RESUMO
Increased production of Prostaglandin D2 (PGD2) is linked to development and progression of asthma and allergy. PGD2 is rapidly degraded to its metabolites, which initiate type 2 innate lymphoid cells (ILC2) migration and IL-5/IL-13 cytokine secretion in a PGD2 receptor 2 (DP2)-dependent manner. Blockade of DP2 has shown therapeutic benefit in subsets of asthma patients. Cellular mechanisms of ILC2 activity in response to PGD2 and its metabolites are still unclear. We hypothesized that ILC2 respond non-uniformly to PGD2 metabolites. ILC2s were isolated from peripheral blood of patients with atopic asthma. ILC2s were stimulated with PGD2 and four PGD2 metabolites (Δ12-PGJ2, Δ12-PGD2, 15-deoxyΔ12,14-PGD2, 9α,11ß-PGF2) with or without the selective DP2 antagonist fevipiprant. Total RNA was sequenced, and differentially expressed genes (DEG) were identified by DeSeq2. Differential gene expression analysis revealed an upregulation of pro-inflammatory DEGs in ILC2s stimulated with PGD2 (14 DEGs), Δ12-PGD2 (27 DEGs), 15-deoxyΔ12,14-PGD2 (56 DEGs) and Δ12-PGJ2 (136 DEGs), but not with 9α,11ß-PGF2. Common upregulated DEGs were i.e. ARG2, SLC43A2, LAYN, IGFLR1, or EPHX2. Inhibition of DP2 via fevipiprant mainly resulted in downregulation of pro-inflammatory genes such as DUSP4, SPRED2, DUSP6, ETV1, ASB2, CD38, ADGRG1, DDIT4, TRPM2, or CD69. DEGs were related to migration and various immune response-relevant pathways such as "chemokine (C-C motif) ligand 4 production", "cell migration", "interleukin-13 production", "regulation of receptor signaling pathway via JAK-STAT", or "lymphocyte apoptotic process", underlining the pro-inflammatory effects of PGD2 metabolite-induced immune responses in ILC2s as well as the anti-inflammatory effects of DP2 inhibition via fevipiprant. Furthermore, PGD2 and metabolites showed distinct profiles in ILC2 activation. Overall, these results expand our understanding of DP2 initiated ILC2 activity.
Assuntos
Asma , Imunidade Inata , Linfócitos , Prostaglandina D2 , Receptores Imunológicos , Receptores de Prostaglandina , Transdução de Sinais , Humanos , Asma/imunologia , Asma/metabolismo , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Prostaglandina D2/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos/metabolismo , Linfócitos/imunologia , Linfócitos/efeitos dos fármacos , Feminino , Masculino , Adulto , Ácidos Indolacéticos , PiridinasRESUMO
BACKGROUND: Non-allergic angioedema is a potentially life-threatening condition caused by accumulation of bradykinin and subsequent activation of bradykinin type 2 receptors (B2). Since COX activity plays a pivotal role in B2 signaling, the aim of this study was to determine which prostaglandins are the key mediators and which COX, COX-1 or COX-2, is predominantly involved. METHODS: We used Miles assays to assess the effects of inhibitors of COX, 5-lipoxygenase, epoxyeicosatrienoic acid generation, cytosolic phospholipase A2α and a variety of prostaglandin receptor antagonists on bradykinin-induced dermal extravasation in C57BL/6 and COX-1-deficient mice (COX-1-/-). In addition, the prostacyclin metabolite 6-keto-PGF1α was quantified by ELISA in subcutaneous tissue from C57BL/6 and human dermal microvascular endothelial cells. In the latter, 6-keto-PGF1α was also quantified and identified by LC-MS/MS. RESULTS: Unspecific COX inhibition by ibuprofen and diclofenac significantly reduced B2-mediated dermal extravasation in C57BL/6 but not COX-1-/-. Likewise, inhibition of cytosolic phospholipase A2α showed similar effects. Furthermore, extravasation in COX-1-/- was generally lower than in C57BL/6. Of the prostaglandin antagonists used, only the prostacyclin receptor antagonist RO1138452 showed a significant reduction of dermal extravasation. Moreover, 6-keto-PGF1α concentrations were increased after bradykinin treatment in subcutaneous tissue from C57BL/6 as well as in human dermal microvascular endothelial cells and this increase was abolished by diclofenac. CONCLUSION: Our findings suggest that COX-1-dependent prostacyclin production is critically involved in dermal extravasation after activation of B2 in small dermal blood vessels. Targeting prostacyclin production and/or signaling appears to be a suitable option for acute treatment of non-allergic angioedema.
