RESUMEN
OBJECTIVE AND DESIGN: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo. MATERIALS AND TREATMENT: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively. METHODS: 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA. RESULTS: Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models. CONCLUSIONS: Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Inhibidores de la Ciclooxigenasa/toxicidad , Indoles/toxicidad , Inhibidores de la Lipooxigenasa , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Araquidonato 5-Lipooxigenasa/sangre , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Ácido Araquidónico/toxicidad , Calcimicina/toxicidad , Factores Quimiotácticos/metabolismo , Cromatografía Líquida de Alta Presión , Inhibidores de la Ciclooxigenasa/administración & dosificación , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Técnicas para Inmunoenzimas , Indoles/administración & dosificación , Ionóforos/toxicidad , Leucemia Basofílica Aguda/enzimología , Leucemia Basofílica Aguda/patología , Leucotrieno B4/metabolismo , Leucotrieno E4/metabolismo , Activación Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Oxindoles , Conejos , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Tromboxano B2/metabolismo , Zimosan/toxicidadRESUMEN
Tenidap is a new anti-rheumatic agent which has clinical properties characteristic of a disease modifying drug combined with acute antiinflammatory and analgesic activity. This paper details tenidap's cyclooxygenase (COX) inhibitory activity and the resulting pharmacological properties in experimental animals. Tenidap inhibited calcium ionophore-stimulated prostaglandin D2 synthesis by rat basophilic leukemia cells (COX-1) with an IC50 of 20 nM. In two different in vitro human test systems, tenidap inhibited COX-1 activity more potently than COX-2, although the relative potency ratio (COX-1/COX-2) differed markedly between the two systems. Tenidap inhibited the COX pathway when added to human blood in vitro (IC50, 7.8 mu M) and when administered orally to monkeys, rats and dogs (at 5, 2.5 and 10 mg/kg p.o., respectively) and COX activity measured ex vivo in blood collected 2 to 4 hours post dose. After oral administration to rats, tenidap inhibited carrageenan-induced paw edema with an ED50 of 14 mg/kg and inhibited the glucocorticoid-resistant UV erythema in guinea pigs with an ED50 of 1.4 mg/kg. It retained antiinflammatory activity in adrenalectomized rats indicating that this property is independent of adrenal stimulation. Oral administration of tenidap inhibited the development of adjuvant-induced polyarthritis in the rat and exhibited antinociceptive activity in the murine phenylbenzoquinone and rat acetic acid abdominal constriction tests. These data indicate that tenidap is an effective antiinflammatory and analgesic agent in animal models. These cyclooxygenase-dependent pharmacologic activities do not explain tenidap's disease modifying anti-arthritic properties but add a useful symptom modifying component to its clinical profile.
Asunto(s)
Analgésicos no Narcóticos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Artritis/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Indoles/farmacología , Animales , Ácido Araquidónico/metabolismo , Perros , Cobayas , Haplorrinos , Humanos , Masculino , Ratones , Oxindoles , Ratas , Ratas Sprague-DawleyRESUMEN
Inhaled PAF provokes bronchoconstriction, causes peripheral blood neutropenia with rebound neutrophilia, and generates urinary production of the bronchoconstrictor eicosanoids, thromboxane (TX)A2, and the cysteinyl leukotrienes. We examined the effects of an oral PAF antagonist UK,74505 on each of these responses to a single 36 micrograms dose of inhaled PAF. In a double-blind randomized placebo-controlled crossover study, 12 normal male subjects inhaled PAF on two consecutive days, 3 and 24 h after intake of two doses of UK,74505 25 mg and 100 mg, or matched placebo (P). After P, inhalation of PAF provoked bronchoconstriction, measured at regular time points for 60 min as a change in sGaw from baseline and computed as area under the curve (AUC), induced a neutropenia at 5 min and rebound neutrophilia at 2 h, and stimulated production of urinary eicosanoids. Bronchoconstriction was maximal at 5 min but had receded at 1 h; (AUC mean [95% Cl]; 20.0 [13.2, 26.8] at 3 h; 11.0 [5.3, 16.6] at 24 h) and was completely abolished by both doses of UK,74505 at 3 h and by the higher 100 mg dose at 24 h. PAF-induced neutropenia and rebound neutrophilia were abolished by both doses of drug; neutropenia at 5 min (expressed as mean [95% Cl] change from baseline; -2.5 x 10(9)/L [-2.9, -2.1] after P; -0.3 [-0.7, 0.1] after 25 mg; 0.1 [-0.3, 0.4] after 100 mg), neutrophilia at 2 h (2.0 [-1.3, 2.6] after P; -0.2 [-0.8, 0.5] after 25 mg; -0.1 [-0.8, 0.5] after 100 mg).