RESUMEN
Inefficient wound healing poses a global health challenge with a lack of efficient treatments. Wound healing issues often correlate with low endogenous nitric oxide (NO) levels. While exogenous delivery with NO-releasing compounds represents a promising therapeutic strategy, controlling the release of the highly reactive NO remains challenging. Phosphodiesterase 5 (PDE5) inhibitors, like sildenafil, have also been shown to promote wound healing. This study explores hybrid compounds, combining NO-releasing diazeniumdiolates with a sildenafil-derived PDE5 inhibitor. One compound demonstrated a favorable NO-release profile, triggered by an esterase (prodrug), and displayed inâ vitro nanomolar inhibition potency against PDE5 and thrombin-induced platelet aggregation. Both factors are known to promote blood flow and oxygenation. Thus, our findings unveil promising prospects for effective wound healing treatments.
Asunto(s)
Compuestos Azo , Donantes de Óxido Nítrico , Inhibidores de Fosfodiesterasa 5 , GMP Cíclico , Óxido Nítrico , Donantes de Óxido Nítrico/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología , Cicatrización de HeridasRESUMEN
Chronic non-healing wounds impose a huge burden on patients and health care providers. In spite of improvements in standards of care there are no effective and safe treatments that promote new tissue formation and wound closure in ailments such as diabetic foot ulcer, pressure ulcer, venous leg ulcer or digital ulcer in systemic sclerosis. Endothelial dysfunction, which associates with impaired endogenous nitric oxide formation is assumed to be a main disease mechanism in chronic, non-healing wounds in diabetic and elderly patients as well as in digital ulcers in systemic sclerosis. Topadur Pharma has invented small molecular weight nitric oxide-releasing PDE5 inhibitors, which by modulating a key enzyme system of intracellular signaling may address chronic non-healing wounds. The promising first drug candidate TOP-N53 is currently in early clinical development. Here we describe for the first time the design of TOP-N53 and the synthesis of the clinical GMP batch.
Asunto(s)
Pie Diabético , Cicatrización de Heridas , Anciano , Pie Diabético/tratamiento farmacológico , HumanosRESUMEN
Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC50 values on both TbrPDEB1 (IC50: 4nM) and TbrPDEB2 (IC50: 3nM) (J. Infect. Dis.2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC50 and EC50 against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Piridazinas/farmacología , Tetrazoles/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Inhibidores de Fosfodiesterasa/química , Proteínas Protozoarias/metabolismo , Piridazinas/síntesis química , Piridazinas/química , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/química , Tripanocidas/síntesis química , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
BACKGROUND AND PURPOSE: Lung macrophages (LMs) are critically involved in respiratory diseases. The primary objective of the present study was to determine whether or not an adenosine analog (NECA) and prostaglandin E2 (PGE2) affected the interleukin (IL)-4- and IL-13-induced release of M2a chemokines (CCL13, CCL17, CCL18, and CCL22) by human LMs. EXPERIMENTAL APPROACH: Primary macrophages isolated from resected human lungs were incubated with NECA, PGE2, roflumilast, or vehicle and stimulated with IL-4 or IL-13 for 24 h. The levels of chemokines and PGE2 in the culture supernatants were measured using ELISAs and enzyme immunoassays. KEY RESULTS: Exposure to IL-4 (10 ng/mL) and IL-13 (50 ng/mL) was associated with greater M2a chemokine production but not PGE2 production. PGE2 (10 ng/mL) and NECA (10-6 M) induced the production of M2a chemokines to a lesser extent but significantly enhanced the IL-4/IL-13-induced production of these chemokines. At either a clinically relevant concentration (10-9 M) or at a concentration (10-7 M) that fully inhibited phosphodiesterase 4 (PDE4) activity, roflumilast did not increase the production of M2a chemokines and did not modulate their IL-13-induced production, regardless of the presence or absence of PGE2. CONCLUSIONS: NECA and PGE2 enhanced the IL-4/IL-13-induced production of M2a chemokines. The inhibition of PDE4 by roflumilast did not alter the production of these chemokines. These results contrast totally with the previously reported inhibitory effects of NECA, PGE2, and PDE4 inhibitors on the lipopolysaccharide-induced release of tumor necrosis factor alpha and M1 chemokines in human LMs.