Assuntos
Angioedema/patologia , Ciclo-Oxigenase 1/metabolismo , Epoprostenol/metabolismo , Angioedema/induzido quimicamente , Animais , Araquidonato 5-Lipoxigenase/efeitos dos fármacos , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Bradicinina/farmacologia , Diclofenaco/farmacologia , Células Endoteliais/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/metabolismo , Ibuprofeno/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases/efeitos dos fármacos , Oxigenases/metabolismo , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Prostaglandina/antagonistas & inibidoresRESUMO
Senescence in vascular smooth muscle cells (VSMCs) is involved in vascular remodeling of aged mice. ProstaglandinF2α- (PGF2α-) FP receptor plays a critical role in cardiovascular diseases (CVDs), hypertension, and cardiac fibrosis. However, its role in senescence-induced arteriosclerosis is yet to be fully elucidated. In this study, we found that FP receptor expression increased in aged mouse aortas and senescence VSMCs. FP receptor gene silencing can ameliorate vascular aging and inhibit oxidative stress, thereby reducing the expression of PAI-1, inhibiting the activation of MMPs, and ultimately improving the excessive deposition of ECM and delaying the process of vascular fibrosis. FP receptor could promote VSMC senescence by upregulated Src/PAI-1 signal pathway, and inhibited FP receptor/Src/PAI-1 pathway could ameliorate VSMCs aging in vitro, evidenced by the decrease of senescence-related proteins P16, P21, P53, and GLB1 expressions. These results suggested that FP receptor is a promoter of vascular aging, by inducing cellular aging, oxidative stress, and vascular remodeling via Src and PAI-1 upregulation.
Assuntos
Senescência Celular , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Remodelação Vascular , Quinases da Família src/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Colágeno/genética , Colágeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Quinases da Família src/genéticaRESUMO
Prostaglandin D2 (PGD2) released from immune cells or other cell types activates its receptors, D prostanoid receptor (DP)1 and 2 (DP1 and DP2), to promote inflammatory responses in allergic and lung diseases. Prostaglandin-mediated inflammation may also contribute to vascular diseases such as abdominal aortic aneurysm (AAA). However, the role of DP receptors in the pathogenesis of AAA has not been systematically investigated. In the present study, DP1-deficient mice and pharmacological inhibitors of either DP1 or DP2 were tested in two distinct mouse models of AAA formation: angiotensin II (AngII) infusion and calcium chloride (CaCl2) application. DP1-deficient mice [both heterozygous (DP1+/-) and homozygous (DP1-/-)] were protected against CaCl2-induced AAA formation, in conjunction with decreased matrix metallopeptidase (MMP) activity and adventitial inflammatory cell infiltration. In the AngII infusion model, DP1+/- mice, but not DP1-/- mice, exhibited reduced AAA formation. Interestingly, compensatory up-regulation of the DP2 receptor was detected in DP1-/- mice in response to AngII infusion, suggesting a potential role for DP2 receptors in AAA. Treatment with selective antagonists of DP1 (laropiprant) or DP2 (fevipiprant) protected against AAA formation, in conjunction with reduced elastin degradation and aortic inflammatory responses. In conclusion, PGD2 signaling contributes to AAA formation in mice, suggesting that antagonists of DP receptors, which have been extensively tested in allergic and lung diseases, may be promising candidates to ameliorate AAA.
