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PURPOSE OF REVIEW: Lipid emulsions are an integral part of parenteral nutrition. Enteral nutrition is the preferred route to feed critically ill patients and parenteral nutrition is used in case of contraindications or when enteral nutrition does not reach the nutritional goals. n-3 Lipids are included into some newer lipid emulsions including fish oil or may be added by a fish oil-based lipid emulsion to lipid emulsion without fish oil. This review focuses on recent clinical trials, metaanalyses, and guidelines of parenteral nutrition with n-3 lipids in critically ill patients. RECENT FINDINGS: Two single-center studies report a mortality benefit of adding fish oil-based lipid emulsions to the parenteral nutrition. Metaanalyses performed without these two studies had demonstrated beneficial effects of n-3 lipids regarding infections, length of stay, and time of mechanical ventilation but not on mortality. However, all metaanalyses judged the database derived from the underlying studies as not sufficient for a firm recommendation. Consecutively, guidelines and expert groups issue very cautious recommendations for the use of n-3 lipids in parenteral nutrition. SUMMARY: Beneficial effects of n-3 lipids in trials and metaanalyses became available; however, high-quality multicenter randomized controlled trials are needed before more endorsing recommendation will be available.
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Cuidados Críticos/métodos , Emulsões Gordurosas Intravenosas/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Nutrição Parenteral/métodos , Administração Intravenosa , Estado Terminal , Ácidos Graxos Ômega-3/administração & dosagem , HumanosRESUMO
BACKGROUND: Anti-inflammatory n-3 fatty acids (FA) like docosahexaenoic acid (DHA) opposed to the pro-inflammatory n-6 FA arachidonic acid (AA) might modulate lipid rafts within the cell membrane by differential incorporation. In inflammation, monocyte adhesion to endothelial cells is a crucial step mediated by intracellular calcium changes. We investigated whether lipid rafts mediate FA-induced modulation of adhesion and intracellular calcium. METHODS: In isolated human monocytes and monocytic U937 cells we measured adhesion to human umbilical vein endothelial cells (HUVEC) using a parallel flow chamber and a static assay, adhesion molecules by FACScan, and intracellular calcium by fluorescence. Monocyte lipid rafts were isolated by ultracentrifugation and submitted to gas chromatography for FA analysis. RESULTS: Pre-incubation with AA or DHA resulted in a predominant incorporation of the respective FA into raft compared to non-raft fraction. DHA as compared to AA significantly reduced monocyte adhesion and calcium release after stimulation with TNF-α while expression of adhesion molecules remained unchanged. Pre-treatment with a calcium chelator abolished the effect of FA on calcium and adhesion. Disruption of lipid rafts prevented FA-induced modulations. CONCLUSION: Incorporation of FA into lipid rafts seem to be crucial for modulation of adhesion under inflammatory conditions.
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Ácido Araquidônico/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Antígenos CD/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Monócitos/metabolismo , Monócitos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a major cause of mortality in intensive care units. Patients with ARDS often require parenteral nutrition with lipid emulsions as essential components. Besides being an energy supply, these lipid emulsions might display differential modulatory effects on lung integrity and inflammation. METHODS: In a pre-emptive strategy, we investigated the impact of three different intravenously infused lipid emulsions on lung morphology, leukocyte invasion, protein leakage and cytokines in a murine model of ARDS. Mice received an infusion of normal saline solution, a pure long-chain triglycerides (LCT) emulsion, a medium-chain triglycerides (MCT) containing mixed emulsion (LCT/MCT), or a fish oil (FO) containing mixed emulsion (LCT/MCT/FO) before lipopolysaccharide (LPS) challenge. RESULTS: Mice pre-infused with fish oil-containing lipid emulsion showed decreased leukocyte invasion, protein leakage, myeloperoxidase activity, and cytokine production in their alveolar space after LPS challenge compared to mice receiving LCT or LCT/MCT. In line with these findings, lung morphology assessed by histological staining after LPS-induced lung injury improved faster in the LCT/MCT/FO group. Concerning the above mentioned parameters, no significant difference was observed between mice infused with LCT or the combination of LCT and MCT. CONCLUSION: Fish oil-containing lipid emulsions might exert anti-inflammatory and pro-resolving effects in the murine model of acute lung injury. Partial replacement of n-6 fatty acids with n-3 fatty acids may thus be of benefit for critically ill patients at risk for ARDS which require parenteral nutrition.
