Small-molecule-induced Rho-inhibition: NSAIDs after spinal cord injury.

Limited axonal plasticity within the central nervous system (CNS) is a major restriction for functional recovery after CNS injury. The small GTPase RhoA is a key molecule of the converging downstream cascade that leads to the inhibition of axonal re-growth. The Rho-pathway integrates growth inhibitory signals derived from extracellular cues, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein, Ephrins and repulsive guidance molecule-A, into the damaged axon. Consequently, the activation of RhoA results in growth cone collapse and finally outgrowth failure. In turn, the inhibition of RhoA-activation blinds the injured axon to its growth inhibitory environment resulting in enhanced axonal sprouting and plasticity. This has been demonstrated in various CNS-injury models for direct RhoA-inhibition and for downstream/upstream blockade of the RhoA-associated pathway. In addition, RhoA-inhibition reduces apoptotic cell death and secondary damage and improves locomotor recovery in clinically relevant models after experimental spinal cord injury (SCI). Unexpectedly, a subset of "small molecules" from the group of non-steroid anti-inflammatory drugs, particularly the FDA-approved ibuprofen, has recently been identified as (1) inhibiting RhoA-activation, (2) enhancing axonal sprouting/regeneration, (3) protecting "tissue at risk" (neuroprotection) and (4) improving motor recovery confined to realistic therapeutical time-frames in clinically relevant SCI models. Here, we survey the effect of small-molecule-induced RhoA-inhibition on axonal plasticity and neurofunctional outcome in CNS injury paradigms. Furthermore, we discuss the body of preclinical evidence for a possible clinical translation with a focus on ibuprofen and illustrate putative risks and benefits for the treatment of acute SCI.

In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and Salvia sahendica.

Phytochemical investigations of the n-hexane extract from the roots of Peltodon longipes (Lamiaceae) resulted in the isolation of 12 known abietane diterpenes (1-12). Structures were established on the basis of one and two dimensional nuclear magnetic resonance spectroscopic data ((1)H and (13)C, COSY, HSQC and HMBC), electron ionization mass spectrometric analysis (EIMS) as well as comparison with data from literature. These compounds, as well as eight known diterpenes (13-19) from Salvia miltiorrhiza, and two from Salvia sahendica (20 and 21) were evaluated for their cytotoxic effects in human pancreatic (MIAPaCa-2) and melanoma (MV-3) tumor cell lines using the MTT assay. Tanshinone IIa (13), 7α-acetoxyroyleanone (1), 1,2-dihydrotanshinone (16) and cryptotanshinone (14) had the highest cytotoxic effects in MIAPaCa-2, displaying IC(50) of 1.9, 4.7, 5.6, and 5.8 µM, respectively. Structure-activity relationships of abietane diterpenoid quinones are discussed.

Pharmacophore design of p38α MAP kinase inhibitors with either 2,4,5-trisubstituted or 1,2,4,5-tetrasubstituted imidazole scaffold.

Synthetic compounds with a tri- and tetra-substituted imidazole scaffold are known as selective inhibitors of the p38 mitogen-activated protein (MAP) kinase responsible for proinflammatory cytokine release. The scope is to review the literature describing their design, synthesis and activity studies. To date a great plethora of crystal structures of p38 in complex with small organic ligands have been published. Cocrystallized ligand information is of particular interest to our review study, i.e. ATP itself, the reference inhibitor SB203580 with its aryl-pyridinyl-imidazoles and related imidazole and pyrimidine-based derivatives. The selective inhibitors bind to the pocket of adenosine 5'-triphoshate (ATP) replacing the latter. The hydrophobic region II, however, is not occupied by the natural binder ATP, but accommodates the pyridine substituents preserving the 4-fluorophenyl ring occupation in pocket I as a prerequisite to gain higher binding selectivity and potency than the reference compound SB203580 (4-[5-(4-fluoro-phenyl)-2-(4-methanesulfinyl-phenyl)-3himidazol-4-yl]-pyridine). Experimental and computed work is reviewed which evidence that the 2 position of the pyrimidine ring is amenable to the introduction of a side chain and the replacement of pyridine in SB203580 by a pyrimidine ring improves both inhibitory activity and selectivity for p38 over other kinases. All ligands with a pyridyl C2 side chain occupy the hydrophobic pocket II and in some cases a double hydrogen bond is reported between methionine 109 and glycine 110 of the hinge region, following an observed backbone shift. The substituted pyridine ring binds stronger than the two other side chains on the imidazole scaffold.