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Broncoconstricción/efectos de los fármacos , Dihidropiridinas/farmacología , Eicosanoides/orina , Imidazoles/farmacología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/farmacología , Administración por Inhalación , Administración Oral , Resistencia de las Vías Respiratorias/efectos de los fármacos , Dihidropiridinas/administración & dosificación , Dihidropiridinas/farmacocinética , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacocinética , Recuento de Leucocitos/efectos de los fármacos , Leucotrieno E4/orina , Masculino , Neutropenia/inducido químicamente , Factor de Activación Plaquetaria/administración & dosificación , Tromboxano B2/análogos & derivados , Tromboxano B2/orinaRESUMEN
Ampiroxicam is a nonacidic ether carbonate prodrug of piroxicam. Our results demonstrate that, in contrast to piroxicam, ampiroxicam does not possess detectable prostaglandin synthesis inhibitory activity in vitro. Ampiroxicam, however, has similar in vivo potency to piroxicam in suppressing paw swelling in rat adjuvant arthritis. In an acute model of paw inflammation in rats, ampiroxicam is less potent than piroxicam itself: the ED50's of ampiroxicam are 9- and 3.5-fold higher than those of piroxicam following a single or multiple (5) daily oral doses, respectively. Using the phenylbenzoquinone stretching test as a method of evaluating acute analgetic activity, the ED50 for ampiroxicam is about 3-fold higher than that of piroxicam. These tests of activity share the property of being partially prostaglandin-dependent. Ampiroxicam itself is not observed in plasma after oral dosing to man, nor in the rat, dog, and monkey as reported here. Bioavailability studies show that conversion to piroxicam is about 100%, 90%, 70%, and 50% in these four species, respectively. These results indicate that ampiroxicam's anti-inflammatory activity is produced in vivo by conversion to piroxicam and support its credentials as an efficacious prodrug of piroxicam.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Profármacos/farmacología , Tiazinas/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Artritis Experimental/tratamiento farmacológico , Benzoquinonas/farmacología , Disponibilidad Biológica , Biotransformación , Calcimicina/farmacología , Células Cultivadas , Perros , Edema/inducido químicamente , Edema/patología , Haplorrinos , Humanos , Absorción Intestinal , Masculino , Ratones , Profármacos/farmacocinética , Antagonistas de Prostaglandina/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Ratas Sprague-Dawley , Tiazinas/farmacocinéticaRESUMEN
Mouse peritoneal macrophages stimulated with LPS produce large amounts of pro-IL-1 beta. When these cells were pulse-labeled with [35S]methionine, however, little labeled cytokine appeared in the medium after a chase, and that which was externalized was not processed to its mature biologically active form. In an effort to promote proteolytic maturation of IL-1 beta, macrophages were treated with agents that were expected to compromise their viability. The calcium ionophore A23187 and the detergent saponin caused complete release of nonprocessed 35-kDa pro-IL-1 beta and liberation into the extracellular medium of the cytoplasmic marker enzyme LDH and the lysosomal enzyme beta-N-acetylglucosaminidase. Hypotonic lysis resulted in the release of a 20-kDa IL-1 beta species that was distinct from the 17-kDa mature species. Importantly, incubation of the murine macrophages with the potassium/proton ionophore nigericin led to a quantitative conversion of pro-IL-1 beta to a 17-kDa species. The N-terminus of this nigericin-derived product possessed the amino acid sequence expected for mature biologically active IL-1 beta. Monensin, an ionophore similar to nigericin, did not induce release or proteolysis of IL-1 beta. Complete release of mature IL-1 beta required concentrations of nigericin in excess of 2 microM and a minimum of 10 min of treatment. Mature 17-kDa IL-1 beta was observed within the nigericin-treated cells before their lysis. Nigericin's effect was not limited to mouse peritoneal macrophages, inasmuch as the ionophore also induced release and proteolytic maturation of IL-1 beta produced by LPS-stimulated human peripheral blood monocytes. Treatment of macrophages with LPS and nigericin, therefore, results in a unique series of intracellular events that promote formation of mature 17-kDa IL-1 beta.
