RESUMEN
Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Ciclohexilaminas/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Pulmón/efectos de los fármacos , Triazinas/farmacología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Administración Oral , Adulto , Animales , Quimiocina CXCL1/biosíntesis , Relación Dosis-Respuesta a Droga , Epóxido Hidrolasas/metabolismo , Exotoxinas/metabolismo , Femenino , Humanos , Inflamación/enzimología , Inflamación/etiología , Inflamación/patología , Inflamación/prevención & control , Recuento de Leucocitos , Leucocitos/metabolismo , Leucocitos/patología , Pulmón/enzimología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Ratas , Ácidos Esteáricos/metabolismo , Contaminación por Humo de Tabaco/efectos adversosRESUMEN
Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production by human peripheral blood mononuclear cells was roflumilast (IC(50) = 5 nM) > EPPA-1 (38) > rolipram (269) > cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D(50) = 0.042 mg/kg) > roflumilast (0.24) > rolipram (3.34) > cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D(50) = 0.495 mg/kg) > roflumilast (1.6) > cilomilast (6.4) > EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of alpha(2)-adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D(50))/(neutrophilia D(50))] was EPPA-1 (578) > roflumilast (6.4) > cilomilast (1.4) > rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D(50))/(neutrophilia D(50))]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.
Asunto(s)
Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/farmacología , Pica/psicología , Piperazinas/farmacología , Piridinas/farmacología , Vómitos/inducido químicamente , Aminopiridinas/farmacología , Animales , Benzamidas/farmacología , Ácidos Carboxílicos/farmacología , Ácidos Ciclohexanocarboxílicos , Ciclopropanos/farmacología , Hurones , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Nitrilos/farmacología , Pica/inducido químicamente , Ratas , Ratas Endogámicas Lew , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Adrenérgicos alfa 2/fisiología , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Macrophages release cytokines that may contribute to the chronic inflammation observed in pulmonary conditions such as asthma and chronic obstructive pulmonary disease. Thus, inhibition of macrophage cytokine production may have a therapeutic benefit. Human lung macrophages are a rich source of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, and IL-8, that are elevated in the bronchoalveolar lavage and sputum of subjects with respiratory diseases. Cytokine production from both monocytes and macrophages is mediated by mitogen-activated protein kinase (MAPK) pathways. This study compared the effects of a novel p38 MAPK inhibitor, N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl]amino}ethyl)-guanidine (PCG), and an extracellular signal-regulated kinase (ERK) pathway inhibitor, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059), on cytokine release from lipopolysaccharide (LPS)-stimulated human monocytes, monocyte-derived macrophages (MDM), and lung macrophages. Lung macrophages, MDM, and monocytes were stimulated with LPS, and cytokine release was measured by enzyme-linked immunosorbent assay. Immunoblots were performed to confirm p38 and ERK1/2 MAPK expression and activity. PCG inhibited TNF-alpha release more effectively from monocytes compared with MDM or macrophages (maximal inhibition was 99.3 +/- 1.4, 62.7 +/- 4.3, and 58.6 +/- 6.6%, respectively; n = 7-9). PD098059 was less effective at suppressing TNF-alpha release from monocytes compared with MDM and lung macrophages (maximal inhibition was 37.4 +/- 2.8, 70.1 +/- 4.5, and 68.7 +/- 5.1%, respectively; n = 7-9). The pattern of GM-CSF, IL-6, and IL-8 release was comparable with that of TNF-alpha. These data suggest a differential involvement for each of these MAPK pathways in macrophage cytokine production compared with monocytes.
Asunto(s)
Citocinas/inmunología , Macrófagos/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , Monocitos/inmunología , Diferenciación Celular , Células Cultivadas , Flavonoides/farmacología , Humanos , Lipopolisacáridos , Pulmón/citología , Macrófagos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/citología , Monocitos/efectos de los fármacosRESUMEN
PURPOSE: The purposes of this study are (1) to develop an efficient aerosol inhalation system for the delivery of [(99m)Tc]DTPA aerosol into guinea pig airways with high uniformity for measuring lung clearance using SPECT/CT imaging, as a measure of lung permeability, and (2) to use [(99m)Tc]DTPA studies in guinea pig model of chronic obstructive pulmonary disease (COPD) to determine its usefulness in studying pathogenesis of COPD. PROCEDURE: We developed an aerosol delivery system and single-photon emission computed tomography (SPECT) imaging method to measure the pulmonary clearance rate in anesthetized guinea pigs. In this system, an 830-cc rebreather exposure chamber was filled with oxygen immediately before a 5 min [(99m)Tc]DTPA (4-5 mCi/mL) aerosol exposure. The rebreather included a top mounted AeroNeb micro pump nebulizer, a nose-only exposure tube, and rear evacuation port. An 830-cc rebreather exposure chamber was filled with oxygen immediately before 5 min [(99m)Tc]DTPA (4 â¼ 5 mCi/mL) aerosol exposure. One control and one cigarette smoking group were studied. RESULTS: Images showed high and uniform lung deposition and the mean clearance rate was increased by 37% in smoking group. CONCLUSIONS: The combined SPECT/CT imaging method developed here can be used for serial evaluation of lung function and its response to drug therapy in guinea pig model of COPD.