Asunto(s)
Adenosina , Aminopiridinas , Benzamidas , Dinoprostona , Humanos , Dinoprostona/farmacología , Adenosina/farmacología , Interleucina-4/farmacología , Interleucina-13/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Quimiocinas , Macrófagos , Factor de Necrosis Tumoral alfa/farmacología , Quimiocina CCL17 , Pulmón , Células Cultivadas , CiclopropanosRESUMEN
Lung fibroblasts are believed to be a major source of vascular endothelial growth factor (VEGF), which supports the survival of lung endothelial cells and modulates the maintenance of the pulmonary microvasculature. VEGF has been related to the pathogenesis of lung diseases, including chronic obstructive pulmonary disease (COPD). Prostaglandin E2 (PGE2) stimulates VEGF production from lung fibroblasts via the E-prostanoid (EP)-2 receptor. The EP2 signaling pathway uses cyclic adenosine monophosphate (cAMP) as a second messenger, and cAMP is degraded by phosphodiesterases (PDEs). This study investigates whether phosphodiesterase inhibition modulates the human lung fibroblast VEGF production induced by PGE2. Human fetal lung fibroblasts were cultured with PGE2 and PDE inhibitors. The PDE4 inhibitors roflumilast, roflumilast N-oxide, and rolipram with PGE2 increased VEGF release, as quantified in supernatant media by ELISA. In contrast, PDE3, PDE5, and PDE7 inhibitors did not affect VEGF release. Roflumilast increased VEGF release with either an EP2 or an EP4 agonist. Roflumilast augmented the cytosolic cAMP levels induced by PGE2 and VEGF release with other agents that use the cAMP signaling pathway. Roflumilast-augmented VEGF release was completely inhibited by a protein kinase A (PKA) inhibitor. Roflumilast with PGE2 increased VEGF mRNA levels, and the blockade of mRNA synthesis inhibited the augmented VEGF release. The stimulatory effect of roflumilast on VEGF release was replicated using primary healthy and COPD lung fibroblasts. These findings demonstrate that PDE4 inhibition can modulate human lung fibroblast VEGF release by PGE2 acting through the EP2 and EP4 receptor-cAMP/PKA signaling pathway. Through this action, PDE4 inhibitors such as roflumilast could contribute to the survival of lung endothelial cells.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Dinoprostona/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Aminopiridinas/farmacología , Benzamidas/farmacología , Células Cultivadas , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclopropanos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Pulmón/citología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Mensajero/genética , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulate in vitro the oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that have per se fibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2α was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [(3)H]-thymidine and [(3)H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4.
Asunto(s)
Aminopiridinas/farmacología , Benzamidas/farmacología , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Pulmón/citología , NADPH Oxidasas/metabolismo , Proliferación Celular , Células Cultivadas , Colágeno/química , Ciclopropanos/farmacología , ADN/metabolismo , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibrosis , Humanos , Pulmón/patología , NADPH Oxidasa 4 , Estrés Oxidativo , Oxígeno/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Piridazinas/farmacología , Tetrazoles/farmacología , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Ratones , Estructura Molecular , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/genética , Piridazinas/química , Piridazinas/uso terapéutico , Tetrazoles/química , Tetrazoles/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Chronic, non-healing wounds constitute a major health problem, and the current therapeutic options are limited. Therefore, pharmaceuticals that can be locally applied to complicated wounds are urgently needed. Such treatments should directly target the underlying causes, which include diabetes mellitus, chronic local pressure and/or vascular insufficiency. A common consequence of these disorders is impaired wound angiogenesis. Here, we investigated the effect of topical application of a nitric oxide-releasing phosphodiesterase 5 inhibitor (TOP-N53)-containing liquid hydrogel on wound repair in mice. The drug-loaded hydrogel promoted re-epithelialization and angiogenesis in wounds of healthy and healing-impaired diabetic mice. Using a non-invasive label-free optoacoustic microscopy approach combined with automated vessel analysis, we show that the topical application of TOP-N53 formulation increases the microvascular network density and promotes the functionality of the newly formed blood vessels, resulting in enhanced blood perfusion of the wounds. These results demonstrate a remarkable healing-stimulating activity of topically applied TOP-N53 formulation, supporting its further development as a wound therapeutic.
