RESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) were investigated for their ability to affect the chlorinating activity of human myeloperoxidase and to scavenge HOCl, the main myeloperoxidase system product. Fourteen drugs representative of various NSAIDs families were tested with the chlorination of taurine used as a detection system. All were unable to inhibit taurine chlorination in a system without myeloperoxidase. In contrast, most of them induced a dose-dependent inhibition of the taurine chlorination mediated by a myeloperoxidase/H2O2/Cl- system. This took place at variable drug concentrations and IC50 were calculated. The inhibitory effect was therefore due to a direct interaction with the enzyme rather than to HOCl scavenging. A spectroscopic method used to measure the myeloperoxidase compound II lifetime in presence of the different drugs showed that all the drugs, which inhibited chlorination activity were able to induce accumulation of compound II. The extent of chlorinating activity inhibition (IC50) was inversely related to the duration of the block of enzyme in compound II form. This further demonstrates that myeloperoxidase is an interesting target for anti-inflammatory therapy. The recombinant myeloperoxidase used for the first time in this kind of study was as convenient for pharmacological purposes as the purified one.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Cloro/metabolismo , Peroxidasa/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/química , Relación Dosis-Respuesta a Droga , Humanos , Ácido Hipocloroso/química , Peroxidasa/genética , Peroxidasa/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Taurina/metabolismoRESUMEN
A panel of non-steroidal anti-inflammatory drugs commonly used for therapeutic purposes was assessed for their effects on the respiratory burst of isolated human polymorphonuclear neutrophils. Cells were stimulated with opsonised yeast and the production of reactive oxygen species was measured by amplified chemiluminescence with luminol and lucigenin which are two luminogenic agents measuring different cellular events. A special attention was devoted to the establishment of dose-effect curves and calculation of ED50. Some of the drugs tested (acemetacine, diclofenac, flufenamic acid and niflumic acid) were able to decrease both luminol and lucigenin chemiluminescence in a dose-dependent manner reflecting an inhibitory effect on the respiratory burst. The most potent derivative was flufenamic acid (ED50 8 and 78 microM, respectively, with luminol and lucigenin), followed by diclofenac (21 and 98 microM), niflumic acid (97 and 227 microM) and acemetacine (585 and 427 microM). In contrast, several other drugs (flurbiprofen, ibuprofen, ketoprofen, piroxicam) stimulated both luminol and lucigenin chemiluminescence, suggesting a pro-oxidant activity. Acetylsalicylic acid (up to 1250 microM) was a modest inhibitor (maximum 25% inhibition) showing no dose-dependent effect and tolmetin (up to 125 microM) had no significant effect in both systems. The results were in agreement using both luminogenic agents, except for indomethacin, naproxen and tenoxicam which showed different kinds of effects. The unspecific and complex nature of the measurement systems used did not allow to give a complete mechanistic interpretation of the results, but the comparison with literature data gave some pertinent explanations for both anti- and pro-oxidant effects.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Neutrófilos/efectos de los fármacos , Acridinas , Humanos , Técnicas In Vitro , Mediciones Luminiscentes , Luminol , Neutrófilos/metabolismoRESUMEN
In lung diseases such as chronic obstructive pulmonary disease (COPD) or cystic fibrosis, the activation of phagocytic cells produces high amounts of cytotoxic reactive oxygen species (ROS) that are partly implicated in the pathogenic process. In this study, the ex vivo antioxidant activity of nacystelyn (NAL), a recently developed mucoactive thiol-containing agent, was investigated using the respiratory burst of human blood polymorphonuclear neutrophils (PMNs). The ROS generation was induced by serum-opsonized zymosan and assessed with luminol- and lucigenin-enhanced chemiluminescence (ECL). The activity of NAL was compared with N-acetylcysteine (ACC) and captopril, other thiol-containing pharmacological agents having documented antioxidant properties. The three drugs significantly inhibited the ECL response of activated PMNs in the presence of luminol, a luminogenic agent which mostly reflects the production of hydroxyl and hypohalite radicals. NAL was more efficient than the other two drugs: the concentrations producing a 50% inhibition (IC50) of total luminol-ECL were 290 microM, 1580 microM and 760 microM for NAL, ACC and captopril, respectively. The inhibition of the lucigenin-ECL response of activated PMNs was less marked for all compounds suggesting a poorer reactivity with superoxide radicals. These findings demonstrate that NAL, at concentrations obtainable in vivo by inhalation, impairs the PMNs chemiluminescence response related to hydroxyl and hypohalite radicals production. As those radicals are highly cytotoxic, NAL appears as a promising agent in the prevention of oxidative lung damage caused by an active inflammatory response.