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Modelos Animais de Doenças , Emulsões Gordurosas Intravenosas/administração & dosagem , Óleos de Peixe/administração & dosagem , Imunomodulação/fisiologia , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/imunologia , Animais , Imunomodulação/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Síndrome do Desconforto Respiratório/patologiaRESUMO
The matrix metalloprotease ADAMTS7 has been identified by multiple genome-wide association studies as being involved in the development of coronary artery disease. Subsequent research revealed the proteolytic function of the enzyme to be relevant for atherogenesis and restenosis after vessel injury. Based on a publicly known dual ADAMTS4/ADAMTS5 inhibitor, we have in silico designed an ADAMTS7 inhibitor of the catalytic domain, which served as a starting point for an optimization campaign. Initially our inhibitors suffered from low selectivity vs MMP12. An X-ray cocrystal structure inspired us to exploit amino acid differences in the binding site of MMP12 and ADAMTS7 to improve selectivity. Further optimization composed of employing 5-membered heteroaromatic groups as hydantoin substituents to become more potent on ADAMTS7. Finally, fine-tuning of DMPK properties yielded BAY-9835, the first orally bioavailable ADAMTS7 inhibitor. Further optimization to improve selectivity vs ADAMTS12 seems possible, and a respective starting point could be identified.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Humanos , Proteína ADAMTS7/genética , Proteína ADAMTS7/metabolismo , Estudo de Associação Genômica Ampla , Metaloproteinase 12 da MatrizRESUMO
The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC's salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (ß1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-Nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.
Assuntos
Proteínas de Bactérias/química , Benzoatos/química , Compostos de Bifenilo/química , Guanilato Ciclase/química , Hidrocarbonetos Fluorados/química , Nostoc/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Proteínas de Bactérias/metabolismo , Benzoatos/metabolismo , Compostos de Bifenilo/metabolismo , Cristalografia por Raios X , Guanilato Ciclase/metabolismo , Hidrocarbonetos Fluorados/metabolismo , Modelos Moleculares , Óxido Nítrico/metabolismo , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase SolúvelRESUMO
Activated coagulation factor XI (FXIa) is a highly attractive antithrombotic target as it contributes to the development and progression of thrombosis but is thought to play only a minor role in hemostasis so that its inhibition may allow for decoupling of antithrombotic efficacy and bleeding time prolongation. Herein, we report our major efforts to identify an orally bioavailable, reversible FXIa inhibitor. Using a protein structure-based de novo design approach, we identified a novel micromolar hit with attractive physicochemical properties. During lead modification, a critical problem was balancing potency and absorption by focusing on the most important interactions of the lead series with FXIa while simultaneously seeking to improve metabolic stability and the cytochrome P450 interaction profile. In clinical trials, the resulting compound from our extensive research program, asundexian (BAY 2433334), proved to possess the desired DMPK properties for once-daily oral dosing, and even more importantly, the initial pharmacological hypothesis was confirmed.
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Fator XIa , Fibrinolíticos , AnticoagulantesRESUMO
Herein, we describe the identification, chemical optimization, and preclinical characterization of novel soluble guanylate cyclase (sGC) stimulators. Given the very broad therapeutic opportunities for sGC stimulators, new tailored molecules for distinct indications with specific pharmacokinetics, tissue distribution, and physicochemical properties will be required in the future. Here, we report the ultrahigh-throughput (uHTS)-based discovery of a new class of sGC stimulators from an imidazo[1,2-a]pyridine lead series. Through the extensive and staggered optimization of the initial screening hit, liabilities such as potency, metabolic stability, permeation, and solubility could be substantially improved in parallel. These efforts resulted ultimately in the discovery of the new sGC stimulators 22 and 28. It turned out that BAY 1165747 (BAY-747, 28) could be an ideal treatment alternative for patients with hypertension, especially those not responding to standard anti-hypertensive therapy (resistant hypertension). BAY-747 (28) demonstrated sustained hemodynamic effects up to 24 h in phase 1 studies.