Assuntos
Aneurisma da Aorta Abdominal/etiologia , Receptores Imunológicos/fisiologia , Receptores de Prostaglandina/fisiologia , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/prevenção & controle , Masculino , Camundongos , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidoresRESUMO
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe respiratory disorders that are caused by aspiration, sepsis, trauma, and pneumonia. A clinical feature of ALI/ARDS is the acute onset of severe hypoxemia, and the mortality rate, which is estimated at 38-50%, remains high. Although prostaglandins (PGs) are detected in the bronchoalveolar lavage fluid of patients with ALI/ARDS, the role of PGF2α in ALI remains unclear. We aimed to clarify the role of PGF2α/PGF2α receptor (FP) signaling in acid-induced ALI using an FP receptor antagonist, AL8810. Intratracheal injection of hydrochloric acid (HCl) increased neutrophil migration into the lungs, leading to respiratory dysfunction. Pre-administration of AL8810 further increased these features. Moreover, pre-treatment with AL8810 enhanced the HCl-induced expression of pro-inflammatory cytokines and neutrophil migratory factors in the lungs. Administration of HCl decreased the gene expression of lung surfactant proteins, which was further reduced by co-administration of AL8810. Administration of AL8810 also increased lung edema and reduced mRNA expression of epithelial sodium channel in the lungs, indicating that AL8810 reduced fluid clearance. Furthermore, AL8810 also increased lipopolysaccharide-induced expression of adhesion molecules such as intracellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells. These results indicate that inhibition of FP receptors by AL8810 exacerbated HCl-induced ALI.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Pulmão/efeitos dos fármacos , Pneumonia/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Síndrome do Desconforto Respiratório/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Modelos Animais de Doenças , Feminino , Ácido Clorídrico/toxicidade , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Pneumonia/patologia , Prostaglandinas F/metabolismo , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/patologiaRESUMO
Human type 2 cytotoxic T (Tc2) cells are enriched in severe eosinophilic asthma and can contribute to airway eosinophilia. PGD2 and its receptor PGD2 receptor 2 (DP2) play important roles in Tc2 cell activation, including migration, cytokine production, and survival. In this study, we revealed novel, to our knowledge, functions of the PGD2/DP2 axis in Tc2 cells to induce tissue-remodeling effects and IgE-independent PGD2 autocrine production. PGD2 upregulated the expression of tissue-remodeling genes in Tc2 cells that enhanced the fibroblast proliferation and protein production required for tissue repair and myofibroblast differentiation. PGD2 stimulated Tc2 cells to produce PGD2 using the routine PGD2 synthesis pathway, which also contributed to TCR-dependent PGD2 production in Tc2 cells. Using fevipiprant, a specific DP2 antagonist, we demonstrated that competitive inhibition of DP2 not only completely blocked the cell migration, adhesion, proinflammatory cytokine production, and survival of Tc2 cells triggered by PGD2 but also attenuated the tissue-remodeling effects and autocrine/paracrine PGD2 production in Tc2 induced by PGD2 and other stimulators. These findings further confirmed the anti-inflammatory effect of fevipiprant and provided a better understanding of the role of Tc2 cells in the pathogenesis of asthma.
Assuntos
Ácidos Indolacéticos/farmacologia , Inflamação/tratamento farmacológico , Prostaglandina D2/antagonistas & inibidores , Piridinas/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Humanos , Inflamação/imunologia , Prostaglandina D2/biossíntese , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
The primary means of intestinal absorption of nutrients by villus cells is via Na-dependent nutrient co-transporters located in the brush border membrane (BBM). These secondary active co-transport processes require a favorable transcellular Na gradient that is provided by Na-K-ATPase. In chronic enteritis, malabsorption of essential nutrients is partially due to inhibition of villus Na-K-ATPase activity mediated by specific immune inflammatory mediators that are known to be elevated in the inflamed mucosa. However, how Prostaglandin E2 (PGE2), a specific mediator of nutrient malabsorption in the villus BBM, may mediate the inhibition of Na-K-ATPase is not known. Therefore, this study aimed to determine the effect of PGE2 on Na-K-ATPase in villus cells and define its mechanism of action. In vitro, in IEC-18 cells, PGE2 treatment significantly reduced Na-K-ATPase activity, accompanied by a significant increase in the intracellular levels of cyclic Adenosine Monophosphate (cAMP). The treatment with cAMP analog 8-Bromo-cAMP mimicked the PGE2-mediated effect on Na-K-ATPase activity, while Rp-cAMP (PKA inhibitor) pretreatment reversed the same. The mechanism of inhibition of PGE2 was secondary to a transcriptional reduction in the Na-K-ATPase α1 and ß1 subunit genes, which was reversed by the Rp-cAMP pretreatment. Thus, the PGE2-mediated activation of the PKA pathway mediates the transcriptional inhibition of Na-K-ATPase activity in vitro.
