Purine Metabolism and Clinical Status of Patients with Rheumatoid Arthritis Treated with Dipyridamole

The anti-inflammatory activities of methotrexate and sulphasalazine may be mediated by increases in endogenous adenosine levels. Since the vascular protective drug dipyridamole inhibits the uptake and metabolism of adenosine we have now tested this compound in patients with rheumatoid arthritis to assess its effects on their symptoms. Forty patients (aged 18–75 years) received dipyridamole 400 mg/day or placebo. The levels of adenosine and its major metabolites were determined by high performance liquid chromatography (HPLC) in blood samples taken at baseline and at monthly intervals during treatment for 6 months. After three months of treatment there was a significant reduction in the modified Health Assessment Questionnaire (mHAQ) score, but these effects were not maintained, and dipyridamole did not modify disease severity scores or the levels of adenosine and its metabolites. We conclude that the symptoms of rheumatoid arthritis were not modified by treatment with dipyridamole.     

Effects of PDE4 inhibitors on lipopolysaccharide-induced priming of superoxide anion production from human mononuclear cells.

AIMS: Phosphodiesterase 4 (PDE4) inhibitors have been described as potent anti-inflammatory compounds, involving an increase in intracellular levels of cyclic 3'',5''-adenosine monophosphate (AMP). The aim of this study was to compare the effects of selective PDE4 inhibitors, rolipram and RP 73-401 with the cell permeable analogue of cyclic AMP, dibutyryl-cyclic AMP (db-cAMP) and the anti-inflammatory cytokine interleukin-10 (IL-10) on superoxide anion production from peripheral blood mononuclear cells preincubated with lipopolysaccharide (LPS). MAJOR FINDINGS: We report that, after incubation of the cells with LPS, a large increase in superoxide anion production was observed. Rolipram or RP 73-401 (10(-8) to 10(-5) M) induced significant reductions of fMLP-induced superoxide anion production in cells incubated with or without LPS. The db-cAMP (10(-5) to 10(-3) M) also elicited dose-dependent inhibitions of the fMLP-induced superoxide anion production. In contrast, IL-10 (1 or 10 ng/ml) did not elicit a reduction in fMLP-induced superoxide anion production in both conditions. PRINCIPAL CONCLUSION: These results suggest that the inhibitory activity of PDE4 inhibitors on fMLP-induced production of superoxide anion production is mediated by db-cAMP rather than IL-10.     

Purine metabolism and clinical status of patients with rheumatoid arthritis treated with dipyridamole

The anti-inflammatory activities of methotrexate and sulphasalazine may be mediated by increases in endogenous adenosine levels. Since the vascular protective drug dipyridamole inhibits the uptake and metabolism of adenosine we have now tested this compound in patients with rheumatoid arthritis to assess its effects on their symptoms. Forty patients (aged 18-75 years) received dipyridamole 400 mg/day or placebo. The levels of adenosine and its major metabolites were determined by high performance liquid chromatography (HPLC) in blood samples taken at baseline and at monthly intervals during treatment for 6 months. After three months of treatment there was a significant reduction in the modified Health Assessment Questionnaire (mHAQ) score, but these effects were not maintained, and dipyridamole did not modify disease severity scores or the levels of adenosine and its metabolites. We conclude that the symptoms of rheumatoid arthritis were not modified by treatment with dipyridamole.     

Is Nitric Oxide Decrease Observed with Naphthoquinones in LPS Stimulated RAW 264.7 Macrophages a Beneficial Property?

The search of new anti-inflammatory drugs has been a current preoccupation, due to the need of effective drugs, with less adverse reactions than those used nowadays. Several naphthoquinones (plumbagin, naphthazarin, juglone, menadione, diosquinone and 1,4-naphthoquinone), plus p-hydroquinone and p-benzoquinone were evaluated for their ability to cause a reduction of nitric oxide (NO) production, when RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS). Dexamethasone was used as positive control. Among the tested compounds, diosquinone was the only one that caused a NO reduction with statistical importance and without cytotoxicity: an IC(25) of 1.09±0.24 μM was found, with 38.25±6.50% (p<0.001) NO reduction at 1.5 μM. In order to elucidate if this NO decrease resulted from the interference of diosquinone with cellular defence mechanisms against LPS or to its conversion into peroxynitrite, by reaction with superoxide radical formed by naphthoquinones redox cycling, 3-nitrotyrosine and superoxide determination was also performed. None of these parameters showed significant changes relative to control. Furthermore, diosquinone caused a decrease in the pro-inflammatory cytokines: tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Therefore, according to the results obtained, diosquinone, studied for its anti-inflammatory potential for the first time herein, has beneficial effects in inflammation control. This study enlightens the mechanisms of action of naphthoquinones in inflammatory models, by checking for the first time the contribution of oxidative stress generated by naphthoquinones to NO reduction.     

