RESUMEN
Background Selinexor, a first-in-class, oral selective inhibitor of nuclear export (SINE) compound inhibits Exportin-1(XPO1), had demonstrated synergistic activity with many chemotherapies and conferred in vivo antitumor efficacy in hematologic as well as solid tumors. Methods This open-label, single-center, multi-arm phase 1b study used a standard 3 + 3 design and a "basket type" expansion. Selinexor with intravenous topotecan was given in one of the 13 parallel arms. Patients with advanced or metastatic relapsed/refractory solid tumors following prior systemic therapy, or in whom the addition of selinexor to standard chemotherapy deemed appropriate, were eligible. Results Fourteen patients with the median age of 61 years (range, 22-68years) were treated, and the most common cancer types were gynecological cancers; ovarian (n = 5), endometrial (n = 2), and 1 each with fallopian tube and vaginal cancers. Of the 14 patients treated, 12 (86 %) had at least one treatment-related adverse event (TRAE). The most common TRAEs were anemia (71 %), thrombocytopenia (57 %), hyponatremia (57 %), vomiting (57 %), fatigue (50 %), nausea (50 %), and neutropenia (36 %). Two patients had dose limiting toxicities. One patient dosed at selinexor 80 mg had grade 3 nausea and vomiting and one patient dosed at selinexor 60 mg experienced grade 4 neutropenia and thrombocytopenia. Of the 13 efficacy evaluable patients, one (8 %) with endometrial cancer achieved unconfirmed partial response (uPR) and the time-to-treatment failure (TTF) was 48 weeks, whereas 6 of the 13 (46 %) patients had stable disease (SD) contributing to the clinical benefit rate of 46 %. The median TTF for all patients was 9 weeks (range, 2-48weeks). Conclusions Once weekly selinexor in combination with topotecan was viable and showed some preliminary tumor efficacy. The recommend phase 2 dose of selinexor was 60 mg once weekly in combination with IV topotecan.Trial registration: NCT02419495. Registered 14 April 2015, https://clinicaltrials.gov/ct2/show/NCT02419495.
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Hidrazinas/uso terapéutico , Carioferinas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/uso terapéutico , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Hidrazinas/administración & dosificación , Hidrazinas/efectos adversos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/patología , Topotecan/uso terapéutico , Triazoles/administración & dosificación , Triazoles/efectos adversos , Proteína Exportina 1RESUMEN
Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by alpha-keto-gamma-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O2-) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O2- generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.
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Caballos/fisiología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Compuestos de Bifenilo/farmacología , Degranulación de la Célula/efectos de los fármacos , Citocalasina B/farmacología , Etilenos/metabolismo , Enfermedades de los Caballos/fisiopatología , Caballos/inmunología , Técnicas In Vitro , Inflamación/fisiopatología , Inflamación/veterinaria , Mediciones Luminiscentes , Neutrófilos/inmunología , Compuestos Onio/farmacología , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/farmacologíaRESUMEN
The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.
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Caballos/sangre , Interleucina-1beta/farmacología , Neutrófilos/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Acridinas/química , Animales , Mediciones Luminiscentes , Luminol/química , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Estallido Respiratorio/inmunologíaRESUMEN
An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.
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Líquidos Corporales/enzimología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Caballos , Peroxidasa/aislamiento & purificación , Peroxidasa/metabolismo , Animales , Curcumina/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Caballos/enzimología , Neutrófilos/enzimología , Peroxidasa/antagonistas & inhibidores , Resveratrol , Sensibilidad y Especificidad , Estilbenos/farmacología , Temperatura , Factores de TiempoRESUMEN
Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.
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Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedades de los Caballos/enzimología , Obstrucción Intestinal/veterinaria , Neutrófilos/enzimología , Peroxidasa/sangre , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Caballos/sangre , Caballos , Obstrucción Intestinal/sangre , Obstrucción Intestinal/enzimologíaRESUMEN
Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O2 concentration for 14 days, cellular growth kinetics were measured (n=6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (ATP) content was measured. The number of chondrocytes remained stable for the first eight days, then decreased especially at 1% and 21% O2. At 21% O2, normal cells decreased and necrotic cells increased at the end of the 14 day-period. No significant variations were found at 5% O2 except for a decrease in necrotic cells at day 14. Most apoptotic cells were found at 1% O2 from days 5 to 11, and normal cells decreased during the same period. But an unexpected increase in normal cells and decrease in apoptotic cells were observed at day 14. The intracellular ATP content remained stable. It was concluded that, in a three-dimensional culture model of equine articular chondrocytes, O2 tension affected the viability of the cells after an 11-day period, with the most important effects observed at 21% and 1% O2 conditions.