In vivo non-thermal irreversible electroporation impact on rat liver galvanic apparent internal resistance.

Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon which involves application of electric field pulses to cells or tissues, causing certain rearrangements in the membrane structure leading to cell death. The treated tissue ac impedance changes induced by electroporation were shown to be the indicators for NTIRE efficiency. In a previous study we characterized in vitro tissue galvanic apparent internal resistance (GAIR) changes due to NTIRE. Here we describe an in vivo study in which we monitored the GAIR changes of a rat liver treated by NTIRE. Electrical pulses were delivered through the same Zn/Cu electrodes by which GAIR was measured. GAIR was measured before and for 3 h after the treatment at 15 min intervals. The results were compared to the established ac bioimpedance measurement method. A decrease of 33% was measured immediately after the NTIRE treatment and a 40% decrease was measured after 3 h in GAIR values; in the same time 40% and 47% decrease respectively were measured by ac bioimpedance analyses. The temperature increase due to the NTIRE was only 0.5 °C. The results open the way for an inexpensive, self-powered in vivo real-time NTIRE effectiveness measurement.

Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

BACKGROUND AND PURPOSE: A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors. EXPERIMENTAL APPROACH: Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors Birb 796, pamapimod, SB203580 and the new substance CBS-3868. Birb 796- and SB203580-treated cells were analysed in a genome-wide microarray analysis. The efficacy of all inhibitors was characterized by quantitative gene expression analysis and the quantification of PGE(2) and NO release. KEY RESULTS: Microarray analysis revealed inhibitor-specific differences in gene expression. Whereas SB203580 had a broad effect on chondrocytes, Birb 796 counteracted the IL-1beta effect more specifically. All p38MAPK inhibitors significantly inhibited the IL-1beta-induced gene expression of COX-2, mPGES1, iNOS, matrix metalloproteinase 13 (MMP13) and TNFRSF11B, as well as PGE(2) release. Birb 796 and CBS-3868 showed a higher efficacy than SB203580 and pamapimod at inhibiting the expression of COX-2 and MMP13 genes, as well as PGE(2) release. In the case of mPGES1 and TNFRSF11B gene expression, CBS-3868 exceeded the efficacy of Birb 796. CONCLUSIONS AND IMPLICATIONS: Our test system could differentially characterize inhibitors of the same primary pharmaceutical target. It reflects processes relevant in OA and is based on chondrocytes that are mainly responsible for cartilage degradation. It therefore represents a valuable tool for drug screening in between functional in vitro testing and in vivo models.

Identification of rosmarinic acid as the major active constituent in Cordia americana.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparation from leaves of Cordia americana have been widely used in traditional medicine in South Brazil to treat wounds and various inflammations. AIM OF THE STUDY: The objective of this work was to identify the effective compounds in the ethanolic extract prepared from the leaves of Cordia americana, which is used in traditional South Brazilian medicine as anti-inflammatory and wound healing remedy. MATERIALS AND METHODS: Isolation and structure elucidation techniques were performed in order to identify the compounds of Cordia americana and HPLC analysis was used for the quantification. The major constituent and the ethanolic extract were investigated for inhibition of 5-lipoxygenase, p38alpha MAPK, TNFalpha release and NF-kappaB as well as in the fibroblast scratch assay. RESULTS: Rosmarinic acid (1) was identified as the major compound with an amount of 8.44% in the ethanolic extract of the leaves of Cordia americana. The ethanolic extract as well as (1) exhibited the highest inhibitory effects on 5-lipoxygenase (IC(50)=0.69 and 0.97microg/mL, resp., IC50 of BWA4C as reference: 0.3microM) and p38alpha (IC50=3.25 and 1.16microg/mL, resp., IC50 of SB203580 as reference: 0.046microM) and moderate inhibitory effects on TNFalpha release. Slight effects were observed in the fibroblast scratch assay. CONCLUSIONS: This study increases our knowledge on the effective compound in Cordia americana and supports its use in traditional medicine. We demonstrated for the first time pharmacological effects of Cordia americana and we provide evidences for a crucial role of rosmarinic acid as the major key player.

Participation of leukotriene C(4) in the regulation of suicidal erythrocyte death.