Asunto(s)
Interleucina-1/metabolismo , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Nigericina/farmacología , Animales , Exocitosis/efectos de los fármacos , Humanos , Técnicas In Vitro , Interleucina-1/química , Lipopolisacáridos/administración & dosificación , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C3H , Peso Molecular , Monocitos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
We studied the effect of tenidap sodium, a new antiinflammatory/antirheumatic drug (120 mg/day for 7 days), on eicosanoid production and neutrophil degranulation in patients with rheumatoid arthritis. Endogenous prostaglandin E2 levels and ex vivo production of leukotriene B4 (LTB4) were measured in synovial fluid samples obtained at baseline and 1 week later. We measured peripheral blood polymorphonuclear cell (PMN) degranulation following surface-bound IgG stimulation, a possible 5-lipoxygenase product-mediated event, by determining lactoferrin and elastase release into the culture fluid. We found decreased levels of endogenous prostaglandin E2 as measured by radioimmunoassay, and decreased ex vivo production of LTB4 by PMN as measured by high performance liquid chromatography, in synovial fluid samples from patients who took tenidap. Release of the granule proteins lactoferrin and elastase was decreased in PMN obtained from patients receiving tenidap, as well as in the PMN incubated in vitro with tenidap. Improvement in clinical measures paralleled the biochemical changes. The unique 5-lipoxygenase inhibitory property of tenidap, as measured by LTB4 production and degranulation, suggests that it may have clinical activity which differentiates it from nonsteroidal antiinflammatory drugs.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Degranulación de la Célula/efectos de los fármacos , Indoles/uso terapéutico , Inhibidores de la Lipooxigenasa , Neutrófilos/fisiología , Anciano , Artritis Reumatoide/fisiopatología , Células Cultivadas/metabolismo , Cromatografía Líquida de Alta Presión , Inhibidores de la Ciclooxigenasa , Dinoprostona/metabolismo , Humanos , Lactoferrina/metabolismo , Leucotrieno B4/metabolismo , Persona de Mediana Edad , Oxindoles , Elastasa Pancreática/metabolismo , Radioinmunoensayo , Líquido Sinovial/metabolismoRESUMEN
A series of 1,2-dihydro-1-oxopyrrolo[3,2,1-kl]phenothiazine, 1,2-dihydro-1-oxopyrrolo[3,2,1-kl]phenoxazine, and 1,2-dihydro-1-oxopyrrolo[3,2,1-de]acridine-2-carboxamides were prepared by reaction of 1,2-dihydro-1-oxo-pyrrolo[3,2,1-kl]phenothiazine or other corresponding phenoxazine and acridan ethyl or methyl esters with appropriate amines. Several members of this family were found to be potent, dual inhibitors of cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism and to have in vivo antiinflammatory activity in the rat foot edema assay. Structure-activity relationships within this family of compounds are described. 1,2-Dihydro-N-(2-thiazolyl)-1-oxopyrrolo[3,2,1-kl]phenothiazine-1- carboxamide (34) was found to be one of the best compounds to display potent cyclooxygenase/5-lipoxygenase inhibition of arachidonic acid metabolism. Its IC50s against the enzymes sourced from rat basophillic leukemia-1 (RBL-1) cells were 0.07 and 1.4 microM, respectively. It was active in the rat foot edema test for antiinflammatory effect (48% inhibition at 33 mg/kg po) and in the mouse phenylbenzoquinone induced writhing test for analgesic effect (93% inhibition at 32 mg/kg po).