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Cartílago Articular/citología , Técnicas de Cultivo de Célula/veterinaria , Condrocitos/efectos de los fármacos , Caballos/metabolismo , Oxígeno/farmacología , Alginatos/farmacología , Animales , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Condrocitos/citología , Condrocitos/metabolismo , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Oxígeno/administración & dosificaciónRESUMEN
BACKGROUND/AIMS: Pathogenesis of non-alcoholic steatohepatitis (NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1), cytokines, oxidative stress and activation of hepatic stellate cells seem to play a role in this process. The aim was to determine the potential implication of these factors in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS: Animals were fed a standard diet, a 5% orotic acid-diet (OA) developing hepatic steatosis, or the methionine-choline deficient (MCD) diet inducing steatohepatitis for 2 and 6 weeks. Lipid peroxidation, CYP 2E1 expression and activity, expression of UCP-2, interleukin (IL)-6, transforming growth factor (TGF)beta1, KLF6 mRNAs, and activation of hepatic stellate cells were examined by gas chromatography, high-performance liquid chromatography, Western blotting, quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Lipid peroxidation increased in the MCD model whereas only minor changes occurred in the OA model. KLF6 and TGFbeta1 mRNAs were selectively up-regulated in MCD animals. Stellate cell activation, inflammation and collagen deposition only occurred in the MCD group. CYP 2E1 expression and activity increased in the OA group while both decreased in MCD animals. UCP-2 and IL-6 mRNA increased in both groups. CONCLUSIONS: In the context of steatosis, lipid peroxidation is associated with inflammation and stellate cell activation with concomitant increase in TGFbeta1 production, possibly through up-regulation of KLF6.
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Hígado Graso/complicaciones , Hígado Graso/diagnóstico , Cirrosis Hepática/etiología , Estrés Oxidativo , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Actinas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Diagnóstico Diferencial , Progresión de la Enfermedad , Hígado Graso/patología , Inmunohistoquímica , Interleucina-6/genética , Canales Iónicos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Metabolismo de los Lípidos , Peroxidación de Lípido , Hígado/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Microsomas Hepáticos/metabolismo , Proteínas Mitocondriales/genética , Músculo Liso/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transactivadores/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Proteína Desacopladora 2 , Regulación hacia ArribaRESUMEN
Gastrointestinal disorders in horses leading to endotoxic shock could have further consequences on other splanchnic organs such as the pancreas, as can be seen in humans suffering from septic shock. In this study, the range of enzymatically active trypsin (EAT) in healthy horses was established and is similar to the range observed in healthy humans. EAT values were determined in horses with acute abdominal crises on admission as well as during anaesthesia and in the postoperative phase. A significant increase in plasma EAT was found in 59% of the horses with surgical colic when compared to our established reference range. Significantly higher values were found in severe shock cases. When separated in groups according to the duration of colic before referral, significantly higher EAT values were observed in the non-survivor group compared to the survivor group of colics of short duration. EAT plasma values increased significantly during the postoperative phase, and were significantly higher in small intestine obstructions than in large bowel disorders. In human medicine, hypovolaemic or septic shock patients show an increase in pancreatic proteases. Splanchnic hypoperfusion during shock could lead to pancreatic damage resulting in trypsin liberation into the peritoneal space and an increase in plasma levels. Trypsin is able to activate inflammatory cascades and leucocytes and could play a role in multiple organ failure. Further studies are needed to evaluate the implications of changes in plasma trypsin in the disease process of equine acute abdomen and to demonstrate possible pancreatic damage.
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Enfermedades de los Caballos/fisiopatología , Caballos/sangre , Obstrucción Intestinal/veterinaria , Insuficiencia Multiorgánica/veterinaria , Tripsina/sangre , Animales , Biomarcadores/análisis , Inflamación , Obstrucción Intestinal/diagnóstico , Leucocitos , Insuficiencia Multiorgánica/fisiopatología , Páncreas/patología , Valores de ReferenciaRESUMEN
Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1), the cells were differentiated into macrophages by preincubation with C. pneumoniae extract, and further stimulated by phorbol myristate acetate. In these conditions, the differentiated cells oxidized a thiol compound and released superoxide anion as demonstrated respectively by gas liquid chromatography and electron spin resonance. The thiol oxidation and superoxide anion release were inhibited by diphenyliodonium, a NADPH oxidase and NOsynthase inhibitor, proving that the respiratory burst and the NOsynthase were involved in the oxidation processes occurring in the differentiated THP-1. The role of H(2)O(2) (derived from superoxide anion) was indicated by the enhancing effect of a peroxidase on the thiol oxidation. The presence of alpha-tocopherol in the surrounding medium strongly diminished the oxidation of the thiol target.