Eryptosis, the suicidal death of erythrocytes, is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase in cytosolic Ca(2+) concentration upon energy depletion. The present study explored the involvement of leukotrienes. Western blotting was employed to detect the cysteinyl-leukotriene receptor cysLT1, competitive immune assay to determine leukotriene release from erythrocytes, Fluo3 fluorescence to estimate cytosolic Ca(2+) concentration, forward scatter to analyse cell volume and annexin V-binding to disclose phosphatidylserine exposure. As a result, erythrocytes expressed the leukotriene receptor CysLT1. Glucose depletion (24 hours) significantly increased the formation of the cysteinyl-leukotrienes C(4)/D(4)/E(4). Leukotriene C(4) (10 nM) increased Ca(2+) entry, decreased forward scatter, activated caspases 3 and 8, and stimulated annexin V-binding. Glucose depletion similarly increased annexin V-binding, an effect significantly blunted in the presence of the leukotriene receptor antagonist cinalukast (1 microM) or the 5-lipoxygenase inhibitor BW B70C (1 microM). In conclusion, upon energy depletion erythrocytes form leukotrienes, which in turn activate cation channels, leading to Ca(2+) entry, cell shrinkage and cell membrane scrambling. Cysteinyl-leukotrienes thus participate in the signaling of eryptosis during energy depletion.

Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts.

UNLABELLED: PHARMACOLOGICAL RELEVANCE: Presentation of the scratch assay as a convenient and inexpensive in vitro tool to gain first insights in the wound healing potential of plant extracts and natural compounds. AIM OF THE STUDY: The present study deals with the optimization of the scratch assay which can be used as an in vitro model for quantification of fibroblast migration to and proliferation into the wounded area. It is suitable for the first evaluation of the wound re-epithelialization potential of crude herbal extracts, isolated compounds and pharmaceutical preparations. As a proof of concept three preparations from traditional medicinal plants were investigated. MATERIALS AND METHODS: Swiss 3T3 albino mouse fibroblasts were used in monolayers and platelet derived growth factor as positive control. Hexane and ethanolic extracts from Calendula officinalis and Matricaria recutita, Hypericum oil as well as the triterpenoids faradiol myristate and palmitate were studied. To differentiate between proliferation and migration antimitotic mitomycin C was added. RESULTS: Both extracts of Calendula officinalis stimulated proliferation and migration of fibroblasts at low concentrations, e.g. 10 microg/ml enhanced cell numbers by 64.35% and 70.53%, respectively. Inhibition of proliferation showed that this effect is mainly due to stimulation of migration. Faradiol myristate and palmitate gave comparable stimulation rates at an almost 50 microg/ml concentration, indicating that they contribute partially, but not most significantly to the wound healing effects of Calendula preparations. Extracts from Matricaria recutita were only moderately active. Hypericum oil was cytotoxic at concentrations higher than 0.5 microg/ml. CONCLUSIONS: The scratch assay in the present form can be used as a promising scientific approach and platform to differentiate between plant extracts known for their wound healing and their anti-inflammatory properties.

Biological studies on Brazilian plants used in wound healing.

AIM OF THE STUDY: n-Hexanic and ethanolic extracts from twelve plants (Brugmansia suaveolens Brecht. et Presl., Eupatorium laevigatum Lam., Galinsoga parviflora Cav., Iresine herbstii Hook., Kalanchöe tubiflora Hamet-Ahti, Petiveria alliacea L., Pluchea sagittalis (Lam.) Cabrera, Piper regnellii DC., Schinus molle L., Sedum dendroideum Moç et Sessé ex DC., Waltheria douradinha St. Hill., Xanthium cavanillesii Schouw.) used in traditional South Brazilian medicine as wound healing agents were investigated in various biological assays, targeting different aspects in this complex process. MATERIALS AND METHODS: The extracts were investigated on NF-kappaB DNA binding, p38alpha MAPK, TNF-alpha release, direct elastase inhibition and its release as well as on caspase-3. Fibroblasts migration to and proliferation into the wounded monolayers were evaluated in the scratch assay, the agar diffusion test for antibacterial and the MTT assay for cytotoxic effects. RESULTS: The hydrophilic extracts from Galinsoga parviflora, Petiveria alliacea, Schinus molle, Waltheria douradinha and Xanthium cavanillesii as well as the lipophilic extract of Waltheria douradinha turned out to be the most active ones. CONCLUSIONS: These results increase our knowledge on the wound healing effects of the investigated medicinal plants. Further studies are necessary to find out the effective secondary metabolites responsible for the observed effects.

Protective effects of licofelone, a 5-lipoxygenase and cyclo-oxygenase inhibitor, versus naproxen on cartilage loss in knee osteoarthritis: a first multicentre clinical trial using quantitative MRI.