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Guanilato Ciclase , Hipertensão , Humanos , Guanilil Ciclase Solúvel/metabolismo , Guanilato Ciclase/metabolismo , Hipertensão/tratamento farmacológico , Vasodilatadores , Piridinas/farmacologia , Piridinas/uso terapêutico , Óxido Nítrico/metabolismoRESUMO
BACKGROUND: Pulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss of precapillary pulmonary vessel cross-sectional area. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) play important roles in endothelial dysfunction. We investigated whether combined phosphodiesterase (PDE) 3 and 4 inhibition ameliorates endothelial function by regulating the ADMA-DDAH axis. METHODS AND RESULTS: We investigated the effects of the PDE3/4 inhibitor tolafentrine in vitro on endothelial cell survival, proliferation, and apoptosis. Effects of tolafentrine on the endothelial nitric oxide synthase/nitric oxide pathway, DDAH expression, DDAH promoter activity, and cytokine release from endothelial cells and their subsequent influence on DDAH expression were investigated. In monocrotaline-induced pulmonary arterial hypertension in rats, the effects of inhaled tolafentrine on DDAH expression and activity were investigated. Real-time-polymerase chain reaction, immunocytochemistry, and PDE activity assays suggested high expression of PDE3 and PDE4 isoforms in endothelial cells. Treatment of endothelial cells with PDE3/4 inhibitor significantly decreased ADMA-induced apoptosis via a cAMP/PKA-dependent pathway by induction of DDAH2. Chronic nebulization of PDE3/4 inhibitor significantly attenuated monocrotaline-induced hemodynamic, gas exchange abnormalities, vascular remodeling, and right heart hypertrophy. Interestingly, PDE3/4 inhibitor treatment reduced ADMA and elevated nitric oxide/cGMP levels. Mechanistically, this could be attributed to direct modulatory effects of cAMP on the promoter region of DDAH2, which was consequently found to be increased in expression and activity. Furthermore, PDE3/4 inhibitor suppressed apoptosis in endothelial cells and increased vascularization in the lung. CONCLUSION: Combined inhibition of PDE3 and 4 regresses development of pulmonary hypertension and promotes endothelial regeneration by modulating the ADMA-DDAH axis.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Naftiridinas/farmacologia , Óxido Nítrico/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Apoptose/efeitos dos fármacos , Arginina/análogos & derivados , Arginina/antagonistas & inibidores , Arginina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/biossíntese , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Hemodinâmica/efeitos dos fármacos , Humanos , Inibidores de Fosfodiesterase , Regiões Promotoras Genéticas , Troca Gasosa Pulmonar/efeitos dos fármacosRESUMO
Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.
Assuntos
Benzoatos/química , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Heme/química , Mimetismo Molecular , Óxido Nítrico/química , Nostoc/enzimologia , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Ativadores de Enzimas/química , Modelos Moleculares , Mutagênese , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Guanilil Ciclase Solúvel , Relação Estrutura-AtividadeRESUMO
Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.
