Administration of L-arginine reduces the delay of the healing process caused by ibuprofen. Implication of COX and growth factors expression.

The objective of the present study has been to advance knowledge of the gastric role played by the amino acid L-Arginine (L-Arg) in the evolution of a chronic gastric ulcer. In order to clarify it, L-Arg alone or together with Ibuprofen have been administrated in an experimental acetic acid chronic ulcer, analysing characteristic parameters of an active curative process, such as PGE2 production, COX expression, and also angiogenesis, proliferation/apoptosis and growth factors expression. Our results reveal that L-Arg is favourable in the healing process improving the curative course. Ibuprofen caused a delay in ulcer healing, more evident 14 days after ulcer induction; COX-2 expression was increased at the 7th day although no signal of protein could be detected after 14 days; PGE2 production was inhibited in intact and ulcerated areas at both times assayed. In contrast, treatment with L-Arg reduced the delay of the lesion, the increment in COX-2 expression induced by Ibuprofen, and was able to maintain PGE2 levels similar to the control group after 14 days. Additionally, the histological study showed that the healing effects of L-Arg might be associated with an increased angiogenesis and FGF-2 expression. These actions could be considered key factors in the healing response associated with L-Arg administration. However, the proliferation study assayed with the PCNA-immunostaining method did not reveal significant differences, as the same as the apoptosis analysis. In conclusion, the coupling of L-Arg to Ibuprofen is an attractive alternative to Ibuprofen administration alone because it not only attenuates but also improves the evolution of chronic lesions through mechanisms that implicate endogenous PG and FGF-2-associated pathways, which allow an increase of angiogenesis process.

Role of L-arginine in ibuprofen-induced oxidative stress and neutrophil infiltration in gastric mucosa.

It has been proposed that neutrophil and oxygen dependent microvascular injuries may be important prime events in gastrointestinal (GI) toxicity of non-steroidal antiinflammatory drugs (NSAIDs). L-arginine (L-ARG) is an essential amino acid which participates in many important biochemical reactions associated to the normal physiology of the organism. In these experimentations, we studied the role of L-ARG, aminoacid precursor of NO synthesis, on ibuprofen (IB) induced gastric lesions, and also on the inflammatory and oxidative mechanisms related to mucosal damage. Oral administration of IB (100 mg kg(-1)), produced severe damage on gastric mucosa, which was more important after 6 h test-period, and was accompanied by a significant increment in myeloperoxidase (MPO) activity, as index of neutrophil activation, as well as lipid peroxidation (LP) levels and xanthine oxidase (XO) activity. However, no changes were observed in total mucosal glutathione (tGSH), nor glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity. Simultaneous treatment with equimolar doses of L-ARG (oral and i.p.), considerably reduced the number and intensity of lesions, and at the same time (6 h) the maximum protection was also observed. In addition, L-ARG inhibited the IB-induced LP and XO enhancement, but did not produce changes in leukocyte infiltration, tGSH, GSH-Px and SOD activity. These findings suggest that (1) L-ARG protective effect on gastric mucosa against IB-induced mucosal lesions could be explained by a local effect and also might be due to the systemic action of the aminoacid; (2) the active oxygen species, derived both from XO and activated neutrophils, could play a role in the pathogenesis of gastric injury induced by IB, (3) L-ARG exhibit a protective effect against IB-induced mucosal damage, probably through the inhibition of oxidative stress derived via xanthine-XO, but it does not block the oxygen free radical production through polymorphe nuclear leukocytes.

Contraction of human airways by oxidative stress protection by N-acetylcysteine.

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 +/- 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was approximately 100 microM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 +/- 0.83 to 26.82 +/- 1.49 nmol mg(-1) protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 +/- 2.6 to 6.9 +/- 1.9 nmol mg(-1) protein) and an increase of oxidized glutathione (from 0.09 +/- 0.03 to 0.18 +/- 0.03 nmol mg(-1) protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 +/- 1.19 nmol mg(-1) protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity. N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.

Absorption of sodium diclofenac after ocular administration in rabbit.

