RESUMO
The use of nanoparticles (NPs) to deliver small inhibiting microRNAs (miRNAs) has shown great promise for treating cancer. However, constructing a miRNA delivery system that targets brain cancers, such as glioblastoma multiforme (GBM), remains technically challenging due to the existence of the blood-tumor barrier (BTB). In this work, a novel targeted antisense miRNA-21 oligonucleotide (ATMO-21) delivery system is developed for GBM treatment. Bradykinin ligand agonist-decorated spermine-modified acetalated dextran NPs (SpAcDex NPs) could temporarily open the BTB by activating G-protein-coupled receptors that are expressed in tumor blood vessels and tumor cells, which increase transportation to and accumulation in tumor sites. ATMO-21 achieves high loading in the SpAcDex NPs (over 90%) and undergoes gradual controlled release with the degradation of the NPs in acidic lysosomal compartments. This allows for cell apoptosis and inhibition of the expression of vascular endothelial growth factor by downregulating hypoxia-inducible factor (HIF-1α) protein. An in vivo orthotopic U87MG glioma model confirms that the released ATMO-21 shows significant therapeutic efficacy in inhibiting tumor growth and angiogenesis, demonstrating that agonist-modified SpAcDex NPs represent a promising strategy for GBM treatment combining targeted gene therapy and antiangiogenic therapy.
Assuntos
Inibidores da Angiogênese , Antagomirs , Antagonistas de Receptor B1 da Bradicinina , Terapia Genética , Glioma , MicroRNAs , Nanopartículas , Espermina , Inibidores da Angiogênese/administração & dosagem , Antagomirs/administração & dosagem , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Linhagem Celular Tumoral , Dextranos , Terapia Genética/métodos , Glioma/terapia , Humanos , MicroRNAs/antagonistas & inibidores , Nanopartículas/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Neuroinflammation has become an important underlying factor in many cardiovascular disorders, including hypertension. Previously we showed that elevated angiotensin II (Ang II) and angiotensin II type I receptor (AT1R) expression levels can increase neuroinflammation leading to hypertension. We also found that kinin B1 receptor (B1R) expression increased in the hypothalamic paraventricular neurons resulting in neuroinflammation and oxidative stress in neurogenic hypertension. However, whether there are any potential interactions between AT1R and B1R in neuroinflammation is not clear. In the present study, we aimed to determine whether Ang II-mediated effects on inflammation and oxidative stress are mediated by the activation of B1R in mouse neonatal primary hypothalamic neuronal cultures. Gene expression and immunostaining revealed that both B1R and AT1R are expressed on primary hypothalamic neurons. Ang II stimulation significantly increased the expression of B1R, decreased mitochondrial respiration, increased the expression of two NADPH oxidase subunits (Nox2 and Nox4), increased the oxidative potential, upregulated several proinflammatory genes (IL-1ß, IL-6, and TNFα), and increased NF-kB p65 DNA binding activity. These changes were prevented by pretreatment with the B1R-specific peptide antagonist, R715. In summary, our study demonstrates a causal relationship between B1R expression after Ang II stimulation, suggesting a possible cross talk between AT1R and B1R in neuroinflammation and oxidative stress.
Assuntos
Angiotensina II/metabolismo , Antagonistas de Receptor B1 da Bradicinina/uso terapêutico , Encefalite/tratamento farmacológico , Hipotálamo/metabolismo , Estresse Oxidativo , Receptor B1 da Bradicinina/metabolismo , Animais , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Hipertensão/prevenção & controle , Hipotálamo/efeitos dos fármacos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.
