Effect of benzydamine on exocytosis and respiratory burst in human neutrophils and mononuclear phagocytes

The effect of benzydamine on stimulus-dependent respiratory burst activity and enzyme release was tested in human neutrophils, monocytes and monocyte-derived macrophages. Established anti-inflammatory compounds, indomethacin, phenylbutazone and bufexamac, were tested for comparison. Care was taken to avoid cytotoxic or cytolytic concentrations of the test compounds, and their effect on release of lactate dehydrogenase was also tested. Release of specific and azurophil granules contents were induced in human neutrophils by A23187, PMA and fMLP with and without cytochalasin B pretreatment. Benzydamine inhibited stimulus-dependent release of vitamin B12-binding proteins, a marker for the specific granules, in a concentration-dependent fashion. By contrast, phenylbutazone and bufexamac were practically inactive. The effect of benzydamine on exocytosis of azurophil granules was tested in cytochalasin B-pretreated neutrophils. Benzydamine, again in contrast to the two reference anti-inflammatory compounds, inhibited release concentration-dependently also under these conditions. The concentration of the compound which inhibited exocytosis by 50% was 30-100 microM in normal and 3-10 microM in cytochalasin B-treated neutrophils. The effect of benzydamine and reference compounds on the respiratory burst was tested by assaying for superoxide formation in neutrophils and H2O2 formation in mononuclear phagocytes. Benzydamine was inactive on neutrophils and inhibited slightly the burst response of monocytes and macrophages. Two reference compounds, bufexamac and phenylbutazone, were generally more active. The strongest inhibitory effect was that of phenylbutazone on fMLP-stimulated cells. Benzydamine lacked activity under these conditions, indicating that it does not bind to the receptor of formylated chemotactic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benzydamine inhibits monocyte migration and MAPK activation induced by chemotactic agonists

1. The present study was aimed to investigate the effect of benzydamine, an anti-inflammatory drug devoid of activity on arachidonic acid metabolism, on monocyte chemotaxis and to define the possible biochemical correlates of activity. 2. Benzydamine inhibited monocyte chemotaxis in response to three classes of chemoattractants: the prototypic CC-chemokine CCL2 (MCP-1), the microbial product fMLP and the complement cascade component C5a. The effect was dose-dependent with IC50's of 100, 50 and 45 microm for MCP-1/CCL2, fMLP and C5a, respectively. At the dose of 100 microm, the effect resulted in a 50+/-10% inhibition of MCP-1/CCL2-induced chemotaxis and 53+/-6 and 54+/-5% inhibitions of chemotaxis in response of fMLP and C5a, respectively (n=3). 3. Receptor expression as well as calcium fluxes in response to chemoattractants were not affected by benzydamine. 4. Benzydamine strongly inhibited chemoattractant-induced activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and of its upstream activator kinase MEK1/2. ERK1/12 activation in response to chemoattractants was 89-98% inhibited by a 100 microm concentration of benzydamine with an IC50 of 30 microm. 5. Under the same experimental conditions, pretreatment with 100 microm benzydamine caused a 75-89% inhibition of p38 activation (IC50 25 microm). 6. These results indicate that the anti-inflammatory activity of benzydamine is exerted at multiple levels, including monocyte migration to chemotactic factors associated to a blockage of ERK and p38 MAPK pathways.     

Human plasma metabolic profiles of benzydamine,a flavin-containing monooxygenase probe substrate,simulated with pharmacokinetic data from control and humanized-liver mice

1.?Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir.

2.?Following oral administration of benzydamine (10?mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide.

3.?Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high.

4.?The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.     

Influence of non-steroid anti-inflammatory and immunosuppressive drugs on hepatic tryptophan pyrrolase activity in rats

High oral doses of the anti-inflammatory drugs indomethacin, acetylsalicyclic acid, sodium salicylate, salicylamide, mefenamic acid, flufenamic acid, amidopyrine, phenylbutazone and benzydamine administered repeatedly, did not influence tryptophan pyrrolase activity in livers of intact rats. The nonresponsiveness of tryptophan pyrrolase was in contrast to a stimulation of tyrosine aminotransferase caused by flufenamic acid.Induction of tryptophan pyrrolase due to hydrocortisone was not inhibited generally by non-steroid drugs; exceptions were flufenamic acid and benzydamine which depressed hormonal induction. The authors' recent statement is confirmed that inhibition of protein synthesis due to induction is no essential property of non-steroid antiinflammatory drugs.The immunosuppressive drugs cyclophosphamide, triaziquone and azathioprine increased tryptophan pyrrolase activity, probably due to an inhibition of enzyme degradation; chloroquine, 6-mercaptopurin, 6-azauridin and amethopterin did not influence enzyme activity. Induction of tryptophan pyrrolase due to hydrocortisone was depressed by azathioprine, 6-mercaptopurin, 6-azauridin and amethopterin.     