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Macrófagos/metabolismo , Monocitos/metabolismo , Oxígeno/metabolismo , Antibacterianos/farmacología , Compuestos de Bifenilo/farmacología , Carcinógenos , Diferenciación Celular , Línea Celular , Chlamydophila pneumoniae/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Cromatografía de Gases y Espectrometría de Masas , Humanos , Peróxido de Hidrógeno/farmacología , Modelos Químicos , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Compuestos Onio/farmacología , Compuestos de Sulfhidrilo/metabolismo , Superóxido Dismutasa/farmacología , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol , Ácido Úrico/farmacología , Vitamina E/metabolismo , Vitamina E/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.
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Biopterinas/análogos & derivados , Biopterinas/metabolismo , Cloro/metabolismo , Radicales Libres/metabolismo , Óxidos/metabolismo , Ácido Peroxinitroso/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Transducción de Señal , Espectrofotometría Ultravioleta , Factores de TiempoRESUMEN
OBJECTIVES: Reactive oxygen species (ROS) are now recognized to play an important role in the pathogenesis of rheumatic diseases and constitute an interesting therapeutic target for drugs. This in vitro study was designed to evaluate the antioxidant properties of nimesulide (NIM), a nonsteroidal antiinflammatory drug of the sulfonanilide class, and its main metabolite 4-OH nimesulide (4-OHNIM). METHODS: The scavenging effects of NIM and 4-OH NIM on hydroxyl radical ((.)OH) and superoxide anions (O(minusd)(2)) were investigated by electron spin resonance (ESR), using 5, 5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. The quenching properties of these drugs on hypochlorite anion was studied by luminol enhanced chemiluminescence. Finally, the effects of NIM and 4-OHNIM on the reactive oxygen species production by human articular chondrocytes were recorded by HRP and luminol-enhanced chemiluminescence. RESULTS: By this method it has been demonstrated that NIM and 4-OHNIM, at concentrations ranging from 10 to 100 microM, are potent scavengers of(.)OH whereas only 4-OHNIM was capable to scavenge O(minusd)(2). Chemiluminescence generated by HOCl was also significantly and dose-dependently inhibited by both NIM and 4-OHNIM. Nevertheless, at each concentration tested, the inhibitory effect of 4-OHNIM was significantly more marked, even at the highest concentration (100 microM). Furthermore, when chondrocytes were pre-incubated for 48-96 h with NIM or 4-OHNIM, the luminol- and HRP-dependent CL produced by the cells was significantly inhibited in a dose-dependent manner. CONCLUSIONS: NIM and 4-OHNIM may protect cartilage against oxidative stress, not only by scavenging ROS but also by inhibiting their production by chondrocytes.
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Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Condrocitos/efectos de los fármacos , Sulfonamidas/farmacología , Anciano , Cartílago Articular/citología , Técnicas de Cultivo de Célula , Condrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Depuradores de Radicales Libres/farmacología , Peroxidasa de Rábano Silvestre/farmacología , Humanos , Radical Hidroxilo/farmacología , Mediciones Luminiscentes , Luminol/farmacología , Masculino , Persona de Mediana Edad , Superóxidos/farmacologíaRESUMEN
We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.
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Citotoxicidad Inmunológica , Endotelio/inmunología , Neutrófilos/inmunología , Animales , Células Cultivadas , Enfermedades de los Caballos/inmunología , Caballos , Peróxido de Hidrógeno/metabolismo , Inflamación/inmunología , Inflamación/veterinaria , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
OBJECTIVES: To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite. METHODS: The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe2+/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps. RESULTS: At 10 microM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe2+/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 microM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO-, suggesting a potential capacity of the molecules to scavenge peroxynitrite. CONCLUSION: The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.
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Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Neutrófilos/efectos de los fármacos , Antiinflamatorios no Esteroideos/metabolismo , Antioxidantes/metabolismo , Ácido Ascórbico , Cloro/metabolismo , Diclofenaco/análogos & derivados , Diclofenaco/metabolismo , Diclofenaco/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Indometacina/metabolismo , Indometacina/farmacología , Hierro , Cinética , Peroxidación de Lípido , Mediciones Luminiscentes , Activación Neutrófila , Neutrófilos/metabolismo , Nitratos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hipoclorito de Sodio/farmacología , Espectrofotometría Ultravioleta , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Acetato de TetradecanoilforbolRESUMEN
OBJECTIVE AND DESIGN: Because high concentrations of histamine are locally released in inflammation, we investigated the effects of supraphysiological doses of histamine on the production of reactive oxygen species (ROS) by neutrophils. MATERIALS AND METHODS: Isolated equine neutrophils were activated by 10(-4) to 5 x 10(-3) M histamine. The production of ROS and free radicals was estimated by luminol-enhanced chemiluminescence (CL) and electron spin resonance (ESR) with spin trapping technique. In this model of histamine-stimulated neutrophils, we tested the antagonists of H1 and H2 histamine receptors, the role of Ca2+ and Mg2+, the role of staurosporine and pertussis toxin (inhibitors of protein kinase C and proteins G) and the effects of superoxide dismutase, catalase, hydroxyl radical scavengers (phenylalanine and mannitol) and N(G)-monomethyl-L-arginine (L-NMMA), inhibitor of NO-synthase. RESULTS: Histamine (from 10(-5) to 10(-3) M) stimulated neutrophils to produce CL and ESR signals characterized by spin adducts of superoxide anion and/or hydroxyl radicals. The CL response was inhibited by 10(-4) and 10(-3) M H1 receptor antagonists (promethazine, pyrilamine, and diphenhydramine), by Ca2+ and Mg2+ depletion and by 10 nmoles staurosporine. CL was partially inhibited by pertussis toxin (4 microg/ mL). The ESR signals were practically suppressed by pyrilamine (an H1 receptor antagonist) and superoxide dismutase, and partially inhibited by catalase, hydroxyl radical scavengers and L-NMMA (respectively 59, +/- 30% and 68% inhibition). CONCLUSIONS: High concentrations of histamine stimulated the neutrophils to product ROS and free radicals via H1 receptors and the NADPH-oxidase pathway.