Asunto(s)
Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Pentetato de Tecnecio Tc 99m , Tomografía Computarizada de Emisión de Fotón Único/métodos , Tomografía Computarizada por Rayos X/métodos , Animales , Modelos Animales de Enfermedad , Cobayas , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico por imagen , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
The anti-inflammatory and utero-relaxant effects of two potent phosphodiesterase 4 (PDE4) inhibitors of the latest generation: cilomilast (one of the most advanced PDE4 inhibitors in clinical development, reportedly more selective for PDE4D) and compound A (which displays 12-fold greater selectivity toward PDE4B and/or PDE4A than toward PDE4D) were evaluated in human uterine smooth muscle. We first established that these compounds exhibit greater efficacy in inhibiting total cAMP-PDE activity in pregnant versus nonpregnant myometrium (E(max) = 78.0% +/- 3.6% and 80.3% +/- 2.2% in pregnant versus 57% +/- 4.7% and 70.5% +/- 5.9% in nonpregnant women for compound A and cilomilast, respectively; P < 0.05 for both compounds), confirming the prominent participation of PDE4 isoforms in cAMP hydrolysis in the near-term pregnant myometrium. Using pregnant myometrial explants, we have shown that both these drugs and also rolipram, the prototype PDE4 inhibitor, produce concentration-dependent inhibition of lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNFalpha) release with similar potency in each case (pD2 = 8.0 +/- 0.5, 7.9 +/- 0.2, and 7.6 +/- 0.2 for compound A, cilomilast, and rolipram, respectively). The maximum inhibition produced is 65%. Pretreatment with forskolin or 8-bromo-cAMP mimics the PDE4 inhibitor effect. Furthermore, compound A and cilomilast both produce concentration-dependent inhibition of the spontaneous contractions of myometrial strips and are more potent in pregnant than in nonpregnant myometrium (pD2 = 7.3 +/- 0.7 and 8.1 +/- 0.3 in pregnant versus 6.2 +/- 0.9 and 6.6 +/- 0.1 in nonpregnant myometrium for compound A and cilomilast, respectively; P < 0.05 for both compounds). This demonstrates that the PDE4 isoforms involved in the mechanism of contraction are different in the pregnant and nonpregnant myometrium. Our study highlights the importance of developing PDE4 inhibitors for the pharmacological management of infection-induced preterm labor.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Broncodilatadores/farmacología , Miometrio/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Contracción Uterina/efectos de los fármacos , Antiinflamatorios/farmacología , Ácidos Carboxílicos , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Ácidos Ciclohexanocarboxílicos , Éteres/farmacología , Femenino , Humanos , Hidrocarburos Fluorados/farmacología , Técnicas In Vitro , Lipopolisacáridos/farmacología , Miometrio/inmunología , Miometrio/metabolismo , Nitrilos , Embarazo , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We have determined the expression of phosphodiesterase (PDE) 7A1 and PDE7A2 in human cells that have been implicated in the pathogenesis of chronic obstructive pulmonary disease and asthma. Messenger RNA transcripts were detected by RT-PCR in T lymphocytes, monocytes, neutrophils, airway and vascular smooth muscle cells, lung fibroblasts, epithelial cells, and cardiac myocytes. Human epithelial, T cell, eosinophil, and lung fibroblast cell lines were also positive for PDE7A1 and PDE7A2 mRNA transcripts. By Western immunoblot analyses the amount of PDE7A1 was greatest in T cell lines, peripheral blood T lymphocytes, epithelial cell lines, airway and vascular smooth muscle cells, lung fibroblasts, and eosinophils but was not detected in neutrophils. In contrast, PDE7A2 protein, which was identified in human cardiac myocytes, was not found in any of the other cell types investigated. Immunoconfocal analyses showed that PDE7A was expressed in neutrophils and alveolar macrophages. As the expression of PDE7A mirrors the distribution of PDE4 we speculate that this enzyme could be a target for novel anti-inflammatory drugs.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Macrófagos Alveolares/enzimología , Neutrófilos/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Western Blotting , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7 , Humanos , Sistema Inmunológico/citología , Inmunohistoquímica , Inflamación/etiología , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Confocal , Neutrófilos/fisiología , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
Fibroblasts, as a major source of extracellular interstitial connective tissue matrix, play an important role in wound healing and the development of fibrosis. The phosphodiesterase (PDE) 4 inhibitor cilomilast inhibits fibroblast chemotaxis and fibroblast-mediated gel contraction. Using the Boyden blindwell chamber chemotaxis assay and the type I collagen gel contraction model, this study investigated whether specific cytokines modulate cilomilast's inhibitory effect through regulation of endogenous PGE(2) production. Human recombinant IL-1beta stimulated PGE(2) production and shifted the cilomilast concentration-dependence curve to the left in both assay systems, indicating increased sensitivity to cilomilast. In contrast, human recombinant IL-4 inhibited PGE(2) production and shifted the cilomilast concentration-dependence curve to the right in both systems. In summary, the inhibitory effect of cilomilast on fibroblast migration and collagen gel contraction is modulated by IL-1beta and IL-4 through regulation of PGE(2) production.