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Araquidonato Lipooxigenasas/antagonistas & inhibidores , Inhibidores de la Ciclooxigenasa , Inhibidores de la Lipooxigenasa , Antagonistas de Prostaglandina/síntesis química , Pirroles/síntesis química , Tiazinas/síntesis química , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Línea Celular , Edema , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Pirroles/farmacología , Pirroles/uso terapéutico , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Tiazinas/farmacología , Tiazinas/uso terapéuticoRESUMEN
A series of 3-substituted indeno[1,2-c]pyrazol-4(1H)-one-2-acetic acids (3a-e) and 3-substituted indeno[1,2-c]pyrazol-4(1H)-one-1-acetic acids (4a-e) were synthesized as semirigid analogues of tolmetin (1). These compounds were evaluated for their anti-inflammatory action by investigating their ability to block arachidonic acid metabolism in vitro as well as the ability to block carrageenan-induced rat foot edema in vivo. No consistent pattern of biological activity was noted.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Indenos/síntesis química , Pirazoles/síntesis química , Animales , Ácidos Araquidónicos/metabolismo , Fenómenos Químicos , Química , Edema/metabolismo , Edema/prevención & control , Indenos/farmacología , Leucemia Experimental/metabolismo , Masculino , Prostaglandina-Endoperóxido Sintasas/metabolismo , Pirazoles/farmacología , Ratas , Ratas Endogámicas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoAsunto(s)
Interleucina-1/metabolismo , Interleucina-1/farmacología , Interleucinas/metabolismo , Interleucinas/farmacología , Animales , Interleucina-1/fisiología , Interleucina-6 , Interleucinas/fisiología , Receptores Inmunológicos/aislamiento & purificación , Receptores Inmunológicos/fisiología , Receptores de Interleucina-1RESUMEN
We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB4, 20-OH-LTB4, and 20-COOH-LTB4 are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB4, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.
Asunto(s)
Lipooxigenasa/sangre , Prostaglandina-Endoperóxido Sintasas/sangre , Calcimicina/farmacología , Cromatografía Líquida de Alta Presión , Ácidos Grasos Insaturados/sangre , Humanos , Técnicas In Vitro , Espectrofotometría UltravioletaRESUMEN
A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described. In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of +/- 12%. Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1-4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 micrograms/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaretic acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 micrograms/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.
Asunto(s)
Araquidonato Lipooxigenasas/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa , Araquidonato 5-Lipooxigenasa/sangre , Ácidos Araquidónicos/sangre , Calcimicina/farmacología , Cromatografía Líquida de Alta Presión , Citocalasina B/farmacología , Ácido Egtácico/farmacología , Humanos , Ácidos Hidroxieicosatetraenoicos/sangre , Técnicas In Vitro , Cinética , Leucotrieno A4 , Leucotrieno B4/sangre , Radioinmunoensayo , Espectrofotometría Ultravioleta , Levadura Seca/farmacologíaRESUMEN
12-Hydroxyheptadecatrienoic acid (HHT), a UV chromophore, has been used to assess cyclooxygenase (CO) pathway metabolism of arachidonic acid (AA) by rat peritoneal cells. Simultaneous monitoring at 235 and 280 nm after HPLC permits the measurement of both CO and 5-lipoxygenase (5-LO) pathway fluxes in a single system.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos Insaturados , Macrófagos/enzimología , Prostaglandina-Endoperóxido Sintasas/análisis , Espectrofotometría Ultravioleta/métodos , Animales , Araquidonato 5-Lipooxigenasa/análisis , Ácidos Araquidónicos/metabolismo , Líquido Ascítico/citología , Calcimicina/farmacología , Inhibidores de la Ciclooxigenasa , Eosinófilos/efectos de los fármacos , Eosinófilos/enzimología , Ácidos Hidroxieicosatetraenoicos/análisis , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Leucotrieno B4/análisis , Leucotrieno B4/biosíntesis , Inhibidores de la Lipooxigenasa , Macrófagos/efectos de los fármacos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Antagonistas de Prostaglandina/farmacología , Ratas , Ratas Endogámicas , Estándares de ReferenciaRESUMEN
The development of a radioimmunoassay for measuring subnanogram amounts of the prostaglandin uterine stimulant, sulprostone (N-methanesulfonyl 16-phenoxy-omega-tetranor PGE2 carboxamide), is described. The 9-carboxymethoxime derivative of sulprostone was coupled to keyhole limpet hemocyanin to prepare the immunogen, and to tyramine to yield a precursor suitable for radioiodination. The antiserum generated from rabbits was specific for sulprostone, showing cross reactivities of less than 0.1% against PGE2, PGF2 alpha, and known sulprostone metabolites. The range for routine assay of sulprostone was 10-300 pg which corresponded to 50-1500 pg/ml of plasma. The coefficient of variation for replicate analyses on the same sample was 8-12%. The assay was used to measure the plasma levels of sulprostone in patients who had received the drug intramuscularly.
