RESUMO
BACKGROUND AND PURPOSE: Translational animal models are essential in the prediction of the efficacy and side effects of new chemical entities. We have carried out a thorough study of three distinct disease-modifying antirheumatic drugs (DMARDs) in an adjuvant-induced arthritis (AIA) model in the rat and critically appraised the results in the context of the reported clinical experience in rheumatoid arthritis (RA) patients. EXPERIMENTAL APPROACH: Teriflunomide - a dihydroorotate dehydrogenase (DHODH) inhibitor; AL8697 - a selective p38 MAPK inhibitor; and tofacitinib - a Janus kinase (JAK) inhibitor; were selected as representatives of their class and dose-response studies carried out using a therapeutic 10-day administration scheme in arthritic rats. Paw swelling and body weight were periodically monitored, and joint radiology and histology, lymph organ weight and haematological and biochemical parameters evaluated at study completion. KEY RESULTS: All three drugs demonstrated beneficial effects on paw swelling, bone lesions and splenomegalia, with p38 inhibition providing the best anti-inflammatory effect and JAK inhibition the best DMARD effect. Leukopenia, body weight loss and gastrointestinal toxicity were dose-dependently observed with teriflunomide treatment. p38 MAPK inhibition induced leukocytosis and increased total plasma cholesterol. JAK inhibition, normalized platelet, reticulocyte and neutrophil counts, and alanine aminotransferase (ALT) levels while inducing lymphopenia and cholesterolemia. CONCLUSIONS AND IMPLICATIONS: This multiparametric approach can reveal specific drug properties and provide translational information. Whereas the complex profile for p38 inhibition in AIA is not observed in human RA, immunosuppressants such as DHODH and JAK inhibitors show DMARD properties and side effects seen in both AIA and RA.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Janus Quinases/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/efeitos adversos , Antirreumáticos/farmacocinética , Artrite Experimental/sangue , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Benzamidas/administração & dosagem , Benzamidas/efeitos adversos , Benzamidas/farmacocinética , Benzamidas/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Crotonatos/administração & dosagem , Crotonatos/efeitos adversos , Crotonatos/farmacocinética , Crotonatos/uso terapêutico , Di-Hidro-Orotato Desidrogenase , Relação Dose-Resposta a Droga , Edema/etiologia , Edema/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Trato Gastrointestinal/efeitos dos fármacos , Meia-Vida , Hidroxibutiratos , Hipercolesterolemia/induzido quimicamente , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Imunossupressores/farmacocinética , Imunossupressores/uso terapêutico , Articulações/efeitos dos fármacos , Articulações/imunologia , Transtornos Leucocíticos/induzido quimicamente , Masculino , Nitrilas , Piperidinas , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Pirróis/administração & dosagem , Pirróis/efeitos adversos , Pirróis/farmacocinética , Pirróis/uso terapêutico , Ratos , Ratos WistarRESUMO
BACKGROUND AND PURPOSE: The chemokine receptor CCR1 is a potential target for the treatment of rheumatoid arthritis. To explore the impact of CCR1 blockade in experimental arthritis and the underlying mechanisms, we used J-113863, a non-peptide antagonist of the mouse receptor. EXPERIMENTAL APPROACH: Compound J-113863 was tested in collagen-induced arthritis (CIA) and three models of acute inflammation; Staphylococcus enterotoxin B (SEB)-induced interleukin-2 (IL-2), delayed-type hypersensitivity (DTH) response, and lipopolysaccharide (LPS)-induced tumour necrosis factoralpha (TNFalpha) production. In the LPS model, CCR1 knockout, adrenalectomised, or IL-10-depleted mice were also used. Production of TNFalpha by mouse macrophages and human synovial membrane samples in vitro were also studied. KEY RESULTS: Treatment of arthritic mice with J-113863 improved paw inflammation and joint damage, and dramatically decreased cell infiltration into joints. The compound did not inhibit IL-2 or DTH, but reduced plasma TNFalpha levels in LPS-treated mice. Surprisingly, CCR1 knockout mice produced more TNFalpha than controls in response to LPS, and J-113863 decreased TNFalpha also in CCR1 null mice, indicating that its effect was unrelated to CCR1. Adrenalectomy or neutralisation of IL-10 did not prevent inhibition of TNFalpha production by J-113863. The compound did not inhibit mouse TNFalpha in vitro, but did induce a trend towards increased TNFalpha release in cells from synovial membranes of rheumatoid arthritis patients. CONCLUSIONS AND IMPLICATIONS: CCR1 blockade improves the development of CIA, probably via inhibition of inflammatory cell recruitment. However, results from both CCR1-deficient mice and human synovial membranes suggest that, in some experimental settings, blocking CCR1 could enhance TNF production.