Asunto(s)
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Antioxidantes/farmacología , Captopril/farmacología , Expectorantes/farmacología , Lisina/análogos & derivados , Neutrófilos/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Adulto , Humanos , Técnicas In Vitro , Lisina/farmacología , Neutrófilos/fisiologíaRESUMEN
The established horseradish peroxidase/guaiacol in an in vitro assay system was used for investigation of the reactivity of nonsteroidal antiinflammatory drugs with hydrogen peroxide. Although the drugs rapidly seemed to react in the selected conditions, difficulties were encountered in attempts to quantify the reaction and an interaction with horseradish peroxidase was suspected. A more specific assay system based on the absolute specificity of the enzyme glutathione peroxidase for glutathione was subsequently used which demonstrated that none of the investigated nonsteroidal antiinflammatory drugs was able to scavenge hydrogen peroxide. An original procedure to further evidence the interaction was developed thereafter, based on the reaction of 5-aminosalicylic acid with similar hemoproteins. This led to the demonstration that nonsteroidal antiinflammatory drugs were substrates for horseradish peroxidase and explained their reactivity in the horseradish peroxidase/guaiacol assay system. The compound 5-aminosalicylic acid showed an unusual behaviour that was attributed to its ability to both scavenge hydrogen peroxide and interact with horseradish peroxidase. It was concluded that the lack of specificity of horseradish peroxidase for its donor substrate may lead to erroneous results in assays for hydrogen peroxide scavenging of some drugs. An alternative method is however available and a simple spectroscopic assay can evidence the interaction with horseradish peroxidase.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Depuradores de Radicales Libres , Glutatión Peroxidasa/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidasa de Rábano Silvestre/química , Técnicas In Vitro , Oxidación-ReducciónRESUMEN
Performing the deoxyribose (DR) assay for determination of the rate constants for reaction of non steroidal antiinflammatory drugs with hydroxyl radicals led to some unusual competition plots. The molecules from the arylpropionic family of drugs: ibuprofen, flurbiprofen, ketoprofen and naproxen produced the linear relationship. However, acemetacin, diclofenac Na, flufenamic acid, indometacin, indometacin, niflumic acid, tolmetin Na and sulindac presented non linear competition plots manifesting at relatively low drug concentrations. This effect was corrected by increasing DR concentrations from 2.8 mM to 15 mM. The modification did not affect rate constants values for those derivatives which already presented a linear plot at 2.8 mM, but allowed to calculate rate constants for other compounds. It is suggested that the experimental conditions have to be adapted particularly for those derivatives with a relatively high rate constant for reaction with the radical species. The oxicam derivatives (tenoxicam and piroxicam) presented another kind of deviation that revealed a prooxidant effect in this system: non linear plots were also observed at relatively low drug concentrations, but in the opposite direction than for the other molecules. This last effect was independent of DR concentration but could be corrected by increasing ascorbate concentration in the system.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Desoxirribosa/química , Unión Competitiva , Depuradores de Radicales Libres , Radical Hidroxilo , Cinética , Modelos Lineales , Modelos Estadísticos , Oxidantes/farmacologíaRESUMEN
Serum thiobarbituric acid reactive substances (TBARS), Zn, Cu, and Se concentrations were determined in 47 healthy adults and in patients with diseases, such as renal insufficiency, insulin-dependent diabetes mellitus, chronic pancreatitis, liver cirrhosis, or cancer, in order to clarify the relationship between this indicator of lipid peroxidation and antioxidative trace element status. TBARS levels were higher than control values in all pathological cases, except in cancer patients. Cu levels in patients highly correlated with ferroxidase ceruloplasmin activity (r = 0.86), but were only statistically different from controls in diabetics. Zn levels were lower than normal in dialysis, liver cirrhosis, and cancer patients. Se levels were significantly decreased in all pathological cases. Half of the subjects with liver cirrhosis or renal insufficiency and 3/4 of chronic pancreatitis or cancer patients had an active inflammatory process. Despite intense modifications in determined indicators, no clear correlation could be demonstrated between the different parameters. Basic antioxidative trace element status and inflammation are therefore not major determinants of TBARS levels in normal and in pathological conditions, despite of the frequent association of low serum Zn and mainly low serum Se with high TBARS levels.