Assuntos
Transtornos de Ansiedade/metabolismo , Bradicinina/metabolismo , Sistema Calicreína-Cinina/fisiologia , Estresse Psicológico/metabolismo , Adulto , Animais , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/genética , Transtornos de Ansiedade/patologia , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Antagonistas de Receptor B2 da Bradicinina/administração & dosagem , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Sistema Calicreína-Cinina/efeitos dos fármacos , Cininogênios/genética , Cininogênios/metabolismo , Masculino , Camundongos , Naftalenos/administração & dosagem , Compostos Organofosforados/administração & dosagem , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Polimorfismo de Nucleotídeo Único , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Especificidade da Espécie , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/patologia , Regulação para CimaRESUMO
BACKGROUND: Tibial fracture is associated with inflammatory reaction leading to severe pain syndrome. Bradykinin receptor activation is involved in inflammatory reactions, but has never been investigated in fracture pain. METHODS: This study aims at defining the role of B1 and B2-kinin receptors (B1R and B2R) in a closed tibial fracture pain model by using knockout mice for B1R (B1KO) or B2R (B2KO) and wild-type (WT) mice treated with antagonists for B1R (SSR 240612 and R954) and B2R (HOE140) or vehicle. A cyclooxygenase (COX) inhibitor (ketoprofen) and an antagonist (SB366791) of Transient Receptor Potential Vaniloid1 (TRPV1) were also investigated since these pathways are associated with BK-induced pain in other models. The impact on mechanical and thermal hyperalgesia and locomotion was assessed by behavior tests. Gene expression of B1R and B2R and spinal cord expression of c-Fos were measured by RT-PCR and immunohistochemistry, respectively. RESULTS: B1KO and B2KO mice demonstrated a reduction in post-fracture pain sensitivity compared to WT mice that was associated with decreased c-Fos expression in the ipsilateral spinal dorsal horn in B2KO. B1R and B2R mRNA and protein levels were markedly enhanced at the fracture site. B1R and B2R antagonists and inhibition of COX and TRPV1 pathways reduced pain in WT. However, the analgesic effect of the COX-1/COX-2 inhibitor disappeared in B1KO and B2KO. In contrast, the analgesic effect of the TRPV1 antagonist persisted after gene deletion of either receptor. CONCLUSIONS: It is suggested that B1R and B2R activation contributes significantly to tibial fracture pain through COX. Hence, B1R and B2R antagonists appear potential therapeutic agents to manage post fracture pain.
Assuntos
Dor/fisiopatologia , Receptor B1 da Bradicinina/fisiologia , Receptor B2 da Bradicinina/fisiologia , Fraturas da Tíbia/fisiopatologia , Animais , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Modelos Animais de Doenças , Inflamação/etiologia , Inflamação/patologia , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dor/tratamento farmacológico , Dor/prevenção & controle , Medição da Dor , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro , Receptor B1 da Bradicinina/deficiência , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/deficiência , Receptor B2 da Bradicinina/genética , Canais de Cátion TRPV/antagonistas & inibidores , Fraturas da Tíbia/complicações , Fraturas da Tíbia/patologia , Pesquisa Translacional BiomédicaRESUMO
Tumour necrosis factor (TNF) and kinins have been associated with neuropathic pain-like behaviour in numerous animal models. However, the way that they interact to cause neuron sensitisation remains unclear. This study assessed the interaction of kinin receptors and TNF receptor TNFR1/p55 in mechanical hypersensitivity induced by an intraneural (i.n.) injection of rm-TNF into the lower trunk of brachial plexus in mice. The i.n. injection of rm-TNF reduced the mechanical withdrawal threshold of the right forepaw from the 3rd to the 10th day after the injection, indicating that TNF1/p55 displays a critical role in the onset of TNF-elicited neuropathic pain. The connection between TNF1/p55 and kinin B1 and B2 receptors (B1R and B2R) was confirmed using both knockout mice and mRNAs quantification in the injected nerve, DRG and spinal cord. The treatment with the B2R antagonist HOE 140 or with B1R antagonist des-Arg9-Leu8-BK reduced both BK- and DABK-induced hypersensitivity. The experiments using kinin receptor antagonists and CPM inhibitor (thiorphan) suggest that BK does not only activate B2R as an orthosteric agonist, but also seems to be converted into DABK that consequently activates B1R. These results indicate a connection between TNF and the kinin system, suggesting a relevant role for B1R and B2R in the process of sensitisation of the central nervous systems by the cross talk between the receptor and CPM after i.n. injection of rm-TNF.