Asunto(s)
Broncodilatadores/farmacología , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Fibroblastos/efectos de los fármacos , Ácidos Carboxílicos , Células Cultivadas , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Ácidos Ciclohexanocarboxílicos , Citocinas/inmunología , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Feto , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Interleucina-1/inmunología , Interleucina-1/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Pulmón/citología , Nitrilos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
The biochemical and pharmacological characteristics in human proinflammatory cells of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene], a novel and selective inhibitor of phosphodiesterase (PDE) 7, are described. BRL 50481 inhibited the activity of hrPDE7A1 expressed in baculovirus-infected Spodoptera frugiperda 9 cells in a competitive manner (Ki value of 180 nM) and was 416 and 1884 times less potent against PDE3 and 38 and 238 times less potent against PDE4 at a substrate concentration of 1 microM and 50 nM cAMP, respectively. Western blotting identified HSPDE7A1 but not HSPDE7A2 in three human cell types that are implicated in the pathogenesis of chronic obstructive lung disease, namely, CD8+ T-lymphocytes, monocytes, and lung macrophages. BRL 50481 had no effect on the proliferation of CD8+ T-lymphocytes and only marginally (approximately 2-11%) reduced the generation of tumor necrosis factor (TNF)alpha from blood monocytes and lung macrophages. However, in the presence of BRL 50481 the inhibitory effect of rolipram was enhanced on all three cell types. The expression of HSPDE7A1 was increased in a time-dependent manner in monocytes that were "aged" in culture medium. Under this condition, BRL 50481 now inhibited TNFalpha generation in a concentration-dependent manner. In aged monocytes, rolipram, Org 9935 (a PDE3 inhibitor), and prostaglandin E2 inhibited TNFalpha generation in a concentration-dependent manner and interacted additively with BRL 50481. BRL 50481 is the first fully documented PDE7 inhibitor that has acceptable selectivity for in vitro studies. Furthermore, although BRL 50481 had only a modest inhibitory effect per se on the proinflammatory cells studied, it acted at least additively with other cAMP-elevating drugs, especially when HSPDE7A1 was up-regulated.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Linfocitos T CD8-positivos/enzimología , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Macrófagos Alveolares/enzimología , Monocitos/enzimología , Nitrocompuestos/farmacología , Sulfonamidas/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Senescencia Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7 , Humanos , Cinética , Leucocitos/enzimología , Activación de Linfocitos , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Therapies that mitigate the fibrotic process may be able to slow progressive loss of function in many lung diseases. Because cyclic adenosine monophosphate is known to regulate fibroblasts, the current study was designed to evaluate the activity of selective phosphodiesterase (PDE) inhibitors on two in vitro fibroblast responses: chemotaxis and contraction of three-dimensional collagen gels. Selective PDE4 inhibitors, rolipram and cilomilast, each inhibited the chemotaxis of human fetal lung fibroblasts (HFL-1) toward fibronectin in the blindwell assay system (control: 100% versus cilomilast [10 microM]: 40.5 +/- 7.3% versus rolipram: [10 microM] 32.1 +/- 2.7% cells/5 high-power fields; P < 0.05, both comparisons). These PDE4 inhibitors also inhibited contraction of three-dimensional collagen gels (control: 100% versus cilomilast: 167.7 +/- 6.9% versus rolipram: 129.9 +/- 1.9% of initial size; P < 0.05, both comparisons). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect in either system. Prostaglandin E(2) (PGE(2)) inhibited both chemotaxis and gel contraction, and the PDE4 inhibitors shifted the PGE(2) concentration-dependence curve to the left in both systems. The inhibition of endogenous PGE(2) production by indomethacin diminished the effects of the PDE4 inhibitors in both chemotaxis and gel contraction, consistent with the concept that the PDE4 inhibitory effects on fibroblasts are related to the presence of cyclic adenosine monophosphate in the cells. In summary, these in vitro results suggest that PDE4 inhibitors may be able to suppress fibroblast activity and, thus, have the potential to block the development of progressive fibrosis.