Assuntos
Artrite/tratamento farmacológico , Receptores de Quimiocinas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Xantenos/uso terapêutico , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Receptores CCR1 , Xantenos/farmacocinéticaRESUMO
A new series of potent and selective cyclooxygenase-2 inhibitors have been prepared. Some of these compounds show good oral anti-inflammatory activity in rats.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/antagonistas & inibidores , Naftalenos/farmacologia , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/uso terapêutico , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Isoenzimas/metabolismo , Naftalenos/síntese química , Naftalenos/química , Naftalenos/uso terapêutico , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Growing evidence indicates that structural cells play a crucial role in the chronic inflammation of autoimmunity by their recruitment of chemokine-dependent cells. Members of the two functional classes of chemokines, inflammatory and homeostatic, seem to be involved in lymphocyte recruitment and survival, and in establishing ectopic lymphoid structures in the target organs of autoimmune diseases. Results from animal models suggest that chemokines are reasonable therapeutic targets in autoimmunity.
Assuntos
Doenças Autoimunes/imunologia , Quimiocinas/fisiologia , Animais , Artrite Reumatoide/imunologia , Doenças Autoimunes/terapia , Quimiocinas/antagonistas & inibidores , Encefalomielite Autoimune Experimental/imunologia , Humanos , Esclerose Múltipla/imunologiaRESUMO
A series of 3,4-diaryloxazolones were prepared and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2). Extensive structure-activity relationship work was carried out within this series, and a number of potent and selective COX-2 inhibitors were identified. The replacement of the methyl sulfone group on the 4-phenyl ring by a sulfonamide moiety resulted in compounds with superior in vivo antiinflammatory properties. In the sulfonamide series, the introduction of a methyl group at the 5-position of the oxazolone ring gave rise to very COX-2-selective compounds but with decreased in vivo activity. Selected 3,4-diaryloxazolones exhibited excellent activities in experimental models of arthritis and hyperalgesia. The in vivo activity of these compounds was confirmed with the evaluation of their antipyretic effectiveness and their ability to inhibit migration of proinflammatory cells. As expected from their COX-2 selectivity, most of the active compounds lacked gastrointestinal toxicity in vivo in rats after a 4-day treatment of 100 mg/kg/day. Within this novel series, sulfonamides 9-11 have been selected for further preclinical evaluation.
Assuntos
Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/farmacologia , Oxazóis/síntese química , Oxazóis/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Febre/tratamento farmacológico , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Oxazóis/uso terapêutico , Prostaglandina-Endoperóxido Sintases/sangue , Ratos , Ratos Wistar , Relação Estrutura-AtividadeAssuntos
Anti-Inflamatórios não Esteroides/toxicidade , Inibidores de Ciclo-Oxigenase/farmacologia , Indometacina/toxicidade , Isoenzimas/antagonistas & inibidores , Isoenzimas/farmacologia , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/farmacologia , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/enzimologia , Sulfonamidas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Aspirina/toxicidade , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/administração & dosagem , Diclofenaco/toxicidade , Dinoprostona/metabolismo , Indometacina/administração & dosagem , Cetorolaco/toxicidade , Masculino , Nitrobenzenos/administração & dosagem , Ratos , Ratos Wistar , Úlcera Gástrica/patologia , Sulfonamidas/administração & dosagem , Fatores de Tempo , Cicatrização/efeitos dos fármacosAssuntos
Endotélio Vascular/metabolismo , Interleucina-1/farmacologia , Ácido Linoleico/metabolismo , Ácidos Linoleicos Conjugados , Ácidos Linoleicos/metabolismo , Fosfolipídeos/biossíntese , Radioisótopos de Carbono , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Humanos , Hidroxilação , Indometacina/farmacologia , Cinética , Técnica de Diluição de Radioisótopos , Veias UmbilicaisRESUMO
This study was focused on the characterization of the metabolism of linoleic acid by human dermal fibroblasts and the effect of interleukin-1 on the biosynthesis of octadecanoids. Dermal fibroblasts untreated and treated with recombinant IL-1beta were incubated with exogenous labeled linoleic acid. A combination of high performance liquid chromatography and gas chromatography-mass spectrometry was used as the analytic technique. We found that dermal fibroblasts convert linoleic acid mainly into 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) and 9-hydroxy-10-trans,12-cis-octadecadienoic acid (9-HODE), 13(S)-HODE and 9(R)-HODE being the predominant enantiomers. IL-1beta increased the formation of both 13-HODE and 9-HODE in a concentration-dependent manner with similar EC50 values as for prostanoid formation. This effect of IL-1beta on HODEs formation was concomitant with the expression of prostaglandin H-synthase-2. Formation of octadecanoids was inhibited in a concentration-dependent manner by acetylsalicylic acid and indomethacin. Dexamethasone, actinomycin D, and cycloheximide abolished the effect of IL-1beta on HODEs biosynthesis. Octadecanoid biosynthetic activity was associated with the microsomal fraction. Dermal fibroblasts incorporated [14C]-9-HODE and [14C]-13-HODE into phospholipids, mainly into phosphatidylcholine. IL-1beta increased significantly the esterification of 13-HODE in all glycerophospholipids, the major increase being observed in phosphatidylinositol. These results indicate that prostaglandin H-synthase-2 is the enzyme responsible for the increase in the ability to form HODEs of dermal fibroblasts stimulated with IL-1beta.
