibited PGE(2) levels in inflamed paws without affecting the content of this eicosanoid in stomachs. Inhibition of PGE(2) production and superoxide scavenging may participate in the mechanism of the antiinflammatory action of these pyrido[1,2-c]pyrimidine derivatives.

Dysidotronic acid, a new sesquiterpenoid, inhibits cytokine production and the expression of nitric oxide synthase.

In a previous study, we reported a new bioactive sesquiterpenoid, named dysidotronic acid, to be a potent, selective human synovial phospholipase A(2) inhibitor. Dysidotronic acid is a novel, non-complex manoalide analogue lacking the pyranofuranone ring. We now investigate the effect of this compound on cytokine, nitric oxide and prostanoid generation on the mouse macrophage cell line RAW 264.7, where it showed a dose-dependent inhibition with inhibitory concentration 50% values in the micromolar range. This effect was also confirmed in the mouse air pouch injected with zymosan. Dysidotronic acid inhibited the production of tumor necrosis factor alpha and interleukin-1 beta as well as the production of nitric oxide, prostaglandin E(2) and leukotriene B(4). Decreased nitric oxide generation was the consequence of inhibition of the expression of nitric oxide synthase, whereas PGE(2) and LTB(4) reduction was due to inhibition of arachidonic acid bioavailability through a direct inhibitory effect of dysodotronic acid on secretory phospholipase A(2).

An anti-inflammatory ditriazine inhibiting leukocyte functions and expression of inducible nitric oxide synthase and cyclo-oxygenase-2.

A ditriazine derivative (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3, 2-d]-1,2,3-ditriazine (DTD)) inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B(4) production, without any effect on 5-lipoxygenase activity. This compound reduced nitric oxide (NO) and prostaglandin E(2) production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase, cyclo-oxygenase-2 or cyclo-oxygenase-1 was observed. DTD significantly reduced mouse paw oedema induced by carrageenan and also markedly reduced NO and prostaglandin E(2) levels in exudates from 24-h zymosan-stimulated mouse air pouch. Western blot analysis showed that DTD reduced the expression of inducible NO synthase and cyclo-oxygenase-2. Our results indicate that DTD exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E(2) production, which could be due to a decreased expression of inducible NO synthase and cyclo-oxygenase-2.

Co-regulation between cyclo-oxygenase-2 and inducible nitric oxide synthase expression in the time-course of murine inflammation.

Many in vitro studies have used cell cultures to focus on the relationships between cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) isoforms. We have investigated the time-course of regulation and the role of COX-2 and iNOS in a model of experimental inflammation in mice, the air pouch injected with zymosan. This study demonstrates that there is an early acute phase (4 h) mediated mainly by eicosanoids, with high levels of prostaglandin E2 (PGE2) produced by cyclo-oxygenase-1. In addition, in the later phase (from 12 h) there is a participation of nitric oxide (NO) and PGE2 accompanied by co-induction of both iNOS and COX-2. These enzymes were detected in migrating leukocytes as well as in macrophages lining the air pouch. Administration of NS398 or indomethacin inhibited PGE2 levels and COX activity, but also nitrite levels and iNOS activity, which was accompanied by a reduction in iNOS expression. Aminoguanidine inhibited nitrite levels and iNOS activity in addition to exerting inhibitory effects on the COX pathway. Treatment of animals with dexamethasone reduced nitrite and PGE2 concentrations in air pouch exudates, as well as iNOS and COX-2 expression in migrating cells. Our results indicate that PGE2 and NO may play in vivo mutual modulatory roles in the inflammatory response caused by zymosan injection into the mouse air pouch, a suitable model to study drugs acting on those pathways.

Cavernolide: a new inhibitor of human sPLA2 sharing unusual chemical features.

The inhibitory effect of cavernolide, a novel C2, terpene lactone isolated from the sponge Fasciospongia cavernosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Cavernolide inhibited human synovial sPLA2 in a concentration-dependent manner with an IC50 value of 8.8 microM. Besides, this compound decreased in the nanomolar range the myeloperoxidase degranulation process using different stimuli. Cavernolide also inhibited TNFalpha, NO and PGE2 production in intact cell experiments. NO and PGE2 reduction was the consequence of the inhibition on iNOS and COX-2 expression because it did not affect inducible nitric oxide synthase and cyclooxygenase-2 activities in intact cells.

Synthesis and pharmacological evaluation of some 8-cyanopyrido[3', 2':4,5]thieno[3,2-d]triazine derivatives as inhibitors of nitric oxide and eicosanoid biosynthesis.

