RESUMO
5-Lipoxygenase (LOX) is an important arachidonic acid-metabolizing enzyme producing leukotrienes and other proinflammatory lipid mediators with potent pathophysiological functions in asthma and other inflammatory diseases. 4-(3-(4-(1-Methyl-1H-pyrazol-5-yl)phenylthio)phenyl)-tetrahydro-2H-pyran-4-carboxamide (PF-4191834) is a novel, selective non-redox 5-lipoxygenase inhibitor effective in inflammation and pain. In vitro and in vivo assays were developed for the evaluation of a novel 5-LOX inhibitor using conditions of maximal enzyme activity. PF-4191834 exhibits good potency in enzyme- and cell-based assays, as well as in a rat model of acute inflammation. Enzyme assay results indicate that PF-4191834 is a potent 5-LOX inhibitor, with an IC(50) = 229 +/- 20 nM. Furthermore, it demonstrated approximately 300-fold selectivity for 5-LOX over 12-LOX and 15-LOX and shows no activity toward the cyclooxygenase enzymes. In addition, PF-4191834 inhibits 5-LOX in human blood cells, with an IC(80) = 370 +/- 20 nM. This inhibitory concentration correlates well with plasma exposures needed for in vivo efficacy in inflammation in models of inflammatory pain. The combination of potency in cells and in vivo, together with a sustained in vivo effect, provides PF-4191834 with an overall pharmacodynamic improvement consistent with once a day dosing.
Assuntos
Inflamação/tratamento farmacológico , Inibidores de Lipoxigenase , Inibidores de Lipoxigenase/farmacologia , Dor/tratamento farmacológico , Pirazóis/farmacologia , Sulfetos/farmacologia , Animais , Asma/sangue , Asma/tratamento farmacológico , Asma/enzimologia , Cromatografia Líquida , Modelos Animais de Doenças , Humanos , Inflamação/sangue , Inflamação/enzimologia , Leucócitos/enzimologia , Leucotrieno B4/sangue , Inibidores de Lipoxigenase/farmacocinética , Inibidores de Lipoxigenase/uso terapêutico , Masculino , Espectrometria de Massas , Oxirredução , Dor/sangue , Dor/enzimologia , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Espectrofotometria , Sulfetos/farmacocinética , Sulfetos/uso terapêuticoRESUMO
Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides , Benzamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Benzamidas/farmacocinética , Disponibilidade Biológica , Western Blotting , Moléculas de Adesão Celular/biossíntese , Movimento Celular/efeitos dos fármacos , Quimiocinas/biossíntese , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Interleucina-8/biossíntese , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Masculino , Cadeias Leves de Miosina/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores CCR2/biossínteseRESUMO
The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.
Assuntos
Hidroxiureia/análogos & derivados , Imidazóis/farmacologia , Inflamação/enzimologia , Leucotrienos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Mastócitos/efeitos dos fármacos , Sulfetos/farmacologia , Ar , Animais , Araquidonato 5-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/metabolismo , Bioensaio/métodos , Calcimicina/farmacologia , Carragenina , Linhagem Celular Tumoral , Cromatografia Líquida , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hidroxiureia/farmacologia , Imunoglobulina E/imunologia , Inflamação/induzido quimicamente , Ionóforos/farmacologia , Leucotrienos/sangue , Masculino , Mastócitos/enzimologia , Mastócitos/imunologia , Oxirredução , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Both nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX) 2-selective inhibitors such as celecoxib are being reported as having potent anticancer activity in laboratory models. Several reports have suggested that the mechanism of action of these agents in reducing tumor volume/burden is unrelated to their inhibition of prostaglandin synthesis. Many in vitro reports use supraphysiological concentrations of these drugs to demonstrate COX-independent activities on apoptosis or proliferation. In vivo, most murine tumor models express COX-2 only in the vasculature and stroma, unlike human tumors that also express COX-2 in the tumor cells. In general, these models have the limitation of having no measurable, COX-2-derived, prostaglandins, the inhibition of which correlates with antitumor efficacy. We report here that 1483 human head and neck xenograft tumors express COX-2 similar to the pattern observed in human solid tumors and that COX-2 activity produces high levels of prostaglandin E2 (PGE2). Inhibition of COX-2 by celecoxib resulted in loss of intratumor PGE2 levels and reduced tumor growth in a dose-dependent manner. In contrast, a selective COX-1 inhibitor, SC-560, did not measurably reduce tumor prostaglandin levels or tumor growth despite the presence of COX-1 in the host and tumor cells. Celecoxib-treated tumors showed reduced proliferation and increased apoptosis of both tumor and stromal cells compared with vehicle controls. Specific inhibition of PGE2 activity by a neutralizing antibody mimicked the reduced tumor growth observed after celecoxib treatment, suggesting growth is PGE2 mediated. These data indicate that a major antitumor mechanism of action of celecoxib is inhibition of COX-2-derived prostaglandins, particularly PGE2, and suggest celecoxib as a novel therapeutic agent for human head and neck cancer.