Pharmacokinetics of benzydamine after intravenous, oral, and topical doses to human subjects

The pharmacokinetics of the anti-inflammatory drug benzydamine were determined after intravenous infusion of 5 mg to six healthy male subjects. Benzydamine was characterized as a drug of relatively low systemic clearance (ca. 160 ml min-1) but high volume of distribution (ca. 1101); the apparent terminal half-life in plasma was ca. 8 h. Benzydamine was well absorbed after oral administration, as indicated by a mean systemic availability of 87 per cent. However, absorption of the drug was low (less than 10 per cent of the dose) after its use by male subjects as a mouthwash, or after its application to female subjects as dermal cream and vaginal douche preparations. The data suggest that benzydamine is generally not well absorbed through the skin and non-specialized mucosae, thereby limiting unrequired systemic exposure to this drug when it is used by these routes.     

A new approach to anti-inflammatory drugs.

All aspirin-like drugs so far tested inhibit prostaglandin biosynthesis but do not prevent the generation of the hydroxy acid 12-l-hydroxyeicosatetraenoic acid (HETE) by arachidonate lipoxygenase. HETE is chemotactic for polymorphonuclear leukocytes, and the failure to inhibit lipoxygenase may explain why the aspirin-like drugs have little or no effect on leukocyte migration at doses which are both anti-inflammatory and inhibit prostaglandin synthesis in vivo. 3-Amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C) inhibits both pathways of arachidonic acid metabolism in vitro and causes a dose-dependent reduction in carrageenin-induced oedema in the rat paw. BW755C also reduces prostaglandin concentration in inflammatory exudates and has a significantly greater effect on leukocyte migration than indomethacin. The dual inhibition of arachidonate cyclo-oxygenase (prostaglandin synthetase) and lipoxygenase could lead, therefore, to increased anti-inflammatory activity.     

The inhibition of histamine release from rat peritoneal mast cells by non-steroid anti-inflammatory drugs and its reversal by calcium.

1 The non-steroid anti-inflammatory drugs, indomethacin, flufenamate and meclofenamate, inhibited the release of histamine from rat peritoneal mast cells induced by pharmacological or immunological challenge in vitro. 2 Anti-inflammatory steroids had little effect on histamine release from the mast cells. 3 Th inhibition of histamine release by the aspirin-like drugs was not prevented by incubation with glucose, unlike the inhibition of 2,4,dinitrophenol or antimycin-A. This suggests that the non-steroid anti-inflammatory compounds do not act by preventing the energy production from oxidative metabolism, required for histamine release. 4 The inhibition of the calcium ionophore A23187-induced histamine release by the aspirin-like drugs was reversed by an increase in the calcium concentration of the incubation medium. 5 The results suggest that the non-steroid anti-inflammatory compounds inhibit histamine release by actions on calcium influx into the mast cell, or on calcium mobilization or utilization within the mast cell.     

A review of the emerging profile of the anti-inflammatory drug oxaprozin

Oxaprozin is a nonsteroidal anti-inflammatory drug characterised by a propionic acid-based structure. It is able to diffuse easily into inflamed synovial tissues after oral administration. Although discovered > 20 years ago, it is now under intensive investigation because of its unusual pharmacodynamic properties. Other than being a nonselective cyclooxygenase inhibitor, the drug is capable of inhibiting both anandamide hydrolase in neurons (median inhibitory concentration [IC50] = 85 micromol/l), with consequent potent analgesic activity, and NF-kappaB activation in inflammatory cells (IC50 = 50 micromol/l). Moreover, oxaprozin induces apoptosis of activated monocytes in a dose-dependent manner, with the effect being detectable at a concentration of 5 micromol/l and reaching the maximum activity at 50 micromol/l. As monocyte-macrophages and NF-kappaB pathways are crucial for synthesis of proinflammatory and histotoxic mediators in inflamed joints, oxaprozin appears to be endowed with pharmacodynamic properties exceeding those presently assumed as markers of classical nonsteroidal anti-inflammatory drug.     

Effects of benzydamine on the lipolytic system of fat cells.