Sodium diclofenac (DCF, CAS 1507-79-6) is a non-steroidal anti-inflammatory drug whose activity is mainly due to an inhibitory effect on the synthesis of the prostaglandins. At present, a tendency towards its topical use in the treatment of the inflammatory state of the anterior segment of the eye as an alternative to the steroid drugs is observed. The pharmacokinetics of DCF was studied in rabbits by assessing the ocular and systemic absorption of DCF after administering DCF eye drops. The chromatographic methods available were not sensitive enough to quantify the extremely low drug concentrations which appeared in the biological fluids after ocular treatment. For this reason, the concentrations of DCF in plasma and aqueous humor were evaluated by a coupled liquid chromatography/gas chromatography (LC/GC) method. DCF was absorbed well through the cornea with the aqueous humor concentration peak being 757.8 ng/ml at 120 min. This good ocular absorption of DCF was confirmed by the concentrations observed in plasma. The presence of tramazoline (TMZ, CAS 74195-73-6) in the eye drops increases the levels of DCF in aqueous humor.

Effect of N-acetylcysteine on the oxidative burst induced by phagocytosis of bacteria in human leukocytes.

The basal peroxide production and the oxidative burst induced by phagocytosis of opsonized E. coli was studied by flow cytometry using dihydrorhodamine 123. The human leukocytes were incubated in the absence and presence of N-acetylcysteine. The oxidative response to the phagocytosis of bacteria differed among cell populations. Thus, 90% of granulocytes and 50% of monocytes showed an oxidative burst in response to opsonized bacteria while less than 1% of lymphocytes showed a fluorescence signal. N-Acetylcysteine (4.7, 9.5, 19, 38 or 76 mM) produced a dose-dependent inhibition of the oxidative response to phagocytosis in the three cellular populations reaching almost complete inhibition for 76 mM. This protective effect of N-acetylcysteine against oxidative stress in leukocytes was obtained without cytotoxicity (assessed by flow cytometry with staining with propidium iodide) or changes in the pH of the medium. These results give further support to the antioxidant effect of N-acetylcysteine in human peripheral blood cells.

Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.

Pseudomonas aeruginosa proteases: purification procedures for an enzymatic standard.

Pseudomonas aeruginosa may cause severe infections in debilitated patients. Strains of this microorganism produce several extracellular space proteins, some of which are believed to be virulence factors. There are experimental correlations between the ability to produce proteases and virulence. Treatment of bacteria with subinhibitory concentrations of antimicrobial agents frequently increases bacterial phagocytosis, intracellular killing, and suppresses the production of bacterial virulence factors, including extracellular enzymes. We suggest a simple method for production, purification and quantitation of Pseudomonas aeruginosa extracellular proteases suitable for use in investigations of their role as virulence factors.

Mitochondrial glutathione oxidation correlates with age-associated oxidative damage to mitochondrial DNA.

Mitochondria may be primary targets of free radical damage associated with aging. We have found that mitochondrial glutathione is markedly oxidized with aging in rats and mice. The oxidized to reduced glutathione ratio rises with aging in the liver, kidney, and brain. The magnitude of these changes is much higher than that previously found in whole cells of any species previously studied. In the liver, this ratio (expressing GSSG as a percent of GSH) changed from 0.77 +/- 0.19% (n=5) in young rats to 2.47 +/- 1.25% (n=5) in old ones, i.e., 320% of the controls. In the brain and kidney, values for old rats were, respectively, 600 and 540% higher than those of young rats. A marked oxidation of mitochondrial glutathione also occurred in mice. Aging also caused an increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in mtDNA in rats and mice. Oral antioxidant administration protected against both glutathione oxidation and mtDNA damage in rats and mice. Finally, we have found a direct relationship between mtDNA damage and mitochondrial glutathione oxidation. This occurs both in rats (r=0.95) and in mice (r=0.98). This relationship, which has been observed for the first time in these studies, underscores the role of glutathione in the protection against free radical damage that occurs upon aging.

Pharmacokinetics of thymoxamine in rabbits after ophthalmic and intravenous administration.

The pharmacokinetics of thymoxamine hydrochloride were studied in rabbits by the assessment of its ocular and systemic absorption after instillation of thymoxamine hydrochloride 0.5% eye drops. Plasma levels were compared with those observed after i.v. bolus administration of thymoxamine hydrochloride at 2.5 mg/kg. Deacetylthymoxamine is the main metabolite of thymoxamine, generated by esterase hydrolysis. It was evaluated, as an indication of the parent drug, in aqueous humor and plasma by an HPLC method with fluorescence detection (detection limit = 5 ng/ml). Thymoxamine was found to permeate the cornea and to be hydrolysed very quickly, showing very good absorption with a maximum aqueous humor concentration of deacetylthymoxamine of 2329 ng/ml 15 min after eye drop instillation. The study of the systemic absorption of thymoxamine allowed the exclusion of the possibility of systemic side effects following ocular treatment. In fact, considering the detection limit of the method, the plasma levels of deacetylthymoxamine are certainly more than 100-times lower than those observed with intravenous treatment.

