Istvan Szelenyi

Possible role of oxygen free radicals in ethanol-induced gastric mucosal damage in rats

The involvement of oxygen free radicals in the development of the ethanol-induced gastric mucosal damage has been investigated. We found that oral administration of superoxide dismutase reduced the incidence of ethanol-induced mucosal lesions. Reduction of superoxide dismutase activity by diethyldithiocarbamate led to a pronounced aggravation of mucosal damage. Inhibition of the chloride-bicarbonate channel by a stilbene derivative also aggravated the ethanol-induced hemorrhagic lesions. Neither glutathione peroxidase, catalase, nor ceruloplasmin were capable of inhibiting the development of mucosal damage. Compounds with scavenging properties such as thiourea, 1-phenyl-3-(2-thiazolyl)-2-thiourea, dimethyl sulfoxide, various inorganic compounds (elements of the first and second subgroups and of the sixth group of the periodic table) and sulfhydrylcontaining substances protected the gastric mucosa against ethanol-induced injury in a dose-related manner. Naturally occurring antioxidants such as α-tocopherol, β-carotene, and coenzyme Q10 were ineffective. The present results suggest that superoxide free radicals are involved in the development of ethanol-induced gastric mucosal lesions, probably via an interaction with cellular membranes.

Mode of antinociceptive action of flupirtine in the rat.

1 Flupirtine is a novel, centrally acting, non‐opioid analgesic agent. The present investigation was undertaken to ascertain which neuronal systems might be responsible for its antinociceptive effect in rodents. The antinociceptive responses to the test compounds were examined in the tail‐flick test. 2 The selective destruction of noradrenergic pathways by 6‐hydroxydopamine considerably reduced the flupirtine‐induced inhibition of nociceptive responses but not the clonidine‐induced antinociception which was significantly enhanced. Depletion of spinal 5‐hydroxytryptaminergic pathways by pretreatment with 5,7‐dihydroxytryptamine failed to affect the action of flupirtine and clonidine. 3 The depletion of neurotransmitters by reserpine totally abolished the antinociceptive action of flupirtine. By contrast, clonidine‐induced inhibition of nociceptive responses remained unchanged. 4 Inhibition of the synthesis of noradrenaline by α‐methyl‐L‐p‐tyrosine attenuated the antinociception induced by flupirtine. In contrast, inhibition of the synthesis of 5‐hydroxytryptamine by (±)−6‐fluorotryptophan did not influence the antinociceptive activity of flupirtine. 5 Inhibition of noradrenaline uptake by imipramine led to a significant augmentation of flupirtine‐induced antinociception. 6 Selective antagonists at α‐adrenoceptors significantly decreased the antinociceptive action of flupirtine. Antinociception induced by clonidine was significantly diminished by idazoxan but not by prazosin. 7 The 5‐hydroxytryptamine (5‐HT) antagonist, ketanserin diminished the antinociceptive activity of flupirtine, probably due to its additional α1‐adrenoceptor antagonist activity. The antinociceptive effect of clonidine was not influenced by ketanserin. 8 Cholinoceptor antagonists such as mecamylamine and pirenzepine did not alter the antinociceptive action of flupirtine. Flupirtine‐induced antinociception also remained unchanged after pretreatment with haloperidol. 9 Flupirtine has no pharmacologically relevant affinity for α1‐, α2‐adrenoceptors, 5‐HT1‐ and 5‐HT2‐receptors as shown in direct binding studies. 10 The present results indicate that the antinociceptive action induced by flupirtine depends on the descending noradrenergic pain‐modulating system.

Regulation of IL-13 synthesis in human lymphocytes: implications for asthma therapy

IL‐13 is an important mediator in inflammatory diseases such as asthma. IL‐13 is mainly produced by T cells. However, signalling pathways leading to induction of this cytokine are not well‐characterized. We analysed the regulation of IL‐13 in human peripheral blood mononuclear cells and CD4+ T cells. Cyclosporine (CsA) and FK‐506 inhibited IL‐13 synthesis, when cells were stimulated by TPA/ionomycin. However, stimulation by α‐CD3/α‐CD28 led to an enhanced IL‐13 synthesis. NF‐κB inhibitor N‐tosyl‐L‐lysine chloromethylketone (TLCK) inhibited IL‐13 synthesis more effectively after TPA/ionomycin stimulation. After α‐CD3/α‐CD28 stimulation, only 300 μM TLCK inhibited IL‐13 synthesis. Dexamethasone inhibited IL‐13 equally effective after α‐CD3/α‐CD28 and TPA/ionomycin stimulation. p38 MAPK inhibitor SB203580 inhibited IL‐13 synthesis only partially. MEK inhibitor U0126 inhibited TPA/ionomycin induced IL‐13 synthesis very effectively, whereas α‐CD3/α‐CD28 stimulated IL‐13 induction was resistant to this drug. These results were confirmed in purified CD4+ T cells. In difference to PBMCs α‐CD3/α‐CD28 stimulated IL‐13 synthesis was effectively inhibited by CsA, FK‐506 and U0126. Therefore U0126 was tested in an animal model of allergic asthma. We could demonstrate for the first time that inhibition of the MEK – ERK cascade is a therapeutic option for asthma. Intraperitoneal administration of 10 mg kg−1 U0126 reduced lung eosinophilia in ovalbumin‐challenged Brown Norway rats by 44%. These results demonstrate that different signalling pathways are involved in regulating IL‐13 synthesis in primary human T cells. Characterizing highly potent inhibitors of IL‐13 synthesis can be exploited to identify new drugs to treat immunological diseases such as asthma.