Assuntos
Desenho de Fármacos , Ativadores de Enzimas/química , Guanilil Ciclase Solúvel/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Cães , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Simulação de Dinâmica Molecular , Ratos , Ratos Endogâmicos SHR , Solubilidade , Guanilil Ciclase Solúvel/metabolismo , Relação Estrutura-AtividadeRESUMO
RATIONALE: Acute lung injury (ALI) remains an important cause of mortality in intensive care units. Inflammation is controlled by cytokines and eicosanoids derived from the n-6 fatty acid (FA) arachidonic acid (AA). The n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and mediators derived from EPA and DHA possess reduced inflammatory potency. OBJECTIVES: To determine whether the ability of fat-1 mice to endogenously convert n-6 to n-3 FA, and thus generate an increased ratio of n-3 to n-6 FA, impacts experimental ALI. METHODS: We investigated ALI induced by intratracheal instillation of endotoxin in fat-1 and wild-type (WT) mice, assessing leukocyte numbers, protein concentration, and prostaglandin and cytokine levels in bronchoalveolar lavage fluid, as well as free FA in plasma, and lung ventilator compliance. Body temperature and motor activity of mice--markers of sickness behavior--were also recorded. MEASUREMENTS AND MAIN RESULTS: In ALI, fat-1 mice exhibited significantly reduced leukocyte invasion, protein leakage, and macrophage inflammatory protein-2 and thromboxane B(2) levels in lavage fluid compared with WT mice. Free AA levels were increased in the plasma of WT mice in response to endotoxin, whereas EPA and DHA were increased in the fat-1 group. Ventilator compliance was significantly improved in fat-1 mice. Body temperature and motor activity were decreased in ALI. fat-1 Mice recovered body temperature and motor activity faster. CONCLUSIONS: fat-1 Mice exhibited reduced features of ALI and sickness behavior. Increasing the availability of n-3 FA may thus be beneficial in critically ill patients with ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/prevenção & controle , Ácido Eicosapentaenoico/metabolismo , Animais , Ácido Araquidônico/metabolismo , Comportamento Animal , Temperatura Corporal , Líquido da Lavagem Broncoalveolar/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quimiocina CXCL2/análise , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Emulsões Gordurosas Intravenosas/farmacologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Óleos de Peixe/farmacologia , Contagem de Leucócitos , Leucócitos/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Atividade Motora , NF-kappa B/metabolismo , Respiração Artificial , Tromboxano B2/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Regardless of the promising results of certain immune checkpoint blockers, current immunotherapeutics have met a bottleneck concerning response rate, toxicity, and resistance in lung cancer patients. Accumulating evidence forecasts that the crosstalk between tumor and immune cells takes center stage in cancer development by modulating tumor malignancy, immune cell infiltration, and immune evasion in the tumor microenvironment (TME). Cytokines and chemokines secreted by this crosstalk play a major role in cancer development, progression, and therapeutic management. An increased infiltration of Tumor-associated macrophages (TAMs) was observed in most of the human cancers, including lung cancer. In this review, we emphasize the role of cytokines and chemokines in TAM-tumor cell crosstalk in the lung TME. Given the role of cytokines and chemokines in immunomodulation, we propose that TAM-derived cytokines and chemokines govern the cancer-promoting immune responses in the TME and offer a new immunotherapeutic option for lung cancer treatment.
RESUMO
Lung cancer is the leading cause of cancer death for both men and women and accounts for almost 18.4% of all deaths due to cancer worldwide, with the global incidence increasing by approximately 0.5% per year. Lung cancer is regarded as a devastating type of cancer owing to its high prevalence, reduction in the health-related quality of life, frequently delayed diagnosis, low response rate, high toxicity, and resistance to available therapeutic options. The highly heterogeneous nature of this cancer with a proximal-to-distal distribution throughout the respiratory tract dramatically affects its diagnostic and therapeutic management. The diverse composition and plasticity of lung epithelial cells across the respiratory tract are regarded as significant factors underlying lung cancer heterogeneity. Therefore, definitions of the cells of origin for different types of lung cancer are urgently needed to understand lung cancer biology and to achieve early diagnosis and develop cell-targeted therapies. In the present review, we will discuss the current understanding of the cellular and molecular alterations in distinct lung epithelial cells that result in each type of lung cancer.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Carcinoma de Células Escamosas/metabolismo , Células Epiteliais/citologia , Neoplasias Pulmonares/metabolismo , Neoplasia de Células Basais/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Plasticidade Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasia de Células Basais/genética , Neoplasia de Células Basais/patologia , Células Neuroendócrinas/citologia , Células Neuroendócrinas/metabolismoRESUMO