Ocular pharmacokinetics of thiamphenicol in rabbits.

The ocular pharmacokinetics of thiamphenicol (TAP, CAS 15318-45-3) was studied in rabbits by means of the assessment of its ocular and systemic absorption, and urinary excretion after instillation of 0.5% TAP eye drops. TAP concentrations in aqueous humor, plasma and urine were evaluated by a coupled LC/GC method (detection limit = 0.1 ng/ml), because the necessity to have a technique much more sensitive than the traditional chromatographic ones available in order to quantify the very low drug concentrations in biological fluids produced by the ocular treatment, and generally by a topical administration. The intravenous route was chosen as reference and allowed the absolute bioavailability to be estimated. TAP proved to be well absorbed through the cornea with the peak aqueous humor concentration of 110 ng/ml at 45 min following the instillation. The good ocular absorption of TAP was confirmed by the plasma concentrations observed after instillation of 0.5% eye drops. In any case, these concentrations were more than 1000 times lower than those observed after the intravenous treatment at the dose normally used for infectious diseases, allowing to exclude any systemic toxicity of TAP eye drops. The absolute ocular bioavailability was 16.2% when estimated from the AUC values and 34.0% from the cumulative urinary excretion values.

Effect of aging on glutathione metabolism. Protection by antioxidants.

The free radical theory of aging suggests that oxygen free radicals may be involved in the aging process. Thus, changes in antioxidant mechanisms may occur with aging. Since glutathione is one of the most effective antioxidant systems in the cell, its metabolism may change with aging. In this chapter we describe experiments which show the involvement of glutathione in the aging process and which provide a rationale for the administration of antioxidants to old organisms to protect them against some of the changes that occur with aging.

Action of metadoxine on isolated human and rat alcohol and aldehyde dehydrogenases. Effect on enzymes in chronic ethanol-fed rats.

Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to accelerate ethanol metabolism. In the present work we have investigated the effect of metadoxine on the activities of isolated alcohol and aldehyde dehydrogenases from rat and man, and on the activity of these enzymes in chronic ethanol-fed rats. Our results indicate that in vitro metadoxine does not activate any of the enzymatic forms of alcohol dehydrogenase (classes I and II) or aldehyde dehydrogenase (low-Km and high-Km, cytosolic and mitochondrial). At concentrations higher than 0.1 mM, metadoxine inhibits rat class II alcohol dehydrogenase, although this would probably not affect the physiological ethanol metabolism. Chronic ethanol intake for 5 weeks results in a 25% decrease of rat hepatic alcohol dehydrogenase (class I) activity as compared with the pair-fed controls. The simultaneous treatment with metadoxine prevents activity loss, suggesting that the positive effect of metadoxine on ethanol metabolism can be explained by the maintenance of normal levels of alcohol dehydrogenase during chronic ethanol intake. No specific effect of chronic exposure to ethanol or to metadoxine was detected on rat aldehyde dehydrogenase activity.

Diuretic effect of ITA 529.

The diuretic and saluretic activity of ITA 529 (Ethyl-beta-[(5-tert-butyl-3-chloro-2-hydroxy)benzylamino]crotonate++ +) in rat, rabbit and monkey is described in this study. In rat, the diuretic ED50 of ITA 529 was 1.23 mg/kg p.o. and its ED100 1.69 mg/kg p.o. The diuretic ED50 of furosemide was 18.48 mg/kg p.o. and its ED100 22.38 mg/kg p.o. Their effect lasted approximately 3 hours. In monkey, the diuretic ED50 of ITA 529 was 2.29 mg/kg p.o. and its ED100 6.54 mg/kg p.o. while for furosemide its ED50 was 2.44 mg/kg p.o. and its ED100 5.72 mg/kg p.o. In rabbit the ED50 of ITA 529 was 1.83 mg/kg p.o. and its ED100 3.37 mg/kg p.o. For furosemide, its ED50 was 2.25 mg/kg p.o. and its ED100 7.13 mg/kg p.o. The natriuretic activity of ITA 529 was reduced by indomethacin.

Activity of plafibride on erythrocyte deformability.