Assuntos
Plexo Braquial/metabolismo , Neuralgia/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Plexo Braquial/efeitos dos fármacos , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/tratamento farmacológicoRESUMO
High nuclear expression of G protein-coupled receptors, including kinin B1 receptors (B1R), has been observed in several human cancers, but the clinical significance of this is unknown. We put forward the hypothesis that these "nuclearized" kinin B1R contribute to tumorigenicity and can be a new target in anticancer strategies. Our initial immunostaining and ultrastructural electron microscopy analyses demonstrated high B1R expression predominantly located at internal/nuclear compartments in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line as well as in clinical samples of patients with TNBC. On the basis of these findings, in the present study, we evaluated the anticancer therapeutic potential of newly identified, cell-permeable B1R antagonists in MDA-MB-231 cells (ligand-receptor binding/activity assays and LC-MS/MS analyses). We found that these compounds (SSR240612, NG67, and N2000) were more toxic to MDA-MB-231 cells in comparison with low- or non-B1R expressing MCF-10A normal human mammary epithelial cells and COS-1 cells, respectively (clonogenic, MTT proliferative/cytocidal assays, and fluorescence-activated cell-sorting (FACS)-based apoptosis analyses). By comparison, the peptide B1R antagonist R954 unable to cross cell membrane failed to produce anticancer effects. Furthermore, the putative mechanisms underlying the anticancer activities of cell-penetrant B1R antagonists were assessed by analyzing cell cycle regulation and signaling molecules related to cell survival and apoptosis (FACS and western blot). Finally, drug combination experiments showed that cell-penetrant B1R antagonists can cooperate with suboptimal doses of chemotherapeutic agents (doxorubicin and paclitaxel) to promote TNBC death. This study provides evidence on the potential value of internally acting kinin B1R antagonists in averting growth of breast cancer.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Receptor B1 da Bradicinina/genética , Receptores Acoplados a Proteínas G/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Células COS , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Chlorocebus aethiops , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Paclitaxel/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.
Assuntos
Bradicinina/química , Hormônios Peptídicos/química , Receptor B1 da Bradicinina/química , Receptor B2 da Bradicinina/química , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases , Humanos , Ligantes , Enzimas Multifuncionais , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Relação Estrutura-AtividadeRESUMO
The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B1R-/-, B2R-/- and double B1R/B2R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B1R and B2R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B2R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B1R/B2R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B1R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg9-BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B1R (des-Arg9-BK and SSR240612) and B2R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis.
Assuntos
Antituberculosos/administração & dosagem , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Dioxóis/administração & dosagem , Pulmão/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Receptor B1 da Bradicinina/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Tuberculose Pulmonar/tratamento farmacológico , Administração Oral , Animais , Carga Bacteriana , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Antagonistas de Receptor B2 da Bradicinina/administração & dosagem , Modelos Animais de Doenças , Feminino , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células RAW 264.7 , Receptor B1 da Bradicinina/deficiência , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/microbiologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
PURPOSE: This study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B1 and B2 receptors on LPS- induced fever and the POA cells involved in this response. MATERIAL AND METHODS: Male Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca++ signaling in POA cells was performed in rat pup brain tissue microcultures. RESULTS: Icatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE2concentration. Effect abolished by indomethacin (i.p.). CONCLUSIONS: LPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B2-receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B2-receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever.