BACKGROUND: Acute respiratory distress syndrome (ARDS) is associated with both high morbidity and mortality in intensive care units worldwide. Patients with ARDS often require parenteral nutrition with lipid emulsions as essential components. In the present study, we assessed the immunomodulatory and apoptotic effects of a modern, n-6-reduced lipid emulsion mixture in murine ARDS. METHODS: Mice received an infusion of either normal saline solution, pure long-chain triglyceride (LCT) emulsion, or SMOF (soybean oil, medium-chain triglycerides, olive oil, and fish oil) before a lipopolysaccharide (LPS) challenge. Mice were sacrificed at different time points (0, 24, or 72 h) after ARDS induction, and an analysis of inflammatory cytokines, protein concentrations, and the cellular composition of the alveolar and interstitial compartments was performed with special focus on alveolar apoptosis and necrosis. RESULTS: Mice infused with SMOF showed decreased leukocyte invasion, protein leakage, myeloperoxidase activity, and cytokine production in alveolar spaces after LPS challenge compared to animals that received LCT. There were fewer cells in the lung interstitium of the SMOF group compared to the LCT group. Both lipid emulsions exerted pro-apoptotic and pro-necrotic properties on alveolar immune cells, with significantly increased necrosis in mice infused with LCT compared to SMOF. CONCLUSION: SMOF has both anti-inflammatory and pro-resolving influences in murine ARDS. Partial replacement of n-6 fatty acids with n-3/n-9 fatty acids may therefore benefit critically ill patients at risk for ARDS who require parenteral nutrition.
RESUMO
Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.
Assuntos
Anticoagulantes/síntese química , Desenho de Fármacos , Trombina/antagonistas & inibidores , Administração Oral , Animais , Anticoagulantes/química , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Benzoxazóis/química , Benzoxazóis/metabolismo , Benzoxazóis/farmacologia , Sítios de Ligação , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Meia-Vida , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Concentração Inibidora 50 , Masculino , Simulação de Acoplamento Molecular , Oxazolidinonas/química , Oxazolidinonas/metabolismo , Oxazolidinonas/farmacologia , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Trombina/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa-BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.
Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Neutralizantes/química , Fator XIa/química , Regulação Alostérica , Sequência de Aminoácidos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Neutralizantes/farmacologia , Coagulação Sanguínea , Catálise , Epitopos/química , Fator XIa/antagonistas & inibidores , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Tromboelastografia , Trombina/metabolismoRESUMO
OBJECTIVE: Ca(2+)-activated K(+)-channels (BK(Ca)) play an important role in lysophosphatidylcholine (LPC)-induced endothelial dysfunction. Aim of our study was to investigate whether LPC-induced activation of BK(Ca) is also involved in monocyte adhesion to endothelial cells (EC). METHODS AND RESULTS: Measurement of membrane potential (MP) was performed using the fluorescence dye DiBAC. Adhesion of the monocytotic cell line U937 to EC was analysed by (3)[H]-thymidine-adhesion-assay. Expression of ICAM-1 and VCAM-1 were analyzed by FACS. LPC induced a hyperpolarization of EC in a dose-dependent manner with the maximum seen with 2 microM. This was prevented by the BK(Ca)-inhibitor iberiotoxin (IBX, 100nM). Adhesion of U937 cells to EC was increased after stimulation of EC with LPC. This effect was time-dependent with the maximum seen after 4h. LPC-induced adhesion was significantly reduced when EC were co-incubated with IBX, or NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI, 5 microM) and also blocked by addition of 2-aminoethoxydiphenylborate (2-APB, 100 microM) or the calcium-chelator BAPTA (10 microM). Stimulation of U937 cells with LPC did not result in an increased adhesion to unstimulated EC. CONCLUSION: Activation of the endothelial BK(Ca) plays an important role in monocyte adhesion to endothelial cells.