N-2-(p-Chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, Idonor, Perifunal, Plafibrinol) is an acid derivative of morpholinomethylurea and clofibric acid. In these studies the effects of plafibride on haemorheological properties have been observed. The studies have been carried out in different animal species (rat, rabbit and dog), and in human volunteers. Under these circumstances, plafibride has shown itself to be highly active in all the animal species and in humans, as well as with the two laboratory methods used for determination of erythrocyte deformability, these being Teitel's method, which evaluates the deformative capacity of erythrocytes when forming compact groups, and the method of Schmid-Schoenbein in which the deformability of individual erythrocytes is measured by passage through fine capillaries. In trials in volunteers, the increase in erythrocyte deformability is notable even 5 days after the last administration. In addition to its microhaemorheological activity, plafibride has been shown to have effects on platelet aggregation and hyperlipaemia.

Microhaemorheological properties of binifibrate. Part I: Experimental studies.

The microhaemorheological properties of 3-pyridine carboxylic acid 2-[2-(4-chlorophenoxy)-2-methyl-1-oxopropoxy]-1,3-propanediyl ester (binifibrate, Biniwas), a hypolipidaemic and anti-arteriosclerotic drug composed of two nicotinic radicals and one clofibric radical united by glycerol, were studied. Binifibrate increased passage of a suspension of red blood cells at 80% haematocrit value through filters of 30-40 micron (Teitel's method), and erythrocyte deformability detectable by increase of flow rate through filters of pore size lesser than erythrocyte diameter (Schmid-Schönbein's method). After treatment, resistance to lysis in hypotonic solution is also augmented, indicating that microhaemorheological activity is due to modification of the elastic properties of the erythrocyte membrane.

Microhaemorheological properties of binifibrate. Part II: Preliminary open studies in patients with peripheral arteriopathies.

3-Pyridinecarboxylic acid 2-[2-(4-chlorophenoxy)-2-methyl-1-oxo-propoxy]-1,3-propanediyl ester (binifibrate, Biniwas) an anti-arteriosclerotic and hypolipidaemic agent which showed an effect on microhaemorheological properties during studies in experimental animals (rat and rabbit), was administered to six hyperlipidaemic patients who suffered from circulatory disturbances in the lower limbs. The treatment with binifibrate was applied at a dosage of 3 X 600 mg/day during one month. Both subjective symptoms such as paraesthesia, dysthaesia, coldness at rest and functional weakness of movements were improved, as well as objective symptoms measured by oscillometry. The microhaemorheological analyses demonstrated a positive variation in relative parameters. The correlation between microhaemorheological parameters and clinical symptoms is discussed.

Anti-ulcer activity and other pharmacological properties of lozilurea.

As a result of trials on a large series of compounds, one of these, N' -3-chlorobenzyl-N'-ethylurea (lozilurea, ITA 312) has shown marked anti-ulcer activity. It has shown itself to be active against chemically and neurogenically induced gastric and duodenal lesions in various experimental animal models. It has no major anti-secretory action. The experimental data obtained suggest that the mechanism of action of lozilurea consists in increasing the protective function of the mucus barrier. In the screening trials carried out in order to detect the side effects of lozilurea, it has shown sedative, antipyretic and vasodilatory actions.

Endothelial damage induced by polyethylene catheter in rat.

A method is described for obtaining endothelial lesions in the aorta by implanting polyethylene catheters in the aorta of rats for two days. Three types of lesions were observed and were quantified by measuring their lengths: (a) the simple disendothelization (b) raised lesions, and (c) tunnel lesions. The effect of a number of antiplatelet drugs on these types of lesions was investigated, and it was observed that these are particularly active in inhibiting tunnel lesions. Using this method, endothelial lesions can be obtained in a short period of time. Moreover, a large number of results can be obtained rapidly, which is important in these types of experiments due to the high dispersion obtained.

Pharmacokinetic approach of plafibride in rat.

The serum concentration of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) and its metabolites was measured in the rat after oral and intravenous administration of the drug. Plafibride was distributed in the rat according to an open two-compartment model. Moreover, 2-(p-chlorophenoxy)isobutyrylurea and clofibric acid were detected. In the urine, 81.7% of the administered dose was recovered in the form of free clofibric acid, 0.8% as plafibride itself and 1.1% as 2-(p-chlorophenoxy)isobutyrylurea. The amount of plafibride and its metabolites present in the feces was practically negligible. A mechanistic scheme for the degradation of plafibride is proposed, which agrees with the observed pharmacological and pharmacokinetic data.