RESUMEN
Cyclic nucleotide phosphodiesterases (PDEs) control the levels of the second messengers cAMP and cGMP in many cell types including endothelial cells. Although PDE2 has the unique property to be activated by cGMP but to hydrolyze cAMP, its role in endothelial function is only poorly understood. Reactive oxygen species (ROS) have been recognized as signaling molecules controlling many endothelial functions. We thus investigated whether PDE2 would link to ROS generation and proliferative responses in human umbilical vein endothelial cells in response to thrombin. Thrombin stimulated the GTPase Rac1, known to activate NADPH oxidases, and enhanced ROS formation, whereas PDE2 inhibition or depletion by short hairpin (sh)RNA prevented these responses. Similar observations were made with 8-Br-cGMP or atrial natriuretic peptide. In agreement, thrombin elevated cGMP but decreased cAMP levels, whereas db-cAMP or forskolin diminished Rac1 activity and ROS production. Subsequently, PDE2 overexpression activated Rac1, increased ROS generation, and enhanced proliferation and in vitro capillary formation. These responses were not observed in the presence of inactive Rac1 or shRNA against the NADPH oxidase subunit NOX2. Inhibition or depletion of PDE2 also prevented thrombin-induced proliferation and capillary formation. Importantly, downregulation of PDE2 by lentiviral shRNA or PDE2 inhibition prevented vessel sprouting from mouse aortic explants and in vivo angiogenesis in a mouse model, respectively. In summary, PDE2 promotes activation of NADPH oxidase-dependent ROS production and subsequent endothelial proliferation and angiogenesis. Targeting PDE2 may provide a new therapeutic approach in diseases associated with endothelial dysfunction, oxidative stress, vascular proliferation, and angiogenesis.
Asunto(s)
Proliferación Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Neovascularización Fisiológica/fisiología , Trombina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Endotelio Vascular/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
The first pharmacological investigations of phosphodiesterase (PDE) inhibitors were developed with the clinical efficacies of drugs isolated from coffee, cacao and tea but only later their relevant ingredients were identified as xanthines that act as PDE. With its diuretic, inotropic and bronchodilating clinical efficacy, use of theophylline anticipated the clinical goals, which were later approached with the first-generation of weakly selective PDE inhibitors in the period from 1980 to 1990. Pharmacological and clinical research with these early compounds provided a vast pool of information regarding desired and adverse actions - although most of these new drugs had to be discontinued due to severe adverse effects. The pharmacological models for cardiac, vascular and respiratory indications were analysed for their PDE isoenzyme profiles, and when biochemical and molecular biological approaches expanded our knowledge of the PDE superfamily, the purified isoenzymes that were now available opened the door for more systematic studies of inhibitors and for generation of highly selective isoenzyme-specific drugs. The development of simple screening models and clinically relevant indication models reflecting the growing knowledge about pathomechanisms of disease are summarised here for today's successful application of highly selective PDE3, PDE4 and PDE5 inhibitors. The interplay of serendipitous discoveries, the establishment of intelligent pharmacological models and the knowledge gain by research results with new substances is reviewed. The broad efficacies of new substances in vitro, the enormous biodiversity of the PDE isoenzyme family and the sophisticated biochemical pharmacology enabled Viagra to be the first success story in the field of PDE inhibitor drug development, but probably more success stories will follow.