Benzydamine dose-dependently increased cyclic 3',5'-AMP levels in isolated fat cells and competitively inhibited phosphodiesterase activity (K_i = 1.1 mM). ATP levels of isolated cells and cyclic 3',5'-AMP-dependent protein kinase activity were not affected. However, benzydamine caused a dose-dependent inhibition of lipolysis in isolated fat cells stimulated by norepinephrine or dibutyryl cyclic 3',5'-AMP. The enhancement of cyclic 3',5'-AMP formation may be due to the local anaesthetic properties of benzydamine. The antilipolytic effect appears to result from the direct inhibition of hormone sensitive triglyceride lipase.     

Effect of benzydamine,a topically administered NSAID onin vitro prostanoid synthesis by human and rat gastric mucosa and rat kidney,aorta and urinary bladder: Lack of effect on cyclooxygenase but potent inhibition of receptor-linked prostanoid synthesis

Orally administered NSAIDs are notorious for their frequent and severe side-effects in the gastrointestinal tract and kidney, whereas topically administered NSAIDs may avoid these untoward effects. Since NSAID-induced side-effects are largely prostaglandin (PG)-mediated, the effects of the topically administered NSAID, benzydamine, onin vitro PGI₂ and PGE₂ synthesis by the rat and human gastric mucosa and rat kidney slices was investigated. The effect on receptor-linked PG synthesis in the isolated rat aorta (adrenergic) and urinary bladder (cholinergic) was also investigated since NSAIDs may disrupt mobilization of calcium therein. Benzydamine was a very weak inhibitor of spontaneous PGI₂ and PGE₂ synthesis by human and rat gastric mucosa and rat kidney. In contrast, benzydamine was a potent inhibitor of noradrenaline- and carbachol-stimulated (but not arachidonate- or trauma-stimulated) PGI₂ synthesis. It is concluded that: a) benzydamine is unlikely to elicit cyclooxygenase-mediated side-effects on the gastrointestinal tract or kidney, b) the anti-inflammatory action of benzydamine is mediated by disruption of calcium linked to receptor-PG synthesis coupling, and c) calcium-dependent inflammatory processes may also be affected by benzydamine.     

Topical immunomodulators for management of oral mucosal conditions, a systematic review; Part II: miscellaneous agents

INTRODUCTION: Topical immunomodulating preparations have utility in inflammatory/immune-mediated oral mucosal disease resistant to topical steroids, in immunologically mediated systemic disease with primary oral involvement or more severe lesions primarily involving the oral mucosa. AREAS COVERED: This paper is the second part of a systematic review of a variety of topical immunomodulators for management of immune/inflammatory oral mucosal conditions. The literature search revealed studies of azathioprine, benzydamine, GM-CSF and G-CSF, tetracyclines, retinoids, imiquimod, amlexanox, sirolimus and bacillus Calmette-Guerin polysaccharide nucleic acid. Weighted conclusions are provided for the topical use of each of the immunomodulators reviewed in the management of these oral diseases. EXPERT OPINION: Topical immunomodulators may be useful as second line treatment in several oral diseases, particularly oral lichen planus and recurrent aphthous stomatitis. Benzydamine was found to be preventive in radiotherapy-induced mucositis; however, it is unclear if this outcome is related to its immunomodulating effects or other mechanisms of action. Topical application of tetracyclines and retinoic acid also shows potential anti-inflammatory actions.     

Current concepts of the actions of paracetamol (acetaminophen) and NSAIDs

There is much uncertainty about the mechanism of action of paracetamol (acetaminophen). It is commonly stated that, unlike the non-steroidal anti-inflammatory drugs (NSAIDs), it is a weak inhibitor of the synthesis of prostaglandins. This conclusion is made largely from studies in which the synthesis of prostaglandins was measured in homogenized tissues. However, in several cellular systems, paracetamol is an inhibitor of the synthesis of prostaglandins with IC₅₀ values ranging from approximately 4 μM to 200 μM. Paracetamol is not bound significantly to plasma proteins and therefore the concentrations in plasma can be equated directly with those used in in vitro experiments. After oral doses of 1 g, the peak plasma concentrations of paracetamol are approximately 100 μM and the plasma concentrations are therefore in the range where marked inhibition of the synthesis of prostaglandins should occur in some cells. Paracetamol is metabolized by the peroxidase component of prostaglandin H synthase but the relationship of this to inhibition of the cyclooxygenase or peroxidase activities of the enzyme is unclear. Paracetamol is also metabolized by several other peroxidases, including myeloperoxidase, the enzyme in neutrophils which is responsible for the production of hypochlorous acid (HOCl). The metabolism of paracetamol by myeloperoxidase leads to the decreased total production of HOC1 by both intact neutrophils and isolated myeloperoxidase, even though the initial rate of production of HOC1 is increased. The IC₅₀ value, derived from inhibition of the total production of HOC1 by isolated myeloperoxidase, is 81 μM. Several NSAIDs inhibit functions of neutrophils in media containing low concentrations of protein but their effects, in contrast to that of paracetamol, are generally produced only at concentrations greater than those of the unbound drug in plasma during treatment with the NSAIDs. However, neutrophils isolated during treatment with NSAIDs, such as piroxicam, ibuprofen and indomethacin show decreased function. Paracetamol has little or no anti-inflammatory activity by itself but may potentiate the clinical activity of NSAIDs in the treatment of rheumatoid arthritis.     