Assuntos
Bradicinina/metabolismo , Febre/metabolismo , Calicreínas/metabolismo , Cininogênios/metabolismo , Receptor B2 da Bradicinina/fisiologia , Animais , Astrócitos/metabolismo , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Antagonistas de Receptor B2 da Bradicinina/administração & dosagem , Sinalização do Cálcio , Captopril/administração & dosagem , Células Cultivadas , Febre/induzido quimicamente , Lipopolissacarídeos , Masculino , Neurônios/metabolismo , Área Pré-Óptica/efeitos dos fármacos , Área Pré-Óptica/metabolismo , Ratos Wistar , Receptor B1 da Bradicinina/fisiologiaRESUMO
Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment strategies that complement conventional chemotherapies. Bradykinin B1 receptor (B1R) is a G protein-coupled receptor that is overexpressed in many cancers, with limited expression in healthy tissues. Previously, we developed 68Ga- and 18F-labeled derivatives of B1R antagonist peptides B9858 and B9958, and successfully targeted B1R-expressing tumor xenografts in vivo. R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-d-2-Nal-Ile), a potent B1R antagonist, is reportedly more stable than B9858 against peptidase degradation. We evaluated two radiolabeled derivatives of R954 (68Ga-HTK01083 and 18F-HTK01146) for B1R PET imaging. Peptides were synthesized via solid phase strategy. Nonradioactive standards were obtain by reacting GaCl3 with DOTA-dPEG2-R954 and by clicking N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate with azidoacetyl-dPEG2-R954. Binding affinity for B1R was determined by an in vitro competition binding assay. 68Ga-HTK01083 was obtained by incubating DOTA-dPEG2-R954 with 68GaCl3 under acidic conditions, while 18F-HTK01146 was prepared via an 18F-19F isotope exchange reaction. Biodistribution and imaging studies were conducted at 1 h postinjection (p.i.) in mice inoculated with B1R-expressing (B1R+) and B1R-nonexpressing (B1R-) cells. HTK01083 and HTK01146 bound B1R with good affinity (Ki = 30.5 and 24.8 nM, respectively). 68Ga/18F-labeled R954 were obtained on average in ≥10% decay-corrected radiochemical yield with >99% radiochemical purity and ≥52 GBq/µmol specific activity. For both tracers, clearance was predominantly renal with minimal involvement of the hepatobiliary system. For PET images, B1R+ tumors, kidneys, and bladder were visible. At 1 h p.i., uptake in B1R+ tumor was comparable between 68Ga-HTK01083 (8.46 ± 1.44%ID/g) and 18F-HTK01146 (9.25 ± 0.69%ID/g). B1R+ tumor-to-blood and B1R+ tumor-to-muscle ratios were 6.32 ± 1.44 and 20.7 ± 3.58 for 68Ga-HTK01083, and 7.24 ± 2.56 and 19.5 ± 4.29 for 18F-HTK01146. Our results indicate R954 is a good lead sequence for optimization of B1R tracers for cancer imaging.
Assuntos
Antagonistas de Receptor B1 da Bradicinina/metabolismo , Fluordesoxiglucose F18/metabolismo , Radioisótopos de Gálio/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Receptor B1 da Bradicinina/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Animais , Linhagem Celular , Células HEK293 , Humanos , Masculino , Camundongos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons/métodosRESUMO
Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.