Asunto(s)
Inhibidores de Fosfodiesterasa/farmacología , Animales , Asma/tratamiento farmacológico , Broncodilatadores/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Humanos , Óxido Nítrico/fisiología , Inhibidores de Fosfodiesterasa/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Teofilina/farmacologíaRESUMEN
Since more than two decades anti-inflammatory effects of inhibitors of phosphodiesterase-4 have been described in numerous cellular and animal studies and were finally confirmed in clinical trials. The path from an early, pioneering study with Ro20-1724 showing reduction of psoriatric plaque size in 1979 to modern PDE4 inhibitors such as oral apremilast in development for psoriasis, the inhaled PDE4 inhibitor GSK256066 in development for asthma and COPD and finally roflumilast, the first PDE4 inhibitor approved and currently marketed as an oral, once-daily remedy for severe COPD was marked by large progress in chemical optimization based on improved understanding of PDE4 biology and drug-like properties determining the appropriate pharmacokinetic profile. In this chapter aspects of the pharmacology and clinical efficacy of PDE4 inhibitors, which have been in clinical development over the years are summarized with specific emphasis on their clinical pharmacokinetic properties.
Asunto(s)
Inhibidores de Fosfodiesterasa 4/farmacología , Humanos , Inhibidores de Fosfodiesterasa 4/efectos adversos , Inhibidores de Fosfodiesterasa 4/farmacocinética , Inhibidores de Fosfodiesterasa 4/uso terapéuticoRESUMEN
Roflumilast is an oral, add-on option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether this phosphodiesterase 4 inhibitor and its active metabolite roflumilast N-oxide affect the tone of human bronchial rings. We also investigated the interactions between roflumilast, roflumilast N-oxide and the long-acting ß2 -agonist formoterol with regard to the relaxation of isolated human bronchial rings at basal tone or pre-contracted with histamine. Our results demonstrated for the first time that at a clinically relevant concentration (1 nm), roflumilast N-oxide and roflumilast induce a weak relaxation of the isolated human bronchus either at resting tone (22% and 16%, respectively) or even weaker on pre-contracted bronchus with histamine (7% and 5%, respectively). In addition, the combination of formoterol with roflumilast or roflumilast N-oxide is more potent than each component alone for relaxing pre-contracted isolated bronchi - the apparent pD2 of formoterol was significantly reduced for the threshold concentration of 1 nm of the phosphodiesterase 4 inhibitors by a factor of 2.4 for roflumilast N-oxide and 1.9 for roflumilast. The full inhibition of phosphodiesterase 4 activity is achieved at 100 nm but this high concentration only caused partial relaxations of the human bronchi. At a clinically relevant concentration, these oral phosphodiesterase 4 inhibitors are not effective direct bronchodilators but could enhance the efficacy of inhaled long-acting ß2-agonists.
Asunto(s)
Aminopiridinas/farmacología , Benzamidas/farmacología , Bronquios/efectos de los fármacos , Broncodilatadores/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Aminopiridinas/administración & dosificación , Aminopiridinas/uso terapéutico , Benzamidas/administración & dosificación , Benzamidas/uso terapéutico , Bronquios/fisiología , Broncodilatadores/administración & dosificación , Broncodilatadores/uso terapéutico , Ciclopropanos/administración & dosificación , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Femenino , Fumarato de Formoterol/administración & dosificación , Fumarato de Formoterol/farmacología , Fumarato de Formoterol/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológicoRESUMEN
Chronic wounds affect a large percentage of the population worldwide and cause significant morbidity. Unfortunately, efficient compounds for the treatment of chronic wounds are yet not available. Endothelial dysfunction, which is at least in part a result of compromised nitric oxide production and concomitant reduction in cGMP levels, is a major pathologic feature of chronic wounds. Therefore, we designed and synthesized a compound with a unique dual-acting activity (TOP-N53), acting as a nitric oxide donor and phosphodiesterase 5 inhibitor, and applied it locally to full-thickness skin wounds in healthy and healing-impaired mice with diabetes. TOP-N53 promoted keratinocyte proliferation, angiogenesis, and collagen maturation in healthy mice without accelerating the wound inflammatory response or scar formation. Most importantly, it partially rescued the healing impairment of mice with genetically determined type II diabetes (db/db) by stimulating re-epithelialization and granulation tissue formation, including angiogenesis. In vitro studies with human and murine primary cells showed a positive effect of TOP-N53 on keratinocyte and fibroblast migration, keratinocyte proliferation, and endothelial cell migration and tube formation. These results demonstrate a remarkable healing-promoting activity of TOP-N53 by targeting the major resident cells in the wound tissue.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Pie Diabético/tratamiento farmacológico , Donantes de Óxido Nítrico/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/genética , Pie Diabético/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Masculino , Ratones , Ratones Transgénicos , Neovascularización Fisiológica/efectos de los fármacos , Donantes de Óxido Nítrico/uso terapéutico , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Repitelización/efectos de los fármacosRESUMEN
Interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) are important regulators of proliferation, and their expression is increased in lungs of patients with asthma, idiopathic pulmonary fibrosis (IPF), or chronic obstructive pulmonary disease (COPD). We investigated the effect of IL-1beta and bFGF on proliferation of human lung fibroblasts and the role of COX-2, PGE(2), and cAMP in this process. Furthermore, the effect of phosphodiesterase (PDE) 3 and 4 inhibition was analyzed. In primary human lung fibroblasts low concentrations of IL-1beta (<10 pg/ml) potentiated the bFGF-induced DNA synthesis, whereas higher concentrations revealed antiproliferative effects. Higher concentrations of IL-1beta-induced COX-2 mRNA and protein associated with an increase in PGE(2) and cAMP, and all of these parameters were potentiated by bFGF. The PDE4 inhibitor piclamilast concentration-dependently reduced proliferation by a partial G1 arrest. The PDE3 inhibitor motapizone was inactive by itself but enhanced the effect of the PDE4 inhibitor. This study demonstrates that bFGF and IL-1beta act in concert to fine-tune lung fibroblast proliferation resulting in amplification or reduction. The antiproliferative effect of IL-1beta is likely attributed to the induction of COX-2, which is further potentiated by bFGF, and the subsequent generation of PGE(2) and cAMP. Inhibition of PDE4 inhibition (rather than PDE3) may diminish proliferation of human lung fibroblasts and therefore could be useful in the therapy of pathological remodeling in lung diseases.
Asunto(s)
AMP Cíclico/metabolismo , Citocinas/metabolismo , Fibroblastos/metabolismo , Sustancias de Crecimiento/agonistas , Pulmón/metabolismo , Inhibidores de Fosfodiesterasa 4 , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Ciclooxigenasa 2/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Citocinas/farmacología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , Dinoprostona/agonistas , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Genes cdc/efectos de los fármacos , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Pulmón/inmunología , Pulmón/fisiopatología , Inhibidores de Fosfodiesterasa 3 , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/metabolismo , Piridazinas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.
Asunto(s)
Aminopiridinas/uso terapéutico , Benzamidas/uso terapéutico , Inhibidores de Fosfodiesterasa/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Administración Oral , Aminopiridinas/efectos adversos , Aminopiridinas/farmacología , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Benzamidas/efectos adversos , Benzamidas/farmacología , Ciclopropanos/efectos adversos , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/efectos adversos , Inhibidores de Fosfodiesterasa/farmacología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatologíaRESUMEN
Background: Roflumilast is an option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether the phosphodiesterase (PDE) 4 inhibitor roflumilast and its active metabolite roflumilast N-oxide affect the release of tumor necrosis factor (TNF)-α and chemokines by lipopolysaccharide (LPS)-stimulated human bronchial explants. We also investigated the interactions between roflumilast, roflumilast N-oxide and the ß2-agonist formoterol with regard to cytokine release by the bronchial preparations. Methods: Bronchial explants from resected lungs were incubated with roflumilast, roflumilast N-oxide and/or formoterol and then stimulated with LPS. An ELISA was used to measure levels of TNF-α and chemokines in the culture supernatants. Results: At a clinically relevant concentration (1 nM), roflumilast N-oxide and roflumilast consistently reduced the release of TNF-α, CCL2, CCL3, CCL4, CCL5 and CXCL9 (but not CXCL1, CXCL5, CXCL8 and IL-6) from human bronchial explants. Formoterol alone decreased the release of TNF-α, CCL2, and CCL3. The combination of formoterol with roflumilast (1 nM) was more potent than roflumilast alone for inhibiting the LPS-induced release of TNF-α, CCL2, CCL3, CCL4, and CXCL9 by the bronchial explants. Conclusions: At a clinically relevant concentration, roflumilast N-oxide and its parent compound, roflumilast, reduced the LPS-induced production of TNF-α and chemokines involved in monocyte and T-cell recruitment but did not alter the release of chemokines involved in neutrophil recruitment. The combination of formoterol with roflumilast enhanced the individual drugs' anti-inflammatory effects.