Asunto(s)
Dinoprostona/análogos & derivados , Prostaglandinas E Sintéticas/análisis , Animales , Reacciones Cruzadas , Humanos , Inyecciones Intramusculares , Microquímica/métodos , Prostaglandinas E Sintéticas/administración & dosificación , Conejos , Radioinmunoensayo/métodosRESUMEN
Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [125I]Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21-80% the binding of antibodies and consequently of [125I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography.
Asunto(s)
Ácidos Araquidónicos/análisis , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animales , Cromatografía Líquida de Alta Presión , Humanos , Conejos/inmunología , Radioinmunoensayo/métodos , Albúmina SéricaRESUMEN
An efficient method for the enzymic preparation of high specific radioactivity [3H]-12-L-hydroxyeicosatetraenoic acid (12-L-HETE) is described. This compound was used as a radiolabeled ligand in the radioimmunoassay (RIA) of 12-L-HETE. The accuracy of the RIA was checked by incubating [14C]arachidonic acid with platelet lipoxygenase, and measuring enzyme activity in the presence of the inhibitor, 1-phenyl-3-pyrazolidinone (phenidone). The amount of 12-L-HETE synthesized, determined by RIA, was found to be in agreement with that analyzed by radiochemical assay after thin layer plate chromatography.
Asunto(s)
Ácidos Araquidónicos/síntesis química , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Ácidos Araquidónicos/análisis , Inhibidores de la Lipooxigenasa , Métodos , Pirazoles/farmacología , RadioinmunoensayoRESUMEN
Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.
Asunto(s)
Antiinflamatorios/farmacología , Prostaglandinas/biosíntesis , Piridinas/farmacología , Tiazinas/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Línea Celular , Inhibidores de la Ciclooxigenasa , Epoprostenol/biosíntesis , Humanos , Técnicas In Vitro , Inhibidores de la Lipooxigenasa , Ratones , Microsomas/metabolismo , Piroxicam , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Tromboxano-A Sintasa/antagonistas & inhibidoresRESUMEN
The new non-steroidal antiinflammatory (NSAI)2 agent, piroxicam [4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide], is a highly active inhibitor of prostaglandin (PG) synthesis by methylcholanthrene transformed mouse fibroblasts (MC5-5) and rabbit synovial cells in culture. Comparison of the PG biosynthesis inhibitory activity of piroxicam with other NSAI drugs in these experiments ranks piroxicam as among the most potent agents of this type now known. Some specific modifications of piroxicam's structure result in significant loss in PG synthesis blocking activity.
Asunto(s)
Antiinflamatorios/farmacología , Prostaglandinas E/biosíntesis , Piridinas/farmacología , Tiazinas/farmacología , Animales , Transformación Celular Neoplásica , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Indometacina/farmacología , Ratones , Piroxicam , Conejos , Relación Estructura-Actividad , Membrana Sinovial/metabolismoRESUMEN
A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.