Treatment of Rheumatoid Arthritis with Fenoprofen: Comparison with Aspirin

Fenoprofen, a compound with analgesic, anti-inflammatory, and antipyretic properties in animals, has been compared with placebo in a double-blind cross-over trial in 60 patients with rheumatoid arthritis. There was a statistically highly significant reduction in pain, duration of morning stiffness, analgesic requirements, and articular index, with increase in grip strength. There was no significant reduction in joint size or temperature. In a subsequent double-blind group-comparative study fenoprofen proved to be as effective as aspirin in relieving the symptoms of rheumatoid arthritis, with strikingly fewer side effects. Almost half of the patients taking aspirin were unable to tolerate the drug in adequate dosage for six months. The remainder were able to take on average only 4 g daily, and at this dose almost half still complained of tinnitus and deafness.Fenoprofen is likely to be useful for patients who cannot tolerate aspirin and other more toxic anti-inflammatory drugs or whose disease is not of sufficient severity to justify their use.     

Trikatu,an herbal compound as immunomodulatory and anti-inflammatory agent in the treatment of rheumatoid arthritis – An experimental study

In the present study, trikatu, an herbal compound was evaluated for its immunomodulatory and anti-inflammatory properties with reference to cell mediated immune responses (delayed type hypersensitivity reaction), humoral immune response (haemagglutination titer and plaque forming assay), macrophage phagocytic index, circulating immune complex and inflammatory mediators in rats. For comparison purposes, indomethacin was used as a reference drug for anti-inflammatory studies. The results obtained in our study showed a significant decrease in cell mediated immune responses, humoral immune responses (haemagglutination titre and plaque forming assay) and macrophage phagocytic index in trikatu treated rats (1000 mg/kg/b.wt.) compared to control animals implying its immunosuppressive property. In addition, significant anti-inflammatory effects were observed in trikatu treated adjuvant induced arthritic rats by a reduction in the levels of circulating immune complexes and inflammatory mediators (TNF-alpha and Interleukin-1beta). Thus, in conclusion, our data suggest that trikatu could be considered as a potential anti-inflammatory agent for treating autoimmune inflammatory disorders like rheumatoid arthritis with immunosuppressive property.     

IL-16 as an anti-inflammatory cytokine in rheumatoid synovitis

T lymphocytes are a major component of the inflammatory infiltrate in rheumatoid synovitis, but their exact role in the disease process is not understood. Functional activities of synovial T cells were examined by adoptive transfer experiments in human synovium-SCID mouse chimeras. Adoptive transfer of tissue-derived autologous CD8+ T cells induced a marked reduction in the activity of lesional T cells and macrophages. Injection of CD8+, but not CD4+, T cells decreased the production of tissue IFN-gamma, IL-1beta, and TNF-alpha by >90%. The down-regulatory effect of adoptively transferred CD8+ T cells was not associated with depletion of synovial CD3+ T cells or synovial CD68+ macrophages, and it could be blocked by Abs against IL-16, a CD8+ T cell-derived cytokine. In the synovial tissue, CD8+ T cells were the major source of IL-16, a natural ligand of the CD4 molecule that can anergize CD4-expressing cells. The anti-inflammatory activity of IL-16 in rheumatoid synovitis was confirmed by treating synovium-SCID mouse chimeras with IL-16. Therapy for 14 days with recombinant human IL-16 significantly inhibited the production of IFN-gamma, IL-1beta, and TNF-alpha in the synovium. We propose that tissue-infiltrating CD8+ T cells in rheumatoid synovitis have anti-inflammatory activity that is at least partially mediated by the release of IL-16. Spontaneous production of IL-16 in synovial lesions impairs the functional activity of CD4+ T cells but is insufficient to completely abrogate their stimulation. Supplemental therapy with IL-16 may be a novel and effective treatment for rheumatoid arthritis.     