OBJECTIVE: In a multicentre study to explore the effects of licofelone as a disease-modifying osteoarthritis drug in comparison with naproxen in patients with knee osteoarthritis (OA), using MRI and x-ray examination. METHODS: Patients with knee OA (n = 355) were randomised to receive either licofelone (200 mg twice a day) or naproxen (500 mg twice a day). MRI and x-ray examinations were performed at baseline, 6 months (MRI only), 12 and 24 months. MRI was used to assess quantitatively changes in cartilage volume, and x-ray examinations (Lyon-Schuss) to measure changes in the mean and minimum joint space width (JSW) in the medial compartment. Questionnaires probing symptoms were completed. Data were presented as intention to treat (ITT) and according to protocol (ATP). RESULTS: Cartilage volume loss in the global joint and medial and lateral compartments was significantly less in the licofelone than in the naproxen group for ITT at 12 and 24 months and for ATP at all times except in the medial compartment. Patients with medial meniscal extrusion had a greater loss of cartilage volume. In these patients, licofelone markedly reduced the cartilage loss for both ITT and ATP at 12 and 24 months. Although licofelone showed less reduction in the JSW than naproxen, this did not reach significance. All clinical variables were improved at 24 months (p<0.001) for both groups, with a good safety profile. CONCLUSION: Licofelone and naproxen were equally effective in reducing OA symptoms; however, licofelone significantly reduced cartilage volume loss over time, thus having a protective effect in patients with knee OA. This study proves the superiority of quantitative MRI over x-ray examinations in a multicentre clinical trial.

Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems.

Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments.

Human whole blood assay for rapid and routine testing of non-steroidal anti-inflammatory drugs (NSAIDs) on cyclo-oxygenase-2 activity.

Aim of this study was to present a simple, fast and reliable method to examine the capacity of NSAIDs to inhibiting COX-2 activity that uses rapid (stimulation takes only 5 h compared to other existing protocols) and routine testing. The assay includes elimination of COX-1-activity using ASS (a selective COX-1 inhibitor) and the thromboxane synthetase inhibitor (TXBSI), COX-2 induction via LPS and measurement of PGE(2). Using TXBSI reduces the amount of LPS and results in higher prostaglandin production. Cremophor EL-EtOH was used as vehicle instead of DMSO because within a defined concentration range, Cremophor EL-EtOH allows even very hydrophobic drugs to be solubilized and applied in vitro without cell damage. Cremophor EL-EtOH at 0.2% was optimal as at this relatively low concentration excellent drug dissolution was obtained whereas many hydrophobic substances precipitate in 0.2% DMSO. Our results demonstrate that the IC(50) values for the tested NSAIDs are in the range of published data.

The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands.

OBJECTIVE: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway. METHODS: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot. RESULTS: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein. CONCLUSIONS: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

In vitro metabolite identification of ML3403, a 4-pyridinylimidazole-type p38 MAP kinase inhibitor by LC-Qq-TOF-MS and LC-SPE-cryo-NMR/MS.

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4- [5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl -(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600 MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.

Antioxidative properties of the gastrointestinal phytopharmaceutical remedy STW 5 (Iberogast).

Since inflammation is a common mechanism of many gastrointestinal diseases, reactive oxygen metabolites may play an important role in their pathophysiology. Therefore it is interesting to know, whether phytopharmaceuticals known to modulate gastrointestinal motor function reveal also antioxidative properties. We tested STW 5 (Iberogast), its constituent nine different plant extracts, and some isolated compounds which are present in STW 5 for characterizing their antioxidative and radical quenching activities. The test assays consisted in pure chemical and complex cellular systems in which different types of reactive species were produced. Quantification of the effects was based on chemiluminescence reactions. The results show that all extracts contribute to the effect of the complete remedy STW 5, in the chemical systems in a strongly additive manner, in the cellular systems in a supraadditive manner. The largest contributions resulted from the extracts from peppermint and melissa leaves. Comparison of effects from isolated phytochemical compounds from the extracts with that of the extracts itself shows that usually the extract is more effective than the monosubstance which indicates also the synergism within the whole plant extracts. This means that the plant extracts present in STW 5 provide strong radical quenching activities that could also be involved in the therapeutic gastrointestinal actions.

High anti-inflammatory activity of harpagoside-enriched extracts obtained from solvent-modified super- and subcritical carbon dioxide extractions of the roots of Harpagophytum procumbens.