RESUMEN
Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg x kg(-1) day(-1)) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg x kg(-1) s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg x kg(-1) day(-1)) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1beta mRNA on days 1 and 21. Roflumilast (1.5 mg x kg(-1) day(-1)) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10(-6) M, potentiated PA-SMC growth inhibition induced by prostacyclin (10(-6) M) or interleukin-1beta (10 ng x ml(-1)) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.
Asunto(s)
Aminopiridinas/uso terapéutico , Benzamidas/uso terapéutico , Hipertensión Pulmonar/enzimología , Hipoxia/enzimología , Monocrotalina/toxicidad , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/uso terapéutico , Aminopiridinas/farmacología , Animales , Benzamidas/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/tratamiento farmacológico , Masculino , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Ratas WistarRESUMEN
Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , Amidas/química , Amidas/farmacología , Dominio Catalítico , Concentración 50 Inhibidora , Modelos Moleculares , Terapia Molecular Dirigida , Proteínas Protozoarias/química , Relación Estructura-ActividadRESUMEN
Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production.
Asunto(s)
Aminopiridinas/farmacología , Antiinflamatorios/farmacología , Benzamidas/farmacología , Células Epiteliales/efectos de los fármacos , Neutrófilos/inmunología , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Línea Celular , Quimiocinas/metabolismo , Quimiotaxis/efectos de los fármacos , Ciclopropanos/farmacología , Dinoprostona/farmacología , Quimioterapia Combinada , Células Epiteliales/fisiología , Humanos , Inmunidad Celular , Elastasa de Leucocito/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/inmunologíaRESUMEN
Pemphigoid diseases such as epidermolysis bullosa acquisita (EBA) may be difficult to treat. In pemphigoid diseases, mucocutaneous blistering is caused by autoantibodies to hemidesmosomal antigens; in EBA the autoantigen is type VII collagen. Despite growing insights into pemphigoid disease pathogenesis, corticosteroids are still a mainstay of treatment. In experimental EBA, myeloid cell activation is a key event leading to blistering. Activation of these cells depends on phosphodiesterase (PDE) 4. We therefore evaluated the potential for PDE4 inhibition in EBA: PDE4 was highly expressed in inflammatory cells and in the epidermis of patients compared with healthy skin samples. PDE4 inhibitors rolipram, roflumilast, and roflumilast N-oxide prevented the release of immune complex-induced reactive oxygen species from polymorphonuclear leukocytes and separation of the dermal-epidermal junction of skin incubated with antibodies to collagen type VII and polymorphonuclear leukocytes. The PDE4 inhibitors also impaired CD62L shedding and decreased CD11b expression on immune complex-stimulated polymorphonuclear leukocytes. For in vivo validation, experimental EBA was induced in mice by transfer of anti-collagen type VII IgG or immunization with collagen type VII. Roflumilast dose-dependently reduced blistering in antibody transfer-induced EBA and also hindered disease progression in immunization-induced EBA. PDE4 inhibition emerges as a new treatment modality for EBA and possibly other neutrophil-driven pemphigoid diseases.