Assuntos
Interleucina-1/farmacologia , Ácidos Linoleicos Conjugados , Ácidos Linoleicos/biossíntese , Ácidos Linoleicos/metabolismo , Prostaglandina-Endoperóxido Sintases/fisiologia , Pele/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Ácido LinoleicoRESUMO
Human umbilical vein endothelial cells (HUVEC) were treated with recombinant interleukin (IL)-1 beta, and the metabolism of exogenous linoleic acid was studied. High performance liquid chromatography, gas chromatography-mass spectrometry, and chiral analysis revealed that HUVEC enzymatically convert linoleic acid mainly into 13-(S)hydroxy-9(Z),11(E)-octadecadienoic (13-HODE) and 9-(R)hydroxy-10(E),12(Z)-octadecadienoic acids, which may isomerize toward all-trans compounds. IL-1 beta increased the formation of all octadecanoids in a time- and dose-dependent manner with similar EC50 (approximately 1 unit/ml). The apparent Km values of linoleic acid were 15.59 +/- 8.39 and 152.9 +/- 84 microM (p < 0.05) in IL-1 beta-treated cells and controls, respectively, indicating a higher substrate affinity in cells stimulated with IL-1 beta. Ratios of S/R enantiomers for the hydroxyoctadecanoids produced by untreated and IL-1 beta-treated cells were similar to those from isolated cyclooxygenases (COXs), whereas isolated 15-lipoxygenase yielded 13-HODE with a strict S configuration. The formation of octadecanoids was inhibited in a dose-dependent manner by several COX inhibitors in both controls and IL-1 beta-treated cells, COX2 selective inhibitors being more effective on IL-1 beta-treated cells than on controls. COX1 and COX2 protein levels increased less than 2-fold and 8-fold, respectively, after IL-1 beta treatment. The specificity of COX inhibitors was proven since they did not inhibit 13-HODE formation by human polymorphonuclear leukocytes. Overall, these results indicate that COXs are responsible for the oxidative metabolism of linoleic acid in HUVEC, and IL-1 beta increases it by inducing the expression of new enzyme, mainly COX2.
Assuntos
Endotélio Vascular/metabolismo , Interleucina-1/farmacologia , Ácidos Linoleicos/metabolismo , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Ácido Linoleico , Oxirredução , Prostaglandina-Endoperóxido Sintases/metabolismo , Veias UmbilicaisRESUMO
Inhibition of the formation of pro-inflammatory eicosanoids such as leukotrienes and 12-hydroxyeicosatetraenoic acid by 15-hydroxyeicosatetraenoic acid (15-HETE) has been reported. Psoriatic dermis synthesizes reduced levels of 15-HETE and it has been postulated to play a role in the pathophysiology of this disease. Interleukin-1 stimulates the production of prostaglandin E2 in fibroblasts, but its effect on the synthesis of 15-HETE is at present unknown. The aim of this study was to investigate the modulation of 15-HETE formation by interleukin-1 in dermal fibroblasts. Cells were treated with recombinant interleukin-1 alpha or beta prior to incubation with exogenous 14C-arachidonic acid, and eicosanoids were analyzed by HPLC. Interleukin-1 significantly increased the production of 15-HETE, but also 12-hydroxy-heptadecatrienoic acid, 11-hydroxyeicosatetraenoic acid, and prostaglandins, in a concentration- and time-dependent fashion. No significant differences between the two types of interleukin-1 were found. Dexamethasone (10 nM), and the protein synthesis inhibitors actinomycin D (1 microM) and cycloheximide (3 micrograms/ml) completely abolished the effect of interleukin-1 on 15-HETE formation. Whereas indomethacin (0.5-25 microM) strongly inhibited the synthesis of 15-HETE, aspirin (100-1000 microM) was unable to significantly inhibit its formation in both untreated and interleukin-treated fibroblasts. Aspirin inhibited the 15-HETE produced by cyclooxygenase from ram seminal vesicles, although to a lesser extent than indomethacin. In cell-free extracts, the activity concerning the synthesis of 15-HETE was associated with the microsomal fraction (100,000 x g pellet). Overall, these results strongly suggest that interleukin-1 increases 15-HETE formation mainly through the expression of new cyclooxygenase.