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Dinoprostona/fisiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma de Células Escamosas/enzimologia , Celecoxib , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Masculino , Proteínas de Membrana , Camundongos , Camundongos Nus , Transplante de Neoplasias , Pirazóis , Sulfonamidas/farmacologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Cyclooxygenase-2 (COX-2) is expressed within neovascular structures that support many human cancers. Inhibition of COX-2 by celecoxib delays tumor growth and metastasis in xenograft tumor models as well as suppresses basic fibroblast growth factor 2 (FGF-2)-induced neovascularization of the rodent cornea. The present studies were undertaken to evaluate possible mechanisms of the antiangiogenic and anticancer effects of celecoxib. Prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)) were increased in rat corneas implanted with slow-release pellets containing FGF-2 (338.6 ng of PGE(2)/g and 17.53 ng of TXB(2)/g) compared with normal rat corneas (63.1 ng of PGE(2)/g and 2.0 ng of TXB(2)/g). Celecoxib at 30 mg/kg/day p.o. inhibited angiogenesis (78.6%) and prostaglandin production by 78% for PGE(2) (72.65 ng/g) and 68% for TXB(2) (5.55 ng/g). Decreased prostaglandin production in corneas was associated with a 2.5-fold cellular increase in apoptosis and a 65% decrease in proliferation. Similar reductions in proliferation were observed in neovascular stroma (65-70%) of celecoxib-treated (dietary 160 ppm/day) xenograft tumors as well as in tumor cells (50-75%). Apoptosis was also increased in the tumor cells (2.2-3.0-fold) in response to celecoxib. Thus, the antitumor activity of celecoxib may be attributable, at least in part, to a direct effect on host stromal elements, such as the angiogenic vasculature.
Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Antineoplásicos/farmacologia , Celecoxib , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Córnea/irrigação sanguínea , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Isoenzimas/biossíntese , Proteínas de Membrana , Camundongos , Camundongos Nus , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/biossíntese , Pirazóis , Ratos , Tromboxano B2/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Combining anticancer drugs with different mechanisms of action has the potential to enhance antitumor effect. CPT-11 (Camptosar, irinotecan), a topoisomerase I inhibitor, has been shown to be highly effective in the treatment of a variety of cancers. However, its clinical usage is often complicated by late diarrhea. A number of studies have shown that cyclooxygenase (COX)-2 is overexpressed in many forms of human tumors, suggesting that COX-2 inhibition may be useful in the treatment of cancer. In this study, we used two mouse tumor models (HT-29 and colon-26 cells) to evaluate the effect of combining CPT-11 with celecoxib on tumor growth. We also assessed the involvement of COX-2 in the pathogenesis of CPT-11-induced late diarrhea using a rat model. Results indicate that celecoxib enhances the antitumor effect of CPT-11 and reduces the severity of late diarrhea in a dose-dependent manner. The extended benefits of combining celecoxib with CPT-11 may significantly improve the outcome of cancer patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Diarreia/prevenção & controle , Isoenzimas/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Celecoxib , Colo/metabolismo , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Diarreia/induzido quimicamente , Dinoprostona/biossíntese , Dinoprostona/fisiologia , Esquema de Medicação , Sinergismo Farmacológico , Células HT29/efeitos dos fármacos , Humanos , Irinotecano , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prostaglandina-Endoperóxido Sintases , Pirazóis , Ratos , Ratos Sprague-Dawley , Sulfonamidas/administração & dosagem , Redução de Peso/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous work has demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors can act synergistically with radiotherapy to improve tumor debulking and control in preclinical models. The underlying mechanism of this remarkable activity has not yet been determined. Here, we report that radiation can elevate intratumoral levels of COX-2 protein and its products, particularly prostaglandin E(2) (PGE(2)). Furthermore, inhibition of COX-2 activity or neutralization of PGE(2) activity enhances radiotherapy even in tumors where COX-2 expression is restricted to the tumor neovasculature. Direct assessment of vascular function by direct contrast enhancement-magnetic resonance imaging showed that the combination of radiation and celecoxib lead to enhanced vascular permeability. These observations suggest that an important mechanism of celecoxib-induced radiosensitization involves inhibition of COX-2-derived PGE(2), thus removing a survival factor for the tumor and its vasculature.