Possible role of sulfhydryls in mucosal protection induced by aluminum hydroxide

The involvement of sulfhydryl-containing compounds was investigated in the cytoprotective effect of certain antacids against ethanol-induced gastric mucosal damage in the rat. Not only the protective effect of a sulfhydryl containing compound,N-acetylcysteine, was abolished after pretreatment withN-ethylmaleimid, but also the adaptive cytoprotection induced by 20% ethanol. Pretreatment of the animals with the cyclooxygenase inhibitor indomethacin or with the thiol reagentN-ethylmaleimide significantly diminished the cytoprotection evoked by aluminum hydroxide. However, the protective effect of aluminium hydroxide could only be totally abolished by pretreatment with the combination of indomethacin andN-ethylmaleimide. The present results suggest the additional role of sulfhydryl-containing compounds localized in the gastric mucosa in the cytoprotection induced by a mild irritant or by aluminium hydroxide and aluminium hydroxidecontaining antacids as well.

Flupirtine, a re-discovered drug, revisited

Flupirtine was developed long before KV7 (KCNQ) channels were known. However, it was clear from the beginning that flupirtine is neither an opioid nor a nonsteroidal anti-inflammatory analgesic. Its unique muscle relaxing activity was discovered by serendipity. In the meantime, broad and intensive research has resulted in a partial clarification of its mode of action. Flupirtine is the first therapeutically used KV7 channel activator with additional GABAAergic mechanisms and thus the first representative of a novel class of analgesics. The presently accepted main mode of its action, potassium KV7 (KCNQ) channel activation, opens a series of further therapeutic possibilities. One of them has now been realized: its back-up compound, the bioisostere retigabine, has been approved for the treatment of epilepsy.

Effect of phosphodiesterase inhibition on IL-4 and IL-5 production of the murine TH2-type t cell clone D10.G4.1

The effect of various phosphodiesterase (PDE) inhibitors on anti-CD3 induced interleukin-(IL)-4 and IL-5 production of the murine T helper cell clone of type 2 phenotype D10.G4.1 (D10) has been investigated in vitro. D10 cells were incubated in the presence of drugs and anti-CD3 mAb for 16 h before measurement of cytokines in the cell supernatants by ELISA. Whereas all PDE inhibitors tested exerted minimal effects on anti-CD3 induced IL-4 production, a marked increase in IL-5 production by the non-selective PDE inhibitors IBMX, theophylline and enprofylline was observed. The action of these non-selective PDE inhibitors was mimicked by the PDE IV-selective inhibitor rolipram and in part by the PDE III-selective inhibitors motapizone and milrinone, whereas the PDE V-selective inhibitor zaprinast was inactive. Rolipram and motapizone enhanced IL-5 production in a synergistic fashion. In support of the functional importance of PDE III and IV for IL-5 synthesis in intact murine D10 cells, we have found PDE III and IV to be the predominant isoenzyme activities in corresponding cell lysates. The stimulatory effect of rolipram on IL-5 production was almost totally reversed by the protein kinase A inhibitor KT-5720. In addition, the membrane-permeable cAMP analogue 8-bromo-cAMP mimicked the stimulatory effect of PDE inhibitors on IL-5 production while leaving IL-4 levels unaffected. Both results support the view that the action of the PDE inhibitors on murine D10 cells is mediated via an elevation of intracellular cAMP.

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors.

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and γ-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10−7 and 10−5 mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.

Nanomedicine: evolutionary and revolutionary developments in the treatment of certain inflammatory diseases

Nanomedicine, although in a nascent stage of development at present, is already a reality. Several pharmaceutical products using this modern technology are already on the market. Nanotechnology offers many potential benefits to medical research. Nanoparticle-based drug carriers can increase the efficacy and safety of drugs by enhancing capacity, improving solubility, combining multiple drugs, protecting against metabolism, and controlling release. Nanoparticles can also form the basis of multifunctional drug delivery vehicles by combining targeting, imaging, and therapeutic moieties. Multifunctional nanoparticles have tremendous potential to treat human diseases. The use of non-viral carriers (nanoparticles) can also improve the cellular/nuclear uptake of corresponding nucleotides. Preclinical characterization of nanoparticles intended for medical applications is complicated—due to the variety of materials used, their unique surface properties, and multifunctional nature. It is hard to predict what precise course nanomedicine will take in years to come. It is, however, very likely that this relatively young research area will become a driving force behind a vast revolution in medical treatment.

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine.

The effect of azelastine, an orally effective antiasthmatic/antiallergic drug, on the generation of oxygenderived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC30=3.9 μM) and AM (IC30=9.8 μM). In AM zymosan-induced CL was inhibited 21.7% by 10 μM azelastine, whereas in PMNL it remained unchanged up to 10 μM azelastine. Azelastine has a significantly stronger inhibitory effect (IC30=4.2 μM) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibitis oxygen-derived free radical generation by interaction with protein kinase C.

Prostaglandin content in the rat gastric mucosa during healing of chronic ulcers induced by acetic acid.

Non-steroidal anti-inflammatory compounds delayed the regeneration of chronic gastric ulcers induced by acetic acid in the rat. The delayed regeneration was connected with a decreased PGE2 content in the proliferating tissue. Glucocorticoid influenced neither the regeneration of mucosal damages nor the gastric PGE2 content. The carboanhydrase inhibitor, acetazolamide, also did not alter the restoration of gastric mucosa after being damaged. From the clinical point of view, the safe use of NOSAC will be emphasized in patients with ulcer anamnese. The findings with prednisolone did not confirm a similar safe use of this drug in patients.