Assuntos
Dinoprostona/farmacologia , Células Epiteliais/enzimologia , Intestinos/citologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Espaço Intracelular/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Xantonas/farmacologiaAssuntos
Asma/sangue , Ácidos Indolacéticos/farmacocinética , Modelos Biológicos , Piridinas/farmacocinética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Asma/tratamento farmacológico , Variação Biológica da População , Criança , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase III como Assunto , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Humanos , Ácidos Indolacéticos/administração & dosagem , Ácidos Indolacéticos/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Adulto JovemRESUMO
Non-hormonal therapeutic strategies for endometriosis are needed. The aim of this study was to characterize the effects of prostaglandin (PG)E2 receptor inhibitors to explore their potential as novel therapeutic strategies for endometriosis. The expression of PGE2 receptors (EP2 and EP4) in donated tissues from human ovarian endometriosis, adenomyosis and peritoneal endometriosis was examined using immunohistochemistry. Human endometriotic stromal cells (ESC) isolated from ovarian endometriotic tissue and peritoneal macrophages were treated with EP2 and EP4 antagonists. cAMP accumulation and the effect of EP antagonists were measured using cAMP assays. DNA synthesis in ESC was detected using bromodeoxyuridine incorporation analysis. Interleukin (IL)-6 and IL-8 protein levels in ESC supernatants were measured using ELISAs. mRNA expression level for aromatase by ESC, and selected cytokines by peritoneal macrophages was measured using RT-PCR. EP2 and EP4 receptors were expressed in cells derived from control and diseased tissue, ovarian endometriotic, adenomyotic and peritoneal lesions. A selective EP2 antagonist reduced DNA synthesis, cAMP accumulation and IL-1ß-induced proinflammatory cytokine secretion and aromatase expression. A selective EP4 antagonist negated IL-1ß-induced IL-6 secretion and aromatase expression. In peritoneal macrophages, EP expression was elevated in endometriosis samples but the EP4 antagonist reduced cAMP levels and expression of vascular endothelial growth factor, chemokine ligand 2 and chemokine ligand 3 mRNA. EP2 and EP4 are functioning in endometriosis lesions and peritoneal macrophages, and their selective antagonists can reduce EP-mediated actions, therefore, the EP antagonists are potential therapeutic agents for controlling endometriosis.
Assuntos
Azetidinas/farmacologia , Benzamidas/farmacologia , Endometriose/tratamento farmacológico , Endométrio/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Receptores de Prostaglandina/antagonistas & inibidores , Células Estromais/efeitos dos fármacos , Adulto , Células Cultivadas , Quimiocinas/biossíntese , AMP Cíclico/metabolismo , Replicação do DNA/efeitos dos fármacos , Endométrio/citologia , Feminino , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidoresRESUMO
Although it is known that prostaglandin (PG) F2α level is elevated in the plasma of patients with sepsis, the roles of PGF2α is still unknown. We aimed to clarify the roles of PGF2α in the regulation of lipopolysaccharide (LPS)-induced systemic inflammation. At 24 hours after LPS administration, neutrophil infiltration in peritoneal cavity, the mRNA expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and macrophage inflammatory protein-2, and tissue damages in lung, liver, and kidney were all increased. Inhibition of FP receptors significantly decreased LPS-induced neutrophil infiltration and lowered the mRNA expression of the pro-inflammatory cytokines. At 6 hour after LPS administration, the level of anti-inflammatory cytokine, IL-10 in peritoneal lavage fluid was higher than that in naïve mice. Inhibition of FP receptors in these mice increased IL-10 level further. Stimulation of isolated peritoneal neutrophils by LPS increased the gene expression of IL-10, which was further increased by AL8810 treatment. Administration of an anti-IL-10 antibody antagonized the AL8810-decreased mRNA expression of pro-inflammatory cytokines and tissue damages. These results indicate that inhibition of FP receptors by AL8810 attenuated LPS-induced systemic inflammation in mice via enhanced IL-10 production.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/metabolismo , Dinoprosta/análogos & derivados , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Neutrófilos/efeitos dos fármacos , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Dinoprosta/farmacologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a rare and devastating chronic lung disease of unknown etiology. Despite the approved treatment options nintedanib and pirfenidone, the medical need for a safe and well-tolerated antifibrotic treatment of IPF remains high. The human prostaglandin F receptor (hFP-R) is widely expressed in the lung tissue and constitutes an attractive target for the treatment of fibrotic lung diseases. Herein, we present our research toward novel quinoline-based hFP-R antagonists, including synthesis and detailed structure-activity relationship (SAR). Starting from a high-throughput screening (HTS) hit of our corporate compound library, multiple parameter improvements-including increase of the relative oral bioavailability Frel from 3 to ≥100%-led to a highly potent and selective hFP-R antagonist with complete oral absorption from suspension. BAY-6672 (46) represents-to the best of our knowledge-the first reported FP-R antagonist to demonstrate in vivo efficacy in a preclinical animal model of lung fibrosis, thus paving the way for a new treatment option in IPF.
Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/efeitos dos fármacos , Quinolinas/síntese química , Receptores de Prostaglandina/antagonistas & inibidores , Administração Oral , Animais , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Estrutura Molecular , Quinolinas/química , Quinolinas/uso terapêutico , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A characteristic feature of COVID-19 disease is lymphopenia. Lymphopenia occurs early in the clinical course and is a predictor of disease severity and outcomes. The mechanism of lymphopenia in COVID-19 is uncertain. It has been variously attributed to the release of inflammatory cytokines including IL-6 and TNF-α; direct infection of the lymphocytes by the virus; and rapid sequestration of lymphocytes in the tissues. Additionally, we postulate that prostaglandin D2 (PGD2) is a key meditator of lymphopenia in COVID-19. First, SARS-CoV infection is known to stimulate the production of PGD2 in the airways, which inhibits the host dendritic cell response via the DP1 receptor signaling. Second, PGD2 is known to upregulate monocytic myeloid-derived suppressor cells (MDSC) via the DP2 receptor signaling in group 2 innate lymphoid cells (ILC2). We propose targeting PGD2/DP2 signaling using a receptor antagonist such as ramatroban as an immunotherapy for immune dysfunction and lymphopenia in COVID-19 disease.
Assuntos
Betacoronavirus , Infecções por Coronavirus/fisiopatologia , Linfopenia/fisiopatologia , Modelos Imunológicos , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/fisiopatologia , Prostaglandina D2/fisiologia , Sistema Respiratório/metabolismo , Adulto , COVID-19 , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Criança , Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Células Dendríticas/imunologia , Humanos , Linfopenia/etiologia , Células Mieloides/imunologia , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Prostaglandina D2/biossíntese , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/fisiologia , SARS-CoV-2 , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Linfócitos T/imunologia , Tromboxano A2/antagonistas & inibidoresRESUMO
AIMS/HYPOTHESIS: Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets. METHODS: Human islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1ß. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets. RESULTS: PGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1ß in human islets. This was accompanied by activation of the Akt-glycogen synthase kinase 3ß signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets. CONCLUSIONS/INTERPRETATION: Inhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Western Blotting , Morte Celular/fisiologia , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina/fisiologia , Prostaglandina D2 , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Macrophages are the effector immune cells with plasticity to differentiate as M1 (classically activated) and M2 (alternatively activated) phenotypes. Prostaglandins (PGs) have various important roles and are involved in the regulation of macrophage activation. However, the role of PGF2α in macrophage activation remains unclear. We investigated the role of PGF2α receptor (FP)-mediated signaling in the M1 macrophage polarization using murine macrophage RAW264.7 cells. Stimulation with lipopolysaccharide (LPS) + interferon (IFN)-γ increased the mRNA expression of the M1 macrophage markers such as inducible nitric oxide synthase, tumor necrosis factor-α, and CD11c. Pre-treatment with AL8810, an FP receptor antagonist, further enhanced the expression of these genes. In contrast, treatment with fluprostenol, an FP receptor agonist, decreased the LPS + IFN-γ-induced expression of M1 markers. LPS-induced M1 macrophage polarization was dependent on the activation of NF-κB p65. Treatment with IκB kinase ß inhibitor reduced AL8810-induced mRNA expression of the M1 markers. Stimulation with LPS + IFN-γ increased the expression of IL-10. Pre-treatment with AL8810 lowered LPS + IFN-γ-induced IL-10 expression, and further enhanced LPS + IFN-γ-stimulated nuclear translocation of NF-κB p65. In contrast, co-treatment with IL-10 reversed AL8810-induced nuclear translocation of NF-κB p65. These results indicate that the FP receptor signaling was involved in the control of M1 polarization of macrophages via IL-10-regulated nuclear translocation of NF-κB p65.