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/radioterapia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/radioterapia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Dinoprostona/antagonistas & inibidores , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulfonamidas/uso terapêutico , Animais , Celecoxib , Terapia Combinada , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Modelos Animais de Doenças , Humanos , Cinética , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Pirazóis , Fatores de TempoRESUMO
Evidence is now available showing that cyclooxygenase (COX)-2, which is involved in prostaglandin production, is overexpressed in many types of tumors including breast. Several reports have indicated that HER-2/neu-positive breast tumors are associated with an increased amount of COX-2 protein. In this study, we evaluated the effectiveness of the select COX-1 and COX-2 inhibitors in preventing mammary tumor development in HER-2/neu transgenic mice. At 4 weeks of age, female HER-2/neu mice were fed a #5020 rodent diet supplemented with 900 ppm celecoxib, a COX-2 inhibitor, 64 ppm of SC560, a COX-1 inhibitor, or the unsupplemented #5001 diet (control). The incidence of mammary tumors was significantly lower in the celecoxib-fed mice (71%; P = 0.001 versus control) than in the control mice (95%) or in the SC560-fed mice (91%). Celecoxib-treated mice also developed fewer tumors (1.3 +/- 1.1 SD; P = 0.039 versus control) than the control mice (2.2 +/- 1.2) or the SC560 treated mice (2.3 +/- 1.3). The median time to tumor development was 266 days in the control group versus 291 days in the celecoxib-treated group (P = 0.003 versus control). Lung metastasis was also reduced by treatment with celecoxib. The COX-1 inhibitor SC560 had no protective effect. The protection offered by celecoxib was associated with significantly lower concentrations of prostacyclin and prostaglandin E(2) in mammary tumors and their adjacent mammary glands. Our findings provide additional preclinical evidence to support the clinical studies to investigate the potential effectiveness of COX-2 inhibitors in protecting woman who are at high risk for breast cancer.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Genes erbB-2/efeitos dos fármacos , Neoplasias Mamárias Experimentais/prevenção & controle , Sulfonamidas/farmacologia , Animais , Biópsia por Agulha , Western Blotting , Celecoxib , Modelos Animais de Doenças , Feminino , Genes erbB-2/genética , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Pirazóis , Sensibilidade e EspecificidadeRESUMO
Prostaglandins (PGs) can be synthetized via two isoforms of cyclooxygenase (COX). COX-1 is constitutively expressed in normal tissues, and its activity represent the normal physiological output of PGs. In inflammatory states, the newly discovered COX-2 is rapidly induced, and its activity accounts for the large amounts of PGs seen in inflammation. The commercially available nonsteroidal anti-inflammatory drugs (NSAIDs) are nonselective inhibitors of both COX isoforms; therefore, they provide anti-inflammatory activity as well as side effects associated with COX-1 inhibition. Selective inhibition of COX-2 expression explains at least in part the potent anti-inflammatory activity of steroids. Anti-inflammatory activity of newly developed COX-2 inhibitors, such as NS-398 or SC-58125, suggest a new approach of inflammatory diseases with more efficacious NSAIDs essentially devoid of side effects such as stomach ulcers.
RESUMO
Elevated hepatic synthesis of fatty acids and triglycerides, driven by hyperactivation of the SREBP-1c transcription factor, has been implicated as a causal feature of metabolic syndrome. SREBP-1c activation requires the proteolytic maturation of the endoplasmic-reticulum-bound precursor to the active, nuclear transcription factor, which is stimulated by feeding and insulin signaling. Here, we show that feeding and insulin stimulate the hepatic expression of PASK. We also demonstrate, using genetic and pharmacological approaches, that PASK is required for the proteolytic maturation of SREBP-1c in cultured cells and in the mouse and rat liver. Inhibition of PASK improves lipid and glucose metabolism in dietary animal models of obesity and dyslipidemia. Administration of a PASK inhibitor decreases hepatic expression of lipogenic SREBP-1c target genes, decreases serum triglycerides, and partially reverses insulin resistance. While the signaling network that controls SREBP-1c activation is complex, we propose that PASK is an important component with therapeutic potential.