Acute overdose due to benzydamine.

Benzydamine is a nonsteroidal anti-inflammatory drug, currently available as mouthwash, aerosol, dermal cream, vaginal douche preparation, pills and otic drops. Up to now no cases of poisoning due to this drug have been reported. A 6-year-old girl with an accidental poisoning with benzydamine is described. The episode consisted of hallucinosis without other symptoms and resolved spontaneously in 17 h.     

Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine.

The flavin-containing monooxygenase (FMO)-dependent N-oxidation of benzydamine has been assessed as a method for monitoring the activity of FMOs in monolayer cultures of hepatocytes from rat, dog, rabbit, hamster and human. The advantage of this substrate is that benzydamine N-oxide formation can be measured directly in extracts of cellular incubations without an intensive work-up procedure. Benzydamine and its N-oxide are readily separated by HPLC with fluorometric detection. This assay proved sensitive enough to monitor FMOs activity in intact monolayer of cultured hepatocytes. The formation of benzydamine N-oxide was inhibited when hepatocytes were coincubated with methimazole (another FMO substrate) in a dose-dependent manner, whereas N-octylamine (an inhibitor of cytochrome P450) had no inhibitory effect. In contrast to cytochrome P450, FMO activity assessed by benzydamine N-oxidation was relatively stable for all species studied during 72-h cultures.     

The amplified chemiluminescence test to characterize antirheumatic drugs as oxygen radical scavengers

High levels of reactive oxygen species (ROS) are generated by phagocytes involved in host defence and inflammation. Thus, it appears highly desirable to learn more about the potential of antirheumatic drugs to scavenge ROS or to inhibit their enzymatic generation. Amplified chemiluminescence (CL) allows detection of O-2 using lucigenin (LgCL) or H2O2 using luminol (LuCL). A total of 43 compounds have been tested quantitatively in vitro (10(-6) to 10(-4) mol/l) with respect to three test parameters; varying cell-activity, and incubation-time employing two different phagocyte populations (neutrophils/macrophages). The most active compounds with LgCL were the known radical scavengers nordihydroguaiaretic acid (NDGA), N-propyl gallate, superoxide dismutase and chloroquine, the non-steroidal anti-inflammatory drugs (NSAID) benzydamine, timegadine, carprofen, enolicam, the known lipoxygenase inhibitors (e.g. CBS 1108/1114, BW 755C) and glucosaminoglucan polysulfate. Inactive in this system were corticosteroids (prednisolone, dexamethasone) most of the tested NSAID (N = 16/20), most disease modifying drugs (D-penicillamine, levamisole, gold-TM) and the anti-gout drugs (sulfinpyrazone, allopurinol, colchicine). Therefore amplified CL with lucigenin appears to be a rapid, kinetic, reproducible means of pharmacological profiling in vitro new anti-inflammatory drugs for radical scavenger activity.     

Nimesulide: an NSAID that preferentially inhibits COX-2, and has various unique pharmacological activities

Nimesulide is a NSAID with good anti-inflammatory, analgesic and antipyretic activities expected of such compounds. However, in addition it has some unique therapeutic and pharmacological activities. The novel therapeutic aspects include a relatively low toxicity to the gastrointestinal tract and kidneys, it can be given to most patients who experience respiratory problems with other NSAIDs, and the onset of analgesia is comparatively quick. The main novel pharmacological actions obtained using nimesulide in vivo at therapeutic doses, or in vitro at concentrations within the therapeutic range of free (unbound) drug, include: a preferential inhibition of prostaglandin synthesis via COX-2, and reductions in cytokine action/release, histamine release, the release of enzymes that degrade cartilage, and the release of superoxide anions and other toxic substances from neutrophils. Interactions with other drugs are few and of little or no clinical significance.     