Assuntos
Aterosclerose/imunologia , Adesão Celular/imunologia , Endotélio Vascular/citologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Monócitos/citologia , Vasculite/imunologia , Aterosclerose/metabolismo , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Inibidores Enzimáticos/farmacologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Lisofosfatidilcolinas/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Peptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células U937 , Veias Umbilicais/citologia , Vasculite/metabolismoRESUMO
The adhesion of monocytes to the endothelium and their proliferation in the subendothelial space play an important role in atherosclerosis. Since the proliferation and migration of cells are influenced by the activity of ion channels, the aim of this study was to examine whether barium chloride (Ba(2+))-sensitive potassium channels (K(iCa)) are involved in lipopolysaccharide (LPS)-induced proliferation of monocytic U937 cells, and in the adhesion of these cells to endothelial cells. The adhesion of LPS-stimulated U937 cells to endothelial cells reached a maximum at a concentration of 5 microg/ml. This effect of LPS was completely abolished in the presence of Ba(2+) (100 micromol/l). In addition, LPS-induced proliferation was significantly reduced by Ba(2+) (control, 100%; LPS 5 microg/ml, 175%; LPS + Ba(2+) 100 micromol/l, 136%; n = 12, P < 0.05). To examine whether K(iCa) are activated by LPS, changes of U937 membrane potential were determined. LPS (5 microg/ml) caused a hyperpolarization of U937 cells indicating a flux of K(+) ions out of the cells. This effect was completely blocked by Ba(2+) (100 micromol/l). In conclusion, we demonstrate that LPS activates K(iCa) in U937 cells, which is responsible for LPS-induced adhesion of these cells to endothelial cells, and to the proliferation of U937 cells.
Assuntos
Compostos de Bário/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cloretos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Endotélio Vascular/fisiologia , Humanos , Monócitos/fisiologia , Células U937 , Cordão Umbilical/citologiaRESUMO
Pathological hypoxia plays an important role in many diseases, such as atherosclerosis, cancer, and rheumatoid arthritis. The aim of the present study was to examine the effects of different statins on hypoxia-induced endothelial cell signalling. Human umbilical cord vein endothelial cells (HUVEC) were treated with NaCN (CN, 2.5 mmol/l) to simulate a transient hypoxia. The CN-induced increase of endothelial cell numbers was significantly (n = 10, p < 0.01) reduced by the Ca(2+) chelator BAPTA (10 micromol/l), or the reactive oxygen species (ROS) scavenger N-acetylcysteine (ACC, 1 mmol/l), or the NAD(P)H-oxidase inhibitor diphenyleneiodonium (DPI, 5 micromol/l). In detail, cell numbers were (in percentage of control): 163.24 (CN), 90.06 (CN+ACC), 92.06 (CN+DPI). Intracellular-Ca(2+) and -ROS, analysed by fluorescence imaging, were significantly increased by CN. Interestingly, the CN-induced increase of ROS was in part Ca(2+)-dependent, whereas the Ca(2+) increase was not ROS-dependent. Simvastatin (5 micromol/l), fluvastatin (2.5 micromol/l), and cerivastatin (0.1 micromol/l) all reduced CN-induced proliferation, ROS generation and Ca(2+) increase. Cell viability was not reduced by the statins and the antiproliferative effect was completely reversed by mevalonate (500 micromol/l). In conclusion our study demonstrates that statins block hypoxia-associated endothelial proliferation by preventing the increase of Ca(2+) and ROS.
Assuntos
Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Óxido Nítrico Sintase/antagonistas & inibidores , Oniocompostos/farmacologia , Cianeto de Sódio/farmacologiaRESUMO
The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in many disease states, including tumor cells, either by growth factor receptor tyrosine kinases or by the genetic mutation and amplification of key pathway components. A variety of PI3K isoforms play differential roles in cancers. As such, the development of PI3K inhibitors from novel compound classes should lead to differential pharmacological and pharmacokinetic profiles and allow exploration in various indications, combinations, and dosing regimens. A screening effort aimed at the identification of PI3Kγ inhibitors for the treatment of inflammatory diseases led to the discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subsequent lead optimization program targeting cancer therapy focused on inhibition of PI3Kα and PI3Kß. Herein, initial structure-activity relationship findings for this class and the optimization that led to the identification of copanlisib (BAY 80-6946) as a clinical candidate for the treatment of solid and hematological tumors are described.