Assuntos
Interleucina-10/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Receptores de Prostaglandina/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Núcleo Celular/metabolismo , Dinoprosta/análogos & derivados , Dinoprosta/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Prostaglandinas F Sintéticas/farmacologia , Células RAW 264.7 , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Tiofenos/farmacologiaRESUMO
BACKGROUND: Adaptive trial designs have the potential to address common challenges in drug development; they decrease timelines and costs of early drug development and efficiently create data that support future trials in target populations. While allowing for flexibility and evolution, adaptive strategies introduce some complexity to the design and implementation of trial protocols. Previously published work by the authors include a retrospective analysis of time savings using adaptive design and a systematic, 3- step methodology for writing early-phase adaptive integrated protocols. METHODS: This article builds on the authors' published work demonstrating the practical implementation of the adaptive protocol writing methodology and discussing the challenges and efficiencies. It describes the integration of an early development program of OBE022, a novel, oral, selective prostaglandin F2a receptor antagonist, intended as a treatment for preterm labor, using 2 interdependent, adaptive trial protocols. The program consisted of first-in-human single and multiple ascending dose parts with assessments of food effect, cardiac safety, proof of concept, and interactions of OBE022 with 4 standard of care medicines. RESULTS: The manuscript shows how the trials were tailored to OBE022's pharmacokinetic and pharmacodynamic characteristics and its therapeutic indication. The use of 2 large interdependent, adaptive protocols was facilitated by the United Kingdom's (UK's) regulatory environment and its acceptance of a rules-guided progression through the program. Changes to the planned trial conduct could be made without impacting on timelines, because they used predefined adaptive options within their authorized boundaries, and could therefore be made as nonsubstantial amendments. The program was successful and achieved its objectives. It was efficient and fast: it required a small number of participants (n=83) and completed from start of protocol writing to first draft of the clinical study report in just 11 months. CONCLUSIONS: This program included all key elements of early drug development in 2 interlinked protocols: the assessment of single and multiple ascending doses, food effect, cardiac safety and proof of concept. The approach described in this article demonstrates how early-phase programs can be designed to be performed, analyzed and reported time- and cost-efficiently.
Assuntos
Ensaios Clínicos Adaptados como Assunto , Desenvolvimento de Medicamentos , Projetos de Pesquisa , Humanos , Receptores de Prostaglandina/antagonistas & inibidores , Estudos RetrospectivosRESUMO
BACKGROUND: GB001 is an oral antagonist of the prostaglandin D2 receptor that may inhibit recruitment and activation of airway eosinophils, reducing airway inflammation. OBJECTIVE: To assess GB001 safety, efficacy and pharmacokinetics from a Phase 2 study and explore the association between type 2 biomarkers (fractional exhaled nitric oxide and blood eosinophils) and asthma control markers following GB001 administration. METHODS: A randomized, placebo-controlled, double-blind study evaluating 36 patients with mild-to-moderate atopic asthma. Patients receiving fluticasone propionate ≤500 mcg/day or equivalent were randomized (2:1) to GB001 (30 mg) or placebo once daily for 28 days. Safety, pharmacokinetics, forced expiratory volume in 1 second, asthma control questionnaire and rescue medication use were assessed. Clinical outcomes were analysed post hoc by baseline fractional exhaled nitric oxide (<35 and ≥35 ppb) and blood eosinophil (<250 and ≥250 cells/µL) subgroups. RESULTS: GB001 was well tolerated and rapidly absorbed with a 14.5-hour terminal half-life. Overall, GB001 demonstrated greater improvement relative to placebo in forced expiratory volume in 1 second at Day 28 (102 mL [95% CI: -110, 314]). Greater effects on forced expiratory volume in 1 second were observed in the high baseline fractional exhaled nitric oxide and blood eosinophil subgroups (207 mL [95% CI: -283, 698];133 mL [95% CI: -422, 687], respectively). These effects were observed as early as Day 2 (229 mL [95% CI: -170, 628]; 163 mL [95% CI: -223, 550] for the high baseline fractional exhaled nitric oxide and blood eosinophil subgroups, respectively) and were sustained through treatment completion. CONCLUSION AND CLINICAL RELEVANCE: GB001 was well tolerated, with the estimated half-life supporting once-daily (QD) dosing. GB001 may have a rapid and sustained effect on lung function, particularly in patients with type 2 phenotype. Further studies are needed to confirm these findings.