Assuntos
Antagonistas de Receptor B1 da Bradicinina/metabolismo , Desenho de Fármacos , Metilaminas/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Receptor B1 da Bradicinina/metabolismo , Animais , Antagonistas de Receptor B1 da Bradicinina/síntese química , Antagonistas de Receptor B1 da Bradicinina/química , Radioisótopos de Flúor/química , Células HEK293 , Humanos , Metilaminas/síntese química , Metilaminas/química , Camundongos , Neoplasias/diagnóstico por imagem , Ligação Proteica , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptor B1 da Bradicinina/química , Distribuição Tecidual , Transplante HeterólogoRESUMO
Bradykinin B1 receptor (B1R), which is upregulated in a variety of malignancies, is an attractive cancer imaging biomarker. In this study we optimized the selection of radiolabel-chelator complex to improve tumor uptake and tumor-to-background contrast of radiolabeled analogues of B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-d-Tic-Cpg), a potent B1R antagonist. Peptide sequences were assembled on solid phase. Cold standards were prepared by incubating DOTA-/NODA-conjugated peptides with GaCl3, and by incubating AlOH-NODA-conjugated peptide with NaF. Binding affinities were measured via in vitro competition binding assays. (68)Ga and (18)F labeling experiments were performed in acidic buffer and purified by HPLC. Imaging/biodistribution studies were performed in mice bearing both B1R-positive (B1R+) HEK293T::hB1R and B1R-negative (B1R-) HEK293T tumors. Z02176 (Ga-DOTA-Pip-B9958; Pip: 4-amino-(1-carboxymethyl)piperidine), Z02137 (Ga-NODA-Mpaa-Pip-B9958; Mpaa: 4-methylphenylacetic acid), and Z04139 (AlF-NODA-Mpaa-Pip-B9958) bound hB1R with high affinity (Ki = 1.4-2.5 nM). (68)Ga-/(18)F-labeled peptides were obtained on average in ≥32% decay-corrected radiochemical yield with >99% radiochemical purity and 100-261 GBq/µmol specific activity. Biodistribution/imaging studies at 1 h postinjection showed that all tracers cleared rapidly from background tissues (except kidneys) and were excreted predominantly via the renal pathway. Only kidneys, bladders, and B1R+ tumors were clearly visualized in PET images. Uptake in B1R+ tumor was higher by using (68)Ga-Z02176 (28.9 ± 6.21 %ID/g) and (18)F-Z04139 (22.6 ± 3.41 %ID/g) than (68)Ga-Z02137 (14.0 ± 4.86 %ID/g). The B1R+ tumor-to-blood and B1R+ tumor-to-muscle contrast ratios were also higher for (68)Ga-Z02176 (56.1 ± 17.3 and 167 ± 57.6) and (18)F-Z04139 (58.0 ± 20.9 and 173 ± 42.9) than (68)Ga-Z02137 (34.3 ± 15.2 and 103 ± 30.2). With improved target-to-background contrast (68)Ga-Z02176 and (18)F-Z04139 are promising for imaging B1R expression in cancers with PET.
Assuntos
Antagonistas de Receptor B1 da Bradicinina/análise , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/análise , Receptor B1 da Bradicinina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Radioisótopos de Flúor/análise , Radioisótopos de Gálio/análise , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos KnockoutRESUMO
Kinins are the endogenous ligands of the constitutive B2 receptor (B2R) and the inducible B1 receptor (B1R). Whereas B2R prevents insulin resistance, B1R is involved in insulin resistance and metabolic syndrome. However, the contribution of B1R in type 2 diabetes associated with obesity remains uncertain. The aim of the present study was to examine the impact of 1-week treatment with a selective B1R antagonist (SSR240612, 10 mg/kg per day, by gavage) on hyperglycemia, hyperinsulinemia, leptinemia, body mass gain, and abnormal plasma fatty acids in obese Zucker diabetic fatty (ZDF) rats. Treatment with SSR240612 abolished the body mass gain and reduced polyphagia, polydipsia, and plasma fatty acid alterations in ZDF rats without affecting hyperglycemia, hyperinsulinemia, and hyperleptinemia. The present study suggests that the upregulated B1R plays a role in body mass gain and circulating fatty acid alterations in ZDF rats. However, mechanisms other than B1R induction would be implicated in glucose metabolism disorder in ZDF rats, based on the finding that SSR240612 did not reverse hyperglycemia and hyperinsulinemia.