Assuntos
Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-1/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Ácido Araquidônico/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Eicosanoides/metabolismo , Epiderme/química , Feminino , Fibroblastos/química , Humanos , Ácidos Hidroxieicosatetraenoicos/análise , Fatores de TempoRESUMO
The cellular origin of Leukotriene B4, a potent pro-inflammatory agent that is present in psoriatic lesions, has not been completely ascertained. The present study was performed in order to assess the possible contribution of epidermal cells to leukotriene B4 synthesis through 5-lipoxygenase or by means of transcellular metabolism of the epoxide intermediate leukotriene A4 from activated polymorphonuclear leukocytes. The metabolism of exogenous arachidonic acid in fresh human epidermal cell, polymorphonuclear leukocyte or mixed suspensions was determined by means of high-performance liquid chromatography. Epidermal cells transformed arachidonic acid mainly into 12-hydroxy-eicosatetraenoic acid and prostaglandin E2. Formation of prostaglandins F2 alpha and D2, 12-hydroxy-eptadecatrienoic acid, and 15- and 11-hydroxy-eicosatetraenoic acids was also detected. We did not detect any eicosanoid derived from 5-lipoxygenase pathway. Mixed suspensions of polymorphonuclear leukocytes and epidermal cells (ratio 1:4) produced 1.72 times more leukotriene B4 than leukocytes alone under the same experimental conditions. Epidermal cells incubated with 5 microM authentic leukotriene A4 for 3 min yielded 2.954 +/- 0.27 pmoles/10(6) cells of leukotriene B4, which was characterized by co-elution with authentic standard and its ultraviolet absorption spectrum. These data demonstrate the existence of a leukotriene A4 epoxide hydrolase activity in human epidermal cells. Our results suggest that epidermal cells could cooperate in leukotriene B4 biosynthesis by transcellular metabolism of leukotriene A4 in lesions of psoriasis, and possibly other inflammatory dermatoses characterized by increased leukotriene B4 levels and prominent polymorphonuclear leukocyte infiltrates.
Assuntos
Comunicação Celular , Leucotrieno B4/biossíntese , Neutrófilos/metabolismo , Pele/metabolismo , Ácido Araquidônico/metabolismo , Calcimicina/farmacologia , Feminino , Humanos , Técnicas In Vitro , Leucotrieno A4 , Leucotrienos/metabolismoRESUMO
Increased levels of eicosanoids in lesions of psoriasis suggest activation of arachidonic acid metabolism in different cell types play a physiopathologic role. The contribution of each cell type present in psoriasis has been the subject of some controversy, which has led us to study the metabolism of arachidonic acid in human epidermal cells suspensions. 12-HETE was found to be the main product, followed by PGE2 and PGF2 alpha; no 5-lypoxygenase products were found. Thus epidermal cell contribution to LTB4 levels present in plaques of psoriasis appears to be irrelevant. Conversely, increased levels of 12-HETE probably do result from arachidonic acid metabolism by epidermal cells.
Assuntos
Ácidos Araquidônicos/metabolismo , Eicosanoides/biossíntese , Epiderme/metabolismo , Células Cultivadas , Humanos , Cinética , Fatores de TempoRESUMO
Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10.5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.
Assuntos
Escherichia coli/genética , Proteínas Hemolisinas/genética , Mutação , Plasmídeos , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos/genética , Fenótipo , Especificidade da Espécie , TetraciclinaRESUMO
Of the different bacterial strains isolated from alfalfa raw material, nine were able to coagulate the protein fraction of alfalfa green juice. The two strains showing the highest efficiency were further used for coagulation experiments. They were classified as Erwinia carotovora and Escherichia coli. Juice samples inoculated (1:10 to 1:100) with stationary-phase cultures were efficiently coagulated. The amount of protein recovered was equivalent to that obtained when the juice was heat treated. A minimal incubation temperature of 30 degrees C was required. The protein coagulum appeared after 8 to 10 h of incubation. During this period no bacterial growth was apparent, but glucose was actively fermented. For both strains no extracellular enzymatic activity could be shown in the culture supernatants. The fermentative metabolism during the incubation period seems to be responsible for protein coagulation.