Assuntos
Dislipidemias/metabolismo , Lipogênese , Obesidade/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Células Cultivadas , Células HEK293 , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E(2) (PGE(2)) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE(2) synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC(50)=16.5+/-3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE(2) synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE(2) synthesis (IC(50) in the range of 0.5-5 microM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF(1alpha) (PGF(1alpha)) and PGF(2alpha). In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE(2), PGF(1alpha) and PGF(2alpha) synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.
Assuntos
Óxidos S-Cíclicos/antagonistas & inibidores , Inibidores de Ciclo-Oxigenase 2/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Tiazinas/antagonistas & inibidores , Animais , Artrite Reumatoide/metabolismo , Carragenina/farmacologia , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Interleucina-1beta/farmacologia , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Prostaglandina-E Sintases , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.
Assuntos
Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Inibidores de Lipoxigenase , Dor/tratamento farmacológico , Sulfetos/administração & dosagem , Sulfetos/farmacologia , Administração Oral , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Adjuvante de Freund/metabolismo , Humanos , Hidroxiureia/análogos & derivados , Hidroxiureia/farmacologia , Imidazóis/uso terapêutico , Imuno-Histoquímica , Inflamação/complicações , Leucotrienos/metabolismo , Masculino , Osteoartrite/complicações , Dor/complicações , Dor/enzimologia , Dor/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Sulfetos/uso terapêuticoRESUMO
The discovery of a second isoform of cyclooxygenase (COX) led to the search for compounds that could selectively inhibit COX-2 in humans while sparing prostaglandin formation from COX-1. Celecoxib and rofecoxib were among the molecules developed from these efforts. We report here the pharmacological properties of a third selective COX-2 inhibitor, valdecoxib, which is the most potent and in vitro selective of the marketed COX-2 inhibitors that we have studied. Recombinant human COX-1 and COX-2 were used to screen for new highly potent and in vitro selective COX-2 inhibitors and compare kinetic mechanisms of binding and enzyme inhibition with other COX inhibitors. Valdecoxib potently inhibits recombinant COX-2, with an IC(50) of 0.005 microM; this compares with IC values of 0.05 microM for celecoxib, 0.5 microM for rofecoxib, and 5 microM for etoricoxib. Unique binding interactions of valdecoxib with COX-2 translate into a fast rate of inactivation of COX-2 (110,000 M/s compared with 7000 M/s for rofecoxib and 80 M/s for etoricoxib). The overall saturation binding affinity for COX-2 of valdecoxib is 2.6 nM (compared with 1.6 nM for celecoxib, 51 nM for rofecoxib, and 260 nM for etoricoxib), with a slow off-rate (t(1/2) approximately 98 min). Valdecoxib inhibits COX-1 in a competitive fashion only at very high concentrations (IC(50) = 150 microM). Collectively, these data provide a mechanistic basis for the potency and in vitro selectivity of valdecoxib for COX-2. Valdecoxib showed similar activity in the human whole-blood COX assay (COX-2 IC(50) = 0.24 microM; COX-1 IC(50) = 21.9 microM). We also determined whether this in vitro potency and selectivity translated to significant potency in vivo. In rats, valdecoxib demonstrated marked potency in acute and chronic models of inflammation (air pouch ED(50) = 0.06 mg/kg; paw edema ED(50) = 5.9 mg/kg; adjuvant arthritis ED(50) = 0.03 mg/kg). In these same animals, COX-1 was spared at doses greater than 200 mg/kg. These data provide a basis for the observed potent anti-inflammatory activity of valdecoxib in humans.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Isoxazóis/farmacologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Humanos , Hiperalgesia/tratamento farmacológico , Inflamação/tratamento farmacológico , Masculino , Proteínas de Membrana , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
Inflammation, which includes the release of growth factors, proinflammatory cytokines and prostaglandins, the infiltration and activation of inflammatory cells, and the induction of oxidative DNA damage, is known to play a role in cancer development. The combination of damage to the skin resulting from chronic ultraviolet light B (UVB) exposure itself and the inflammatory response it induces is a major source of skin cancer development. Cyclooxygenase-2 (COX-2), an inflammatory enzyme responsible for the production of prostaglandins, is now implicated in the development of epithelial cancers, including squamous cell carcinoma in the skin. Previous work conducted in our laboratory has shown that topical treatment with celecoxib following UVB irradiation inhibits several parameters of acute inflammation, including vascular permeability, the infiltration and activation of neutrophils, and the production of prostaglandin E(2) (PGE(2)). The present studies expanded these observations, demonstrating the ability of topical celecoxib to inhibit acute oxidative damage. In addition, long-term studies illustrate the effectiveness of topical treatment with this drug in reducing chronic inflammation and UVB-induced papilloma/carcinoma formation. This data provides compelling evidence to explore the clinical efficacy of topically applied COX-2 inhibitors for the prevention of human skin cancers.