Mechanisms of the anti-inflammatory actions of the angiotensin type 1 receptor antagonist losartan in experimental models of arthritis

Angiotensin (Ang) II and its AT₁ receptors have been implicated in the pathogenesis of rheumatoid arthritis. Activation of the counter-regulatory Ang-(1–7)–Mas receptor axis may contribute to some of the effects of AT₁ receptor blockers (ARBs). In this study, we have used losartan, an ARB, to investigate the role of and the mechanisms by which AT₁ receptors participated in two experimental models of arthritis: antigen-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Treatment with losartan decreased neutrophil recruitment, hypernociception and the production of TNF-α, IL-1β and chemokine (C–X–C motif) ligand 1 in mice subjected to AIA. Histopathological analysis showed significant reduction of tissue injury and inflammation and decreased proteoglycan loss. In addition to decreasing cytokine production, losartan directly reduced leukocyte rolling and adhesion. Anti-inflammatory effects of losartan were not associated to Mas receptor activation and/or Ang-(1–7) production. Anti-inflammatory effects were reproduced in rats subjected to AdIA. This study shows that ARBs have potent anti-inflammatory effects in animal models of arthritis. Mechanistically, reduction of leukocyte accumulation and of joint damage was associated with local inhibition of cytokine production and direct inhibition of leukocyte–endothelium interactions. The anti-inflammatory actions of losartan were accompanied by functional improvement of the joint, as seen by reduced joint hypernociception. These findings support the use of ARBs for the treatment of human arthritis and provide potential mechanisms for the anti-inflammatory actions of these compounds.     

Inhibition of oxidative hemolysis by quercetin,but not other antioxidants

We previously reported that lipid-soluble quercetin, not water-soluble dihydroquercetin, protects human red blood cells against oxidative damage. The objectives of this study were to determine if an antihemolytic effect could be produced by other lipid-soluble antioxidants and if anti-inflammatory activity played a role in antihemolysis by quercetin. This study compared three lipid-soluble polyphenols, muscadine, curcumin and quercetin, and three lipid- (α-tocopherol and α-tocotrienol) or water-soluble (ascorbic acid) vitamins. Among the tested polyphenols, muscadine was the most potent in inhibiting superoxide and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-generated peroxyl radicals, whereas ascorbic acid was the most potent inhibitor of hydrogen peroxide. Activities of the polyphenols after lipid extractions showed that curcumin inhibited superoxide production to a greater extent than quercetin and muscadine. All blood cells were tested 20 min after incubation with the selected compounds. All the polyphenols caused inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced neutrophil oxidative bursts. Quercetin, but not other polyphenols, significantly reduced AAPH-induced oxidative hemolysis. No significant effect on neutrophil oxidative burst or oxidative hemolysis was found with any of the tested vitamins. These results suggest that quercetin enhances the resistance of membrane to destruction by free radicals. This effect of quercetin is not directly mediated through antioxidative or anti-inflammatory actions. Antioxidant or anti-inflammatory potency may not be used as a simple criterion to select polyphenols for cell protection benefits.     

Total body calcium in rheumatoid arthritis: effects of disease activity and corticosteroid treatment.

Rheumatoid arthritis may be associated with generalised as well as periarticular osteoporosis. To assess the extent of bone loss and the influence of corticosteroid treatment total body calcium was measured by in-vivo neutron activation analysis in 63 patients with rheumatoid arthritis treated with non-steroidal anti-inflammatory drugs alone and 31 treated with additional low-dose corticosteroids. The results were compared with those in 40 normal controls matched for age, sex, and menopausal state. There were significant reductions in mean total body calcium in the group treated with non-steroidal anti-inflammatory drugs (5.3% in men; 6.8% in women) and greater reductions in the corticosteroid-treated patients (11.5% in men, 15.5% in women). The reduction was correlated with disease duration and activity in the patients treated with non-steroid anti-inflammatory drugs alone. Measured total body calcium was significantly less than the values predicted when this relation was used in the corticosteroid-treated patients. The data suggest that increased bone loss in patients with rheumatoid arthritis treated with corticosteroids is attributable to drug treatment rather than disease activity. Many patients with rheumatoid arthritis treated with low-dosage corticosteroids and some postmenopausal women with the disease are likely to be at risk from the complications of osteoporosis.     