Solvent-modified carbon dioxide extractions of the roots of Harpagophytum procumbens have been investigated with respect to extraction efficiency and content of harpagoside, and compared with a conventional extract. The effects of pressure, temperature, type and concentration of the modifier have been examined. Two extraction steps were necessary in order to achievehigh anti-inflammatory harpagoside-enriched extracts. The first extraction step was carried out in the supercritical state using carbon dioxide modified with n-propanol to remove undesired lipophilic substances. The main extraction was performed either in the supercritical or in the subcritical state with carbon dioxide modified with ethanol. The supercritical fluid extraction resulted in extracts containing up to 30% harpagoside. The subcritical extracts showed a harpagoside content of ca. 20%, but the extraction yield was nearly three times greater compared with supercritical conditions. The total harpagoside recovery resulting from the sum of the extract and the crude drug residue was greater than 99% in all experiments. The conventional extract and two carbon dioxide extracts were tested for in-vitro inhibition of 5-lipoxygenase or cyclooxygenase-2 biosynthesis. Both carbon dioxide extracts showed total inhibition on 5-lipoxygenase biosynthesis at a concentration of 51.8 mg/L. In contrast, the conventional extract failed to show any inhibition of 5-lipoxygenase biosynthesis.

The effect of single-dose naproxen on eicosanoid formation in human gastroduodenal mucosa.

BACKGROUND: In animal studies, aspirin and non-aspirin non-steroidal anti-inflammatory drugs contribute to gastroduodenal damage via cyclo-oxygenase inhibition and consecutive leucotriene formation (COX-LOX eicosanoid shunt). AIM AND METHODS: Ten Helicobacter pylori-negative healthy volunteers received a single dose of 500 mg naproxen to address two questions: (i) is there a crosstalk between eicosanoids before medication in the human gastroduodenal mucosa and (ii) can we demonstrate a COX-LOX shunt following single-dose naproxen? RESULTS: Significant correlations in the stomach mucosa before medication were obtained between leucotriene B4 (LTB4) and thromboxane B(2) (TxB(2); r = -0.38, P = 0.05), as well as LTB4 and prostaglandin E(2) (PGE(2); r = 0.71, P < 0.0001). In serum, a >90% inhibition of TxB(2) and PGE(2) occurred within 30 min of naproxen administration. In gastric mucosa, a significant decrease of TxB(2) occurred already at 15 min and preferably in the antrum. For LTB4 there was a non-significant trend towards a transient increase. Mucosal PGE(2) was unchanged in all regions; transcript levels of both cyclo-oxygenases/5-lipoxygenase were unaffected (except for a trend of increasing cyclo-oxygenase-2 in the corpus). CONCLUSIONS: Baseline correlations between LTB4-TxB(2) and LTB4-PGE(2) reflect a crosstalk between these eicosanoids. A COX-LOX shunt; however, cannot be demonstrated following single-dose naproxen in a low-risk population.

Licofelone, a novel 5-LOX/COX-inhibitor, attenuates leukocyte rolling and adhesion on endothelium under flow.

The main mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of cycloxygenases COX-1 and COX-2. During recent years, combined 5-LOX/COX-inhibition, interfering with the biosynthesis of both prostaglandins and leukotrienes (LTs), has emerged as a possibility to avoid side effects related to COX-inhibition. The aim of the present study was to investigate if there is a contribution of mechanisms other than the reduction of inflammatory prostaglandins and leukotrienes to the anti-inflammatory effect of the LOX/COX inhibitor licofelone. In a flow chamber assay, licofelone (10-30 microM) dose-dependently decreased both the rolling and adhesion of leukocytes on endothelial cells (EC). In contrast, no effects were found after treatment of EC with the unselective COX-1/COX-2 inhibitor indomethacin (30 microM), the potent and selective 5-LOX inhibitor, ZD-2138 (30 microM), the mainly COX-2 inhibitor aceclofenac (30 microM), the selective COX-2 inhibitor celecoxib (30 microM) and the combination of ZD-2138 with the selective COX-2 inhibitor celecoxib (30 microM). In the presence of licofelone (30 microM) the expression of E-selectin mRNA in cytokine-stimulated EC was attenuated, whereas no NSAID (30 microM) tested showed any effect on E-selectin expression. Moreover, licofelone treatment (30 microM) attenuated expression of VCAM-1 and ICAM-1 on inflammatory EC. The effect of licofelone on leukocyte recruitment was also evaluated in vivo. Using a mouse peritonitis model it was found that leukocyte accumulation was markedly reduced in licofelone treated animals (100mg/kg) compared to untreated mice. Thus, the novel 5-LOX/COX inhibitor licofelone possesses anti-inflammatory activity that, in addition to COX/LOX inhibition, involves effects on leukocyte-endothelial interactions.