Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Some pharmacological properties of tiopronin

Tiopronin (50 mg/kg) andd-penicillamine (50 mg/kg) do not exhibit anti-inflammatory effects in classic animal models (carragenin oedema, granuloma cotton pellets) but suppress pertussis vaccine oedema, an immunological model, when given with a long-lasting dosing regime.Tiopronin andd-penicillamine also fail to inhibit PG release by phagocytosing leucocytes when the concentrations used were in the same range as human blood levels (5–15 g/ml).Indomethacin (1, 3 and 5 mg/kg) instead significantly inhibits both thein vivo andin vitro models considered.This may imply a different mode of action of both tiopronin andd-penicillamine from indomethacin. Tiopronin also possesses similar effects tod-penicillamine suggesting that their overall anti-rheumatic action may have common elements.     

Comparison of in vitro effects of flunixin and tolfenamic acid on human leukocyte and platelet functions

A study was made to compare the effects of two nonsteroidal antiinflammatory drugs (NSAIDs), flunixin and tolfenamic acid, on the leukotriene B₄ (LTB₄) production and migration of human polymorphonuclear leukocytes (PMNs) as well as on platelet aggregation and thromboxane B₂ (TxB₂) production during blood clotting. Tolfenamic acid inhibited LTB₄ production in PMNs as well as FMLP- and LTB₄-induced PMN migration (IC₅₀ values 23 ± 3, 39 ± 11, and 68 ± 13 M, respectively), whereas flunixin inhibited these cell functions only with the highest concentration tested (100 M). On the other hand, flunixin was clearly a more potent inhibitor of TxB₂ production and adrenaline-induced platelet aggregation than tolfenamic acid, the IC₅₀ values in TxB₂ production being 0.28 ± 0.02 M and 2.6 ±0.3 M for flunixin and tolfenamic acid, respectively. We suggest that inhibition of PMN functions may be an additional mechanism in the antiinflammatory action of tolfenamic acid. At least in human PMNs and platelets, flunixin seems to be only an inhibitor of cyclooxygenase.     

Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids

A group of flavonoids isolated from medicinal plants and which are selective inhibitors of lipoxygenase activityin vitro: sideritoflavone, cirsiliol, hypolaetin-8-O--d-glucoside, hypolaetin, oroxindin, quercetagetin-7-O--d-glucoside, gossypin, hibifolin and gossypetin, besides leucocyanidol, have been studied for their effects on acute responses induced by carrageenin in mice. The oral administration of flavonoids to mice inhibited dose-dependently the development of paw oedema at 1, 3 and 5 h after carrageenin injection. A similar administration of flavonoids induced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenin into mice. Some of the flavonoids exhibited a potency against leukocyte infiltration similar to that seen for inhibition of carrageenin oedema at 3 h of induction. In agreement with data reported in rats, indomethacin was much more effective on inhibition of prostaglandin E₂ (PGE₂) formation than on leukocyte infiltration in mice. The selectivity of flavonoids towards lipoxygenase is not retainedin vivo since they behave as dual inhibitors of PGE₂ and leukotriene B₄ (LTB₄) formation in peritoneal exudates. Our data support the inhibition of arachidonic acid metabolism as one of the mechanisms by which flavonoids exert their anti-inflammatory effects.     

Effects ofd-penicillamine,dichlofenac sodium and gold sodium thiomalate upon the selective release of lysosomal enzymes from human polymorphonuclear leucocytes to immune complex

The selective release of -glucuronidase (-Gluc) and -N-acetylglucosaminidase (-Glm) from human polymorphonuclear leucocytes (PMN), initiated with bovine serum albumin/anti-bovine serum albumin (BSA/anti-BSA) immune complex (15 g/ml^–1) was significantly reduced by increasing concentrations (10^–7 M, 10^–6 M and 10^–5 M) ofd-penicillamine (d-PEN) in a dose-dependent fashion. These effects upon the exocytosis of the lysosomal enzymes studied are in accordance with the results obtained previously in rats with adjuvant arthritis. In contrast, Dichlofenac Sodium (DICHL), which has been found to exert inhibitory activity upon extracellular release of -Gluc and -Glm in adjuvant arthritic rats in previous studies, had no significantin vitro effect on the exocytosis of these enzymes at the concentrations identical to those ofd-PEN. Also, Gold Sodium Thiomalate (GST), in the same concentrations ranging from 10^–7 M 10^–5 M, failed to inhibit selective release of -Gluc and -Glm in the present investigations. Additionally BSA/anti-BSA,d-PEN, DICHL and GST did not significantly produce the extracellular release of lactate dehydrogenase (LDH) indicating that under experimental conditions described the cell remained intact. Moreover, neitherd-PEN, DICHL, GST or BSA/anti-BSA significantly changed the activities of lysosomal enzyme markers used in these experiments. The possible mechanism(s) of the observed phenomena are discussed.     