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Anticarcinógenos/farmacologia , Dermatite/prevenção & controle , Neoplasias Induzidas por Radiação/prevenção & controle , Sulfonamidas/farmacologia , Raios Ultravioleta/efeitos adversos , Administração Tópica , Animais , Carcinoma Basocelular , Celecoxib , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Dano ao DNA/efeitos dos fármacos , Feminino , Isoenzimas/antagonistas & inibidores , Camundongos , Camundongos Pelados , Prostaglandina-Endoperóxido Sintases , Pirazóis , Neoplasias Cutâneas/prevenção & controle , Sulfonamidas/administração & dosagem , Proteína Supressora de Tumor p53/metabolismoRESUMO
Over a million nonmelanoma skin cancer cases will be reported in the United States this year alone. Currently the primary form of treatment for these types of skin tumors is excision. However, excision of the initial lesion may not be curative because almost 50% of patients with one nonmelanoma skin cancer lesion develop another tumor within the next 5 yr at the site or adjacent to the site of excision. As with other types of epithelial based cancers, there is mounting evidence for the role of cyclooxygenase-2 (COX-2) and its products, particularly prostaglandin E(2) (PGE(2)), in the development of nonmelanoma skin cancer. To avoid the excision process, the present study was designed to evaluate the possible chemotherapeutic effect of directly treating established tumors with a topical formulation of the specific COX-2 inhibitor celecoxib. Skh/hr hairless mice were irradiated three times per wk for 16 wk to induce tumor formation. The mice were then divided into two groups and treated topically with either 500 microg celecoxib or the vehicle for 6 wk. Our results demonstrated that although topical treatment with celecoxib was not able to induce regression of established tumors, it did prevent new tumor formation after the onset of photocarcinogenesis. Although further studies are warranted, these data suggest that topical celecoxib treatment may prove to be effective in preventing the recurrence of tumors at the site of nonmelanoma skin cancer excision.
Assuntos
Neoplasias Induzidas por Radiação/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Sulfonamidas/farmacologia , Administração Tópica , Animais , Celecoxib , Divisão Celular/efeitos dos fármacos , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Feminino , Isoenzimas/metabolismo , Isoenzimas/efeitos da radiação , Camundongos , Neoplasias Induzidas por Radiação/prevenção & controle , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandina-Endoperóxido Sintases/efeitos da radiação , Pirazóis , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/prevenção & controle , Sulfonamidas/administração & dosagem , Raios UltravioletaRESUMO
There have been a number of reports suggesting inhibition of prostaglandin production may impact tumor-mediated wasting and levels of associated humoral factors such as hypercalcemia. These reductions were achieved using traditional nonsteroidal anti-inflammatory drugs (NSAIDs), which are often contraindicated in cancer patients. This is especially true during chemotherapeutic regimens due to concerns of bleeding from gastrointestinal and hematopoietic toxicities associated with inhibition of the housekeeping cyclooxygenase enzyme COX-1. Here, we report that celecoxib, one of the new class of selective COX-2 inhibitors, has the potential to reverse tumor-mediated wasting and associated humoral factors such as interleukin (IL)-6 and hypercalcemia in preclinical models of cachexia. Tumor bearing mice in late stage cachexia regained weight within days of the start of celecoxib treatment. Two models were tested. The first was the Colon 26 (Col26) syngeneic murine model that induces high levels of circulating IL-6 and hypercalcemia. The second was the human head and neck 1483 HNSCC xenograft model, which is less inflammatory and produces less prostaglandin than Col26. Despite the observation that no significant impact on tumor growth was observed between vehicle and celecoxib-treated animals over the course of the studies, celecoxib rapidly reversed weight loss in both cachectic models. With the added safety of celecoxib over traditional NSAIDs, these results suggest a possible therapeutic use for celecoxib for treating tumor-mediated wasting.