Assuntos
Antagonistas de Receptor B1 da Bradicinina/uso terapêutico , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Ácidos Graxos/sangue , Obesidade/sangue , Obesidade/tratamento farmacológico , Animais , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Dioxóis/farmacologia , Dioxóis/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Masculino , Ratos , Ratos Zucker , Receptor B1 da Bradicinina/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Resultado do TratamentoRESUMO
Kinin B1 receptors are implicated in asthmatic airway inflammation. Here we tested this hypothesis by examining the anti-inflammatory effects of BI113823, a novel non-peptide orally active kinin B1 receptor antagonist in mice sensitized to ovalbumin (OVA). Male Balb-c mice were randomly assigned to four study groups: (1) control, (2) OVA+vehicle, (3) OVA+BI113823, (4) OVA+dexamethasone. Mice were sensitized intraperitoneally with 75µg ovalbumin on days 1 and 8. On days 15-17, mice were challenged intranasally with 50µg of ovalbumin. Mice received vehicle, BI113823, or dexamethasone (positive control) on days 16-18. On day 19, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immuno-histological analysis. Compared to controls treatment with BI113823 significantly reduced the numbers of BAL eosinophils, macrophages, neutrophils and lymphocytes by 58.3%, 61.1%, 66.4% and 56.0%, respectively. Mice treated with dexamethasone showed similar reductions in BAL cells. Treatment with BI113823 and dexamethasone also significantly reduced total protein content, IgE, TNF-α and IL-1ß in lavage fluid, reduced myeloperoxidase activity, mucus secretion in lung tissues, and reduced the expression of B1 receptors, matrix metalloproteinase (MMP)-2 and cyclooxygenase (COX)-2 compared to vehicle-treated mice. Only BI113823 reduced MMP-9 and inducible nitric oxide synthase (iNOS). BI113823 effectively reduced OVA-induced inflammatory cell, mediator and signaling pathways equal to or greater than that seen with steroids in a mouse asthma model. BI113823 might be useful in modulating inflammation in asthma.
Assuntos
Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Antagonistas de Receptor B1 da Bradicinina/uso terapêutico , Alérgenos , Animais , Anti-Inflamatórios/farmacologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Ciclo-Oxigenase 2/imunologia , Dexametasona/farmacologia , Imunoglobulina E/imunologia , Interleucina-1beta/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Metaloproteinase 2 da Matriz/imunologia , Camundongos Endogâmicos BALB C , Muco/metabolismo , Ovalbumina , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Infraorbital nerve constriction (CION) causes hypersensitivity to facial mechanical, heat and cold stimulation in rats and mice and is a reliable model to study trigeminal neuropathic pain. In this model there is evidence that mechanisms operated by kinin B1 and B2 receptors contribute to heat hyperalgesia in both rats and mice. Herein we further explored this issue and assessed the role of kinin receptors in mechanical hyperalgesia after CION. Swiss and C57Bl/6 mice that underwent CION or sham surgery or dynorphin A (1-17) administration were repeatedly submitted to application of either heat stimuli to the snout or mechanical stimuli to the forehead. Treatment of the animals on the fifth day after CION surgery with DALBK (B1 receptor antagonist) or HOE-140 (B2 receptor antagonist), both at 0.01-1µmol/kg (i.p.), effectively reduced CION-induced mechanical hyperalgesia. Knockout mice for kinin B1, B2 or B1/B2 receptors did not develop heat or mechanical hyperalgesia in response to CION. Subarachnoid dynorphin A (1-17) delivery (15nmol/5µL) also resulted in orofacial heat hyperalgesia, which was attenuated by post-treatment with DALBK (1 and 3µmol/kg, i.p.), but was not affected by HOE-140. Additionally, treatment with an anti-dynorphin A antiserum (200µg/5µL, s.a.) reduced CION-induced heat hyperalgesia for up to 2h. These results suggest that both kinin B1 and B2 receptors are relevant in orofacial sensory nociceptive changes induced by CION. Furthermore, they also indicate that dynorphin A could stimulate kinin receptors and this effect seems to contribute to the maintenance of trigeminal neuropathic pain.