Detection and quantitation of cell-surface sugar receptor(s) ofLeishmania donovani by application of neoglycoenzymes

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of -galactosidase fromEscherichia coli: -d-lactose, -d-thiogalactose, -d-mannose, -l-rhamnose, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylglucosamine, the - and -glucosides maltose and cellobiose, -d-xylose, -d-mannose-6-phosphate, the -galactoside melibiose, -l-fucose, and -d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedK _D values of around 100mm and around 10⁴ binding sites for the polyvalent ligands.     

Effects of recombinant human IL-Iβ on production of prostaglandin E2, leukotriene B4, NAG,and superoxide by human synovial cells and chondrocytes

The effects of recombinant human IL-1 on the production of prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄),N-acetyl--D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-1 markedly enhanced PGE₂ production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB₄ into culture medium and IL-1 significantly inhibited LTB₄ production by these cells. IL-1 significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1, but that of chondrocytes was not altered. IL-6, unlike IL-1, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes.These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1 and IL-6.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Effects of some anti-rheumatic agents on copper-catalyzed thermal aggregation of gamma globulin

Gerber has shown that specific anti-rheumatics,d-penicillamine and aurothiomalate, inhibit coppert(II)-catalyzed thermal aggregation of human gamma globulin. Various anti-rheumatics were tested for the activity. Steroidal and non-steroidal anti-inflammatory agents were almost ineffective, while a new non-steroidal anti-inflammatory agent, TAI-284 (6-chloro-5-cyclohexyl-1-indancarboxylic acid), was found to be one half as active as aurothiomalate. The structure-activity relationship of TAI-284 derivatives and the mode of action of TAI-284 were investigated.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

Conjugation of acetaldehyde with cysteinylglycine,the first metabolite in glutathione breakdown byγ-glutamyltranspeptidase

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of -glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by -glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.     

Peptide opioids and morphine effects on inflammatory process

Morphine was found to inhibit human granulocyte aggregation and ATP, thromboxane B₂ (TxB₂), and leukotriene B₄ (LTB₄) secretion during cell aggregation. None of the opioid peptides tested [(d-Ala²,d-Leu⁵)-enkephalin (DADL), (d-Ala², N-Me-Phe⁴, Gly-ol⁵)-enkephalin (DAGO) ordynorphin 1-9 (Dyn 1-9)] was capable of mimicking morphine effects, while Dyn 1-9 per se induced TxB₂ and LTB₄ secretion from granulocytes. Morphine inhibition of both cell aggregation and ATP, but not of arachidonic acid metabolism product secretion, was prevented by naloxone. The naloxone-sensitive impairment by morphine of CD11b-CD18 complex surface expression observed could play a role in opioid inhibition of granulocyte activation.     

Orally administered tolfenamic acid inhibits leukotriene synthesis in isolated human peripheral polymorphonuclear leukocytes

Special interest has been focused on the development of dual inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In contrast to other classic NSAIDs, some fenamates in clinically achievable concentrations have been shown to inhibit synthesis of 5-lipoxygenase productsin vitro. In the present work, we studied the effect of orally administered tolfenamic acid (600 mg) on Ca ionophore A 23187-induced leukotriene synthesis in isolated human polymorphonuclear leukocytes. Leukotriene production was reduced in all 14 subjects studied, the mean inhibition of LTB₄ synthesis being 16±3% and that of LTC₄ 33±7%. The inhibition correlated positively with serum tolfenamic acid concentrations. We suggest that inhibition of leukotriene synthesis is an additional mechanism of the anti-inflammatory, antimigraine and antidysmenorrhoeic effects of tolfenamic acid, and a possible explanation for its rare gastric and bronchoconstrictive side-effects.     