Study on the effect of superoxide dismutase on arachidonic acid metabolism

The effect of orgotein, the drug version of bovine Cu-Zn superoxide dismutase, on PG production by phagocytosing leukocytes has been investigated. Orgotein inhibited PG formation in a dose/dependent manner. Arachidonic acid was able to reverse this inhibitory effect. In the light of these results it is suggested that anti-inflammatory properties of orgotein may depend, at least in part, on the inhibition of phospholipase activation.     

Synthesis and anti-inflammatory structure-activity relationships of thiazine-quinoline-quinones: inhibitors of the neutrophil respiratory burst in a model of acute gouty arthritis

Sixteen new thiazine-quinoline-quinones have been synthesised, plus one bicyclic analogue. These compounds inhibited neutrophil superoxide production in vitro with IC(50)s as low 60 nM. Compounds with high in vitro anti-inflammatory activity were also tested in a mouse model of acute inflammation. The most active compounds inhibited both neutrophil infiltration and superoxide production at doses 2.5 micromol/kg, highlighting their potential for development as novel NSAIDs.     

Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress,Nitric Oxide and Cytokine Production

Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation.     

Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.     

Therapeutic Trial of a Pyrazole Derivative,KB-95, for Patients with Rheumatoid Arthritis

A phenylbutazone-like pyrazole derivative, Sandoz KB-95, claimed to have antiserotonin, anti-inflammatory and analgesic properties, was tested on patients with rheumatoid arthritis. A double-blind study on 17 patients with definite or classical rheumatoid arthritis was performed for two-week periods on both KB-95 and placebo. Grip strength, the time to walk 50 feet, the number of other analgesic tablets taken daily, the erythrocyte sedimentation rate and a subjective rating of pain were the parameters of measurement. KB-95 failed to produce significantly more improvement than the placebo in this trial and was therefore considered not to be a useful drug for the treatment of rheumatoid arthritis. No acute toxic effects were observed to this dose of 300 mg. given three times daily.     

Antioxidant and anti-inflammatory effects of products from Croton celtidifolius Bailon on carrageenan-induced pleurisy in rats

Croton celtidifolius Bailon, commonly known as Sangue-de-Adáve or Pau-Andrade, is a tree found in the Atlantic forest of southern Brazil. It has been popularly used for the treatment of inflammatory and ulcerative disorders. Phytochemical analysis demonstrated the presence of flavonoids and proanthocyanidins in an ethyl acetate fraction (EAF) from C. celtidifolius Bailon. In this study, we have evaluated the effects of EAF and its sub-fractions (35 and 63, catechin) on inflammatory (cell migration and plasma extravasation) and oxidative (lipid peroxidation, superoxide dismutase (SOD) activity and superoxide anion production) parameters in carrageenan-induced pleurisy in rats. NO production was also measured by nitrite/nitrate levels. EAF and sub-fraction 63 (63SF) showed anti-inflammatory activity, as indicated by a reduction in plasma extravasation and cell migration (mainly polymorphonuclear leukocytes) to the pleural cavity. Furthermore, EAF treatment decreased the production of superoxide radical anion by cells isolated from the pleural cavity, while it did not affect the nitrite/nitrate levels in exudates. The results show that C. celtidifolius contains substances with antioxidant and anti-inflammatory activity that, at least in part, act by a modulation of oxidative stress by phenolic compounds.     

4-Hydroxy-oxyphenbutazone is a potent inhibitor of cytokine production

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.     

Enhanced Mitochondrial Radical Production in Patients with Rheumatoid Arthritis Correlates with Elevated Levels of Tumor Necrosis Factor alpha in Plasma

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increase in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p<0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.     

Nebulization of Liposomal rh-Cu/Zn–SOD with a Novel Vibrating Membrane Nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.