Assuntos
Bradicinina/metabolismo , Dinorfinas/metabolismo , Dor Facial/metabolismo , Neuralgia/metabolismo , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Modelos Animais de Doenças , Dinorfinas/farmacologia , Temperatura Alta , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotransmissores/farmacologia , Medição da Dor , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , TatoRESUMO
Bradykinin B1 receptor (B1R) is involved in pain and inflammation pathways and is upregulated in inflamed tissues and cancer. Due to its minimal expression in healthy tissues, B1R is an attractive target for the development of therapeutic agents to treat inflammation, chronic pain, and cancer. The goal of this study is to synthesize and compare two (18)F-labeled peptides derived from potent B1R antagonists B9858 and B9958 for imaging B1R expression with positron emission tomography (PET). Azidoacetyl-B9858 2 and azidoacetyl-B9958 3 were synthesized by a solid-phase approach and subsequently clicked to ammoniomethyl-trifluoroborate (AmBF3)-conjugated alkyne 1 to obtain AmBF3-B9858 and AmBF3-B9958, respectively. AmBF3-B9858 and AmBF3-B9958 bound B1R with high affinity, with Ki values at 0.09 ± 0.08 and 0.46 ± 0.03 nM, respectively, as measured by in vitro competition binding assays. (18)F labeling was performed via an (18)F-(19)F isotope exchange reaction. The radiofluorinated tracers were obtained within a synthesis time of 30 min and with 23-32% non-decay-corrected radiochemical yield, >99% radiochemical purity, and 43-87 GBq/µmol specific activity at the end of the synthesis. PET imaging and biodistribution studies were carried out in mice bearing both B1R-positive (B1R(+)) HEK293T::hB1R and B1R-negative (B1R(-)) HEK293T tumors. Both tracers cleared rapidly from most organs/tissues, mainly through the renal pathway. High uptake in B1R(+) tumors ((18)F-AmBF3-B9858: 3.94 ± 1.24% ID/g, tumor-to-muscle ratio 21.3 ± 4.33; (18)F-AmBF3-B9958: 4.20 ± 0.98% ID/g, tumor-to-muscle ratio 48.6 ± 10.7) was observed at 1 h postinjection. These results indicate that (18)F-AmBF3-B9858 and (18)F-AmBF3-B9958 are promising agents for the in vivo imaging of B1R expression with PET.
Assuntos
Calidina/análogos & derivados , Receptor B1 da Bradicinina/metabolismo , Animais , Biofarmácia , Boratos , Bradicinina/análogos & derivados , Bradicinina/síntese química , Bradicinina/química , Antagonistas de Receptor B1 da Bradicinina/síntese química , Antagonistas de Receptor B1 da Bradicinina/química , Estabilidade de Medicamentos , Radioisótopos de Flúor , Células HEK293 , Humanos , Calidina/síntese química , Calidina/química , Masculino , Camundongos , Camundongos Knockout , Neoplasias Experimentais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Distribuição TecidualRESUMO
BACKGROUND: The kallikrein-kinin system (KKS) is an endogenous pathway involved in angiogenesis and tumourigenesis, both vital for cancer growth and progression. OBJECTIVES: To investigate the effect of two bradykinin receptor (B1R and B2R) agonists on growth and motility of prostate tumour (DU145) and micro-vascular endothelial cells (dMVECs). METHODS: Increasing concentrations of selective B1R and B2R agonists were added to cultured cells. Cell proliferation and migration were assessed using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and modified Boyden Chamber assays, respectively. Where significant stimulation was found, the influence of an antagonist was also investigated. RESULTS: Neither growth nor motility of endothelial cells was affected by either agonist. In DU145 cells, while the B2R agonist was without any significant effect, the B1R agonist stimulated proliferation and migration at concentrations of 10nM and 50nM respectively. Further, this effect was abrogated when cells were pre-incubated with a B1R antagonist. CONCLUSIONS: Unlike the physiologically-active B2R, the pathologically-inducible B1R may be implicated in prostate tumourigenic events. The involvement of the KKS in malignant prostate pathology supports on-going exploration of bradykinin receptor antagonists as target candidates in the development of alternate approaches to cancer therapy.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Neoplasias da Próstata/patologia , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Masculino , Receptores da BradicininaRESUMO
Gliomas are the most common malignant brain tumors in adults. Bradykinin (BK) displays an important role in cancer, although the exact role of kinin receptors in the glioma biology remains unclear. This study investigated the role of kinin B1 and B2 receptors (B1R and B2R) on cell proliferation in human glioblastoma cell lineages. The mRNA expression of B1R and B2R was verified by RT-qPCR, whereas the effects of kinin agonists (des-Arg(9)-BK and BK) were analyzed by cell counting, MTT assay and annexin-V/PI determination. The PI3K/Akt and ERK1/2 signaling activation was assessed by flow cytometry. Our results demonstrated that both human glioblastoma cell lines U-138MG and U-251MG express functional B1R and B2R. The proliferative effects induced by the incubation of des-Arg(9)-BK and BK are likely related to the activation of PI3K/Akt and ERK 1/2 pathways. Moreover, the pre-incubation of the selective PI3Kγ blocker AS252424 markedly prevented kinin-induced AKT phosphorylation. Noteworthy, the selective B1R and B2R antagonists SSR240612 and HOE-140 were able to induce cell death of either lineages, with mixed apoptosis/necrosis characteristics. Taken together, the present results show that activation of B1R and B2R might contribute to glioblastoma progression in vitro. Furthermore, PI3K/Akt and ERK 1/2 signaling may be a target for adjuvant treatment of glioblastoma with a possible impact on tumor proliferation.