d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B₄ (LTB₄), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB₄ by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 M) and different concentrations (0–100 M) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB₄ was dose dependently inhibited by both LA (IC₅₀=45 M) and DGLA (IC₅₀=40M). This inhibition of LTB₄ formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB₄ formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB₄ formation (IC₅₀=7.5 M and IC₅₀=0.2 M). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB₄ formation (IC₅₀=0.75 M). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB₄ formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB₄ formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB₄ formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipiddependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B₄ (LTB₄) in FMLP-stimulated neutrophils, the ID₅₀ was approximately 4.5 g/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB₄ nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB₄ production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Are tolfenamic acid and tenidap dual inhibitors of 5-lipoxygenase and cyclo-oxygenase?

Tolfenamic acid and tenidap have been reported to be dual inhibitors of cyclo-oxygenase and 5-lipoxygenase. In this study inhibition of 5-lipoxygenase by tenidap and tolfenamic acid in plasma-free leukocyte suspensions (IC₅₀ values=10 M) required concentrations more than 100 fold higher than those which inhibited cyclo-oxygenase (IC₅₀ values=0.05 and 0.02 M respectively). The potencies of tolfenamic acid and tenidap as cyclo-oxygenase inhibitors were markedly reduced in blood (IC₅₀=6.5 and 10 M respectively) and neither significantly inhibited 5-lipoxygenase. Since both drugs also failed to inhibit 5-lipoxygenase in rat bloodex vivo, we conclude that, at physiological levels of plasma proteins, tolfenamic acid and tenidap are selective cyclo-oxygenase inhibitors.     

Role of corticosterone in modulation of eicosanoid biosynthesis and antiinflammatory activity by 5-lipoxygenase (5-LO) and cyclooxygenase (CO) inhibitors

The effectiveness of 5-lipoxygenase (LO) and dual LO/cyclooxygenase (CO) inhibitors when administered by the topical or oral routes was significantly decreased in corticosterone depleted (adrenalectomized, Adx) mice as compared to sham mice in the mouse arachidonic acid (AA) induced ear edema model. In contrast, rat carrageenan paw edema was inhibited similarly in sham and Adx animals by 5-LO and dual 5-LO/CO inhibitors. Supplementation of cortisol levels (100 g/dl) in human whole blood for 2 hr increased the observed inhibition of LTB₄ biosynthesis by A-64077, WY-50,295 tromethamine and naproxen while having no effect on thromboxane B₂ (TXB₂) biosynthesis. Thus, corticosteroids may have a permissive effect, by modulating 5-LO inhibitor, effects on mouse AA induced ear edema and human blood leukocytes.     

Effect of Topically Applied Cyclosporin A on Arachidonic Acid (AA)- and Tetradecanoylphorbol Acetate (TPA)-Induced Dermal Inflammation in Mouse Ear

Topical cyclosporin A (CsA) was compared with dexamethasone, indomethacin and phenidone in edema, increases in vascular permeability, eicosanoids and cell-influx induced by arachidonic acid (AA) and tetradecanoylphorbol acetate (TPA) in mouse ears. CsA ED₅₀ on AA-edema (7.7 g/ear) was similar to dexamethasone and lower than indomethacin and phenidone. CsA ED₅₀ in TPA edema (21 g/ear) was higher than dexamethasone and lower than indomethacin or phenidone. All drugs equally reduce the AA-induced increase in vascular permeability, but CsA and dexamethasone had more activity on TPA. AA-increase in 6-keto-PGF₁ was reduced by dexamethasone, indomethacin and phenidone but not by CsA; only phenidone reduced LTB₄. TPA-increase in 6-keto-PGF₁ was reduced by CsA and indomethacin while CsA, dexamethasone and phenidone decreased LTB₄. CsA, indomethacin and phenidone, but not dexamethasone, suppressed AA-neutrophil influx. In TPA-ears all drugs produced similar reduction in neutrophil influx. CsA was shown to be a good topical anti-inflammatory drug.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.