A series of 8-cyanopyrido[3',2':4,5]thieno[3,2-d]-1,2,3-triazines, substituted at C-4 and C-7, were synthesized and evaluated as nitric oxide and prostaglandin E(2) inhibitors in murine peritoneal macrophages stimulated with bacterial endotoxin. Several compounds exhibited considerable activity, compounds 10 and 13 being the most interesting ones with IC(50) values of 11.2 and 3.4 microM on nitrites and 0.9 and 0.6 microM on prostaglandin E(2) production, respectively. None of the examples of pyridothienotriazines that were active at 10 microM showed any effect on inducible nitric oxide synthase, cyclooxygenase-2, and cyclooxygenase-1 enzymes, suggesting that they act by modifiying the level of expression of these inducible enzymes.

Inhibition of human sPLA2 and 5-lipoxygenase activities by two neo-clerodane diterpenoids.

The inhibitory effect of two neo-clerodane diterpenoids, E-isolinaridial (EI) and its methylketone derivative (EIM), isolated from Linaria saxatilis var. glutinosa, on PLA2 and other enzyme activities involved in the inflammatory process was studied. Both compounds inhibited human synovial sPLA2 in a concentration-dependent manner with IC50 values of 0.20 and 0.49 microM, respectively, similar to scalaradial. Besides, these compounds decreased the cell-free 5-lipoxygenase activity and A23187-induced neutrophil LTB4 biosynthesis. Another function of human neutrophils, such as receptor-mediated degranulation, was also significantly reduced. In contrast, none of the compounds affected superoxide generation in leukocytes, or cyclooxygenase-1, cyclooxygenase-2 and inducible nitric oxide synthase activities in cell-free assays.

Manoalide.

Manoalide is a potent analgesic and antiinflammatory sesterterpene isolated in 1980 from a marine sponge. The antiinflammatory activity of manoalide is due to inhibition of PLA2, through irreversible binding to several lysine residues. The binding is realized by means of the two masked aldehyde functions present in the polar part of manoalide. Of the two aldehyde groups, only that present in the g-hydroxybutenolide ring seems to be essential, since cacospongionolides, naturally occurring analogues lacking the second masked aldehyde group, were also shown to be irreversible PLA2 inhibitors. It appears that the minimum structural requirement for exhibiting manoalide-like PLA2 inhibition would be the presence in the inhibitor of functional groups able to seize the amino groups of PLA2 lysine residues with formation of stable covalent bonds. Many manoalide analogues have been isolated from marine sponges, most of them sharing PLA2 inhibitory properties. Other interesting bioactivities have also been reported for some of these compounds.

Release of inflammatory mediators (PGE2, IL-6) by fenofibric acid-photosensitized human keratinocytes and fibroblasts.

Ultraviolet-A radiation has weak effects on the release of inflammatory mediators by skin cells due to the poor overlap between UVA wavelengths and the absorption spectra of the relevant chromophores of key biomolecules. However, this situation could be very different in the presence of a photosensitizing drug. To investigate this issue, we have irradiated human skin cells (keratinocytes and fibroblasts) in the presence of fenofibric acid (the active phototoxic metabolite of fenofibrate). The results of this research show a dual effect on the production/release of inflammatory mediators: the synthesis of the proinflammatory cytokine interleukin-6 becomes strongly inhibited at photosensitizer concentrations that clearly stimulate the production of prostaglandins (PGE2) by skin cells. We have found evidences showing that the de novo synthesis of cytokines is inhibited in photosensitized cells due to the fact that cellular mRNA is degraded. Interestingly, when the medium taken from irradiated cultures is added to nonexposed cells, a significant stimulation of cytokine synthesis is observed that can be inhibited by anti-PGE2 antibodies. These observations may be relevant in vivo, where prostaglandins released by photosensitized skin cells could stimulate cytokine synthesis by underlying, nonirradiated cells.

Suppression of leukotriene B4 and tumour necrosis factor alpha 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 alpha (TNFalpha) 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.

A new cacospongionolide inhibitor of human secretory phospholipase A2 from the Tyrrhenian sponge Fasciospongia cavernosa and absolute configuration of cacospongionolides.

A new inhibitor of human secretory phospholipase A2 (PLA2), cacospongionolide E (4a), has been isolated from the Tyrrhenian sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and by chemical transformations. The absolute configuration of cacospongionolides 2a-4a was established using the modified Mosher's method. Cacospongionolide E was the most potent inhibitor toward human synovial PLA2, showing higher potency than the reference compound manoalide and exerting no signs of toxicity on human neutrophils. It showed high activity in the Artemia salina bioassay and moderate toxicity in the fish (Gambusia affinis) lethality assay.