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Antiasmáticos , Asma , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Adolescente , Adulto , Antiasmáticos/administração & dosagem , Antiasmáticos/efeitos adversos , Antiasmáticos/farmacocinética , Asma/tratamento farmacológico , Asma/imunologia , Asma/patologia , Biomarcadores , Testes Respiratórios , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologiaRESUMO
OBJECTIVE: Prostaglandin 2 (PGD2) mediated signalling of Chemoattractant Receptorhomologous molecule expressed on Th2 cells (CRTh2) receptor has been implicated in the recruitment of inflammatory cells. This explains the design of highly selective compounds with innate abilities to antagonize PGD2-CRTh2 interactions and prevent pro-inflammatory allergies such as rhinitis and uncontrolled asthma. The development of PGD2-competitive CRTh2 binders; CAY10471 and Fevipiprant represent remarkable therapeutic progress even though they elicit disparate pharmacological propensities despite utilizing the same binding pocket. METHODS & RESULTS: In this study, we seek to pinpoint the underlying phenomenon associated with differential CRTh2 therapeutic inhibition by CAY10471 and Fevipiprant using membraneembedded molecular dynamics simulation. Findings revealed that the common carboxylate group of both compounds elicited strong attractive charges with active site Arg170 and Lys210. Interestingly, a distinctive feature was the steady occurrence of high-affinity salt-bridges and an Arg170-mediated pi-cation interaction with the tetrahydrocarbozole ring of CAY10471. Further investigations into the active site motions of both ligands revealed that CAY10471 was relatively more stable. Comparative binding analyses also revealed that CAY10471 exhibited higher ΔG, indicating the cruciality of the ring stabilization role mediated by Arg170. Moreover, conformational analyses revealed that the inhibitory activity of CAY10471 was more prominent on CRTh2 compared to Fevipiprant. CONCLUSIONS: These findings could further advance the strategic design of novel CRTh2 binders in the treatment of diseases related to pro-inflammatory allergies.
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Asma/tratamento farmacológico , Carbazóis/farmacologia , Ácidos Indolacéticos/farmacologia , Piridinas/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Rinite Alérgica/tratamento farmacológico , Sulfonamidas/farmacologia , Asma/metabolismo , Carbazóis/química , Ensaios de Triagem em Larga Escala , Humanos , Ácidos Indolacéticos/química , Simulação de Dinâmica Molecular , Estrutura Molecular , Piridinas/química , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Rinite Alérgica/metabolismo , Sulfonamidas/química , Resultado do TratamentoRESUMO
Fevipiprant, a prostaglandin D2 receptor 2 antagonist, is in clinical development as a treatment for asthma. The goal of this study was to assess the potential of fevipiprant to cause drug-drug interactions (DDI) as a perpetrator, that is, by altering the pharmacokinetics (PK) of co-medications. In vitro drug interaction studies of clinically relevant drug metabolizing enzymes and transporters were conducted for fevipiprant and its acyl glucuronide (AG) metabolite. Comparison of Ki values with unbound systemic or portal vein steady-state plasma exposure of fevipiprant and its AG metabolite revealed the potential for inhibition of organic anion transporting polypeptide 1B1 (OATP1B1) transporters (R-value of 5.99), while other targets including cytochrome P450 enzymes were not, or only marginally, inhibited. Consequently, an open-label, two-part, two-period, single-sequence clinical study assessed the effect of fevipiprant 450â¯mg QD on the pharmacokinetics of simvastatin 20â¯mg and rosuvastatin 20â¯mg, two statins with different dependency in OATP1B1-mediated hepatic uptake, in healthy adult volunteers. The study also assessed the pharmacogenetics of the SLCO1B1 gene, which encodes OATP1B1. Clinically, fevipiprant 450â¯mg QD showed a low potential for interaction and increased the peak concentrations of simvastatin acid and rosuvastatin by 2.23- and 1.87-fold, respectively, with little or no impact on total exposure. Genotype analysis confirmed that SLCO1B1 genotype influences statin pharmacokinetics to a similar extent either with or without fevipiprant co-administration. In summary, fevipiprant at 450â¯mg QD has only minor liabilities as a perpetrator for DDI.