Assuntos
Proliferação de Células , Glioma/metabolismo , Glioma/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Apoptose , Western Blotting , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Dioxóis/farmacologia , Citometria de Fluxo , Glioma/tratamento farmacológico , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor B1 da Bradicinina/química , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
RATIONALE: There are evidences indicating the role of kinins in pathophysiology of traumatic brain injury, but little is known about their action on memory deficits. OBJECTIVES: Our aim was to establish the role of bradykinin receptors B1 (B1R) and B2 (B2R) on the behavioral, biochemical, and histologic features elicited by moderate lateral fluid percussion injury (mLFPI) in mice. METHODS: The role of kinin B1 and B2 receptors in brain damage, neuromotor, and cognitive deficits induced by mLFPI, was evaluated by means of subcutaneous injection of B2R antagonist (HOE-140; 1 or 10 nmol/kg) or B1R antagonist (des-Arg9-[Leu8]-bradykinin (DAL-Bk; 1 or 10 nmol/kg) 30 min and 24 h after brain injury. Brain damage was evaluated in the cortex, being considered as lesion volume, inflammatory, and oxidative damage. The open field and elevated plus maze tests were performed to exclude the nonspecific effects on object recognition memory test. RESULTS: Our data revealed that HOE-140 (10 nmol/kg) protected against memory impairment. This treatment attenuated the brain edema, interleukin-1ß, tumor necrosis factor-α, and nitric oxide metabolites content elicited by mLFPI. Accordingly, HOE-140 administration protected against the increase of nicotinamide adenine dinucleotide phosphate oxidase activity, thiobarbituric-acid-reactive species, protein carbonylation generation, and Na⺠K⺠ATPase inhibition induced by trauma. Histologic analysis showed that HOE-140 reduced lesion volume when analyzed 7 days after brain injury. CONCLUSIONS: This study suggests the involvement of the B2 receptor in memory deficits and brain damage caused by mLFPI in mice.
Assuntos
Bradicinina/análogos & derivados , Lesões Encefálicas/complicações , Lesões Encefálicas/tratamento farmacológico , Transtornos da Memória/etiologia , Transtornos da Memória/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Animais , Bradicinina/metabolismo , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Edema Encefálico/prevenção & controle , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Fatores de TempoRESUMO
UNLABELLED: Cisplatin is a chemotherapeutic agent that causes severe renal dysfunction. The kinin B1 receptor has been associated with the migration of immune cells to injured tissue as well as with renal inflammation. To examine the role of the kinin B1 receptor in cisplatin-induced acute kidney injury, we used kinin B1 receptor knockout mice and treatment with a receptor antagonist before and after cisplatin administration. Cisplatin injection caused exacerbation of renal macrophage and neutrophil migration, higher levels of serum creatinine and blood urea, upregulation of B1 receptor mRNA and an increase in pro-inflammatory cytokines expression. B1 receptor knockout mice exhibited a reduction in serum creatinine and blood urea levels, diminished apoptosis