Asunto(s)
Histamina/farmacología , Caballos/metabolismo , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Calcio/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Radicales Libres/metabolismo , Proteínas de Unión al GTP/metabolismo , Técnicas In Vitro , Indicadores y Reactivos , Mediciones Luminiscentes , Magnesio/metabolismo , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores Histamínicos/efectos de los fármacos , Receptores Histamínicos/metabolismo , Estimulación QuímicaRESUMEN
Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3). The mean peak values of MPO for these phases were 121.6 ng/mL (P1), 168.6 ng/mL (P2), and 107.0 ng/mL (P3) for the non-strangulating group, and 242.6 ng/mL (P1); 426.0 ng/mL (P2), and 379.5 ng/mL (P3) for the strangulation group. The variations of the mean peak values of plasma MPO were significantly different between the 2 groups and between the different phases. A significant increase of the least square means of MPO was observed between P1 and P2. A significant decrease of the least square means of the number of circulating leukocytes was observed between P1 and P3. Polymorphonuclear neutrophil activation could play a major role in the pathogenesis of acute abdominal disease and endotoxic shock.
Asunto(s)
Enfermedades del Colon/veterinaria , Enfermedades de los Caballos/sangre , Obstrucción Intestinal/sangre , Obstrucción Intestinal/cirugía , Neutrófilos/fisiología , Peroxidasa/sangre , Enfermedad Aguda , Animales , Enfermedades del Colon/fisiopatología , Enfermedades del Colon/cirugía , Cuidados Críticos , Enfermedades de los Caballos/fisiopatología , Enfermedades de los Caballos/cirugía , Caballos , Obstrucción Intestinal/fisiopatología , Análisis de los Mínimos Cuadrados , Recuento de LeucocitosRESUMEN
Ultraweak luminescence (uwCL) was coupled with electron spin resonance to study the reactions of 3 heterocyclic compounds (tryptophan, serotonin and imidazole) with peroxynitrite at pH 8.7. Tryptophan and serotonin reacted with emission of a flash peak of light (5 s) followed by a long-living light emission of +/- 80 s. Addition of the spin trap 4-POBN at different intervals, after the beginning of reaction revealed that a short-living free radical was produced in the case of serotonin and imidazole, but that with tryptophan, the initial radical rearranged into a relatively long-living radical, which was still formed when 4-POBN was added after 55 s (decreasing phase of uwCL).
Asunto(s)
Compuestos Heterocíclicos/química , Nitratos/química , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Imidazoles/química , Cinética , Mediciones Luminiscentes , Estructura Molecular , Óxidos de Nitrógeno/química , Piridinas , Serotonina/química , Marcadores de Spin , Triptófano/químicaAsunto(s)
Acepromazina/farmacología , Antipsicóticos/farmacología , Caballos/sangre , Neutrófilos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Histamina/farmacología , Caballos/metabolismo , Lipopolisacáridos/farmacología , Mediciones Luminiscentes , Especies Reactivas de Oxígeno , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
To address the question of whether the increased plasma concentration of interleukin 6 (IL-6) following strenuous muscular work could be related to exercise-induced muscle damage, 5 moderately active male volunteers underwent two isokinetic exercise sessions in the eccentric mode, separated by a period of 3 weeks during which the subjects underwent five training sessions. Before training, exercise was followed by severe muscle pain (delayed-onset muscle soreness; DOMS), and by significant increases in plasma IL-6 level and serum myoglobin concentration (SMb) (P < 0.001). After training, postexercise DOMS and SMb values were significantly lower than those measured before training. There was no significant difference between plasma IL-6 levels measured at the same time points before and after training. We conclude that the hypothetical relationship between exercise-induced muscle damage and increased postexercise levels of circulating IL-6 is not substantiated by the present results.