Immunosuppressive actions of prostaglandins and the possible increase in chronic inflammation after cyclo-oxygenase inhibitors

A method is described to examine the activity of potential antirheumatic drugs on the release and activity of lymphokines and interleukins in vitro, using human peripheral blood mononuclear cells and synovial cells. The enhancement of lymphocyte-mediated effects brought about by non-steroid anti-inflammatory drugs has been shown to be the result of inhibition of a prostaglandin negative-feedback mechanism. Since the underlying features of rheumatoid arthritis and related diseases are almost certainly brought about by mononuclear cell activation, their enhancement by non-steroid anti-inflammatory drugs might well have serious clinical implications. The possibility is discussed that aspirin-like drugs, administered in large doses to patients suffering slight joint pain, might well exacerbate, perpetuate or even initiate a chronic arthritic condition. We suggest that, as soon as the disease has been diagnosed, patients should be treated with a disease-modifying drug and, if necessary, an analgesic which does not inhibit cyclo-oxygenase.     

Topical immunomodulators for management of oral mucosal conditions,a systematic review; part II: miscellaneous agents

Introduction: Topical immunomodulating preparations have utility in inflammatory/immune-mediated oral mucosal disease resistant to topical steroids, in immunologically mediated systemic disease with primary oral involvement or more severe lesions primarily involving the oral mucosa.

Areas covered: This paper is the second part of a systematic review of a variety of topical immunomodulators for management of immune/inflammatory oral mucosal conditions. The literature search revealed studies of azathioprine, benzydamine, GM-CSF and G-CSF, tetracyclines, retinoids, imiquimod, amlexanox, sirolimus and bacillus Calmette-Guerin polysaccharide nucleic acid. Weighted conclusions are provided for the topical use of each of the immunomodulators reviewed in the management of these oral diseases.

Expert opinion: Topical immunomodulators may be useful as second line treatment in several oral diseases, particularly oral lichen planus and recurrent aphthous stomatitis. Benzydamine was found to be preventive in radiotherapy-induced mucositis; however, it is unclear if this outcome is related to its immunomodulating effects or other mechanisms of action. Topical application of tetracyclines and retinoic acid also shows potential anti-inflammatory actions.     

Dopamine autoreceptor agonists as potential antipsychotics. 1. (Aminoalkoxy)anilines

The synthesis and pharmacological properties of a novel type of [(arylpiperazinyl)alkoxy]anilines with dopaminergic properties are described. One of these compounds, 3-[3-(4-phenyl-1-piperazinyl)propoxy]benzenamine (4c), has been identified as a selective dopamine (DA) autoreceptor agonist in tests that include [3H]haloperidol binding, inhibition of striatal DA synthesis, inhibition of DA neuronal firing, inhibition of spontaneous locomotor activity, and reversal of reserpine-induced depression in rats. In addition, 4c possesses good oral activity in the Sidman conditioned avoidance test in squirrel monkeys, which is indicative of antipsychotic activity. In a primate model, 4c was found to lack the liability for extrapyramidal side effects usually associated with antipsychotic drugs.     

In vitro activities of transition metal derivatives of ketoconazole and clotrimazole against a wild type strain of Saccharomyces cerevisiae in absence or presence of human neutrophils

In vitro activities of a series of gold, copper and ruthenium clotrimazole (CTZ, CAS 23593-75-1) and ketoconazole (KTZ, CAS 65277-42-1) derivatives were investigated individually and in combination with human neutrophils (PMNs) against a wild type strain of Saccharomyces cerevisiae. For 11 out of 12 tested metal complexes, the minimal inhibitory concentrations (MICs) at which 100 % of yeast growth was inhibited ranged from 0.75 to 3.0 micromol/L. The complex RuCl3(CTZ)3 x 2CH3OH (1f) (MIC = 0.75 micromol/L) was, although modestly, the only one able to increase the fungistatic activity of the parental drug (MIC = 1 micromol/L). On the other hand, at a sub-MIC concentration (0.5 micromol/L), the complexes [Cu(KTZ)Cl2]2 x 2H2O (2c) and RuCl2(KTZ)2 (2e) displayed synergistic fungicidal effects with PMNs whereas phagocytic capacity was enhanced by complexes [Cu(KTZ)3Cl2] (2b) and RuCl2(KTZ)2 (2e). The findings suggest that the metal-based agents may give rise to drugs with improved antifungal properties.