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Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Ácidos Indolacéticos/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Piridinas/farmacologia , Rosuvastatina Cálcica/farmacocinética , Sinvastatina/farmacocinética , Adulto , Interações Medicamentosas , Feminino , Genótipo , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Transportadores de Ânions Orgânicos , Farmacogenética , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidoresRESUMO
INTRODUCTION: MK-7246 is a potent and selective antagonist for chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Within the pancreas CRTH2 is selectively expressed in pancreatic ß-cells where it is believed to play a role in insulin release. Reduction in ß-cell mass and insufficient insulin secretion in response to elevated blood glucose levels is a hallmark for type 1 and type 2 diabetes. Reported here is the synthesis of [11C]MK-7246 and initial preclinical evaluation towards CRTH2 imaging. The aim is to develop a method to quantify ß-cell mass with PET and facilitate non-invasive studies of disease progression in individuals with type 2 diabetes. METHODS: The precursor N-desmethyl-O-methyl MK-7246 was synthesized in seven steps and subjected to methylation with [11C]methyl iodide followed by hydrolysis to obtain [11C]MK-7246 labelled in the N-methyl position. Preclinical evaluation included in vitro radiography and immune-staining performed in human pancreatic biopsies. Biodistribution studies were performed in rat by PET-MRI and in pig by PET-CT imaging. Saturable tracer binding was examined in pig by scanning before and after administration of MK-7246 (1â¯mg/kg). Predicted dosimetry of [11C]MK-7246 in human males was estimated based on the biodistribution in rat. RESULTS: [11C]MK-7246 was obtained with activities sufficient for the current investigations (270⯱â¯120â¯MBq) and a radiochemical purity of 93⯱â¯2%. The tracer displayed focal binding in areas with insulin positive islet of Langerhans in human pancreas sections. Baseline uptake in pig was reduced in tissues with known expression of CRTH2 after administration of MK-7246; pancreas (66% reduction) and spleen (88% reduction). [11C]MK-7246 exhibited a safe human predicted dosimetry profile as extrapolated from the rat biodistribution data. CONCLUSIONS: Initial preclinical in vitro and in vivo evaluations of [11C]MK-7246 show binding and biodistribution properties suitable for PET imaging of CRTH2. Further studies are warranted to assess its potential in ß-cell mass imaging and CRTH2 drug development.
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Carbolinas/síntese química , Carbolinas/farmacologia , Radioisótopos de Carbono , Tamanho Celular , Células Secretoras de Insulina/citologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Carbolinas/química , Carbolinas/farmacocinética , Técnicas de Química Sintética , Humanos , Masculino , Traçadores Radioativos , Radioquímica , Ratos , Suínos , Distribuição TecidualRESUMO
Mechanistic studies have improved our understanding of molecular and cellular components involved in asthma and our ability to treat severe patients. An mAb directed against IgE (omalizumab) has become an established add-on therapy for patients with uncontrolled allergic asthma and mAbs specific for IL-5 (reslizumab, mepolizumab), IL-5R (benralizumab), and IL-4R (dupilumab) have been approved as add-on treatments for uncontrolled eosinophilic (type 2) asthma. While these medications have proven highly effective, some patients with severe allergic and/or eosinophilic asthma, as well as most patients with severe non-type-2 disease, have poorly controlled disease. Agents that have recently been evaluated in clinical trials include an antibody directed against thymic stromal lymphopoietin, small molecule antagonists to the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) and the receptor for stem cell factor on mast cells (KIT), and a DNA enzyme directed at GATA3. Antibodies to IL-33 and its receptor, ST2, are being evaluated in ongoing clinical studies. In addition, a number of antagonists directed against other potential targets are under consideration for future trials, including IL-25, IL-6, TNF-like ligand 1A, CD6, and activated cell adhesion molecule (ALCAM). Clinical data from ongoing and future trials will be important in determining whether these new medications will offer benefits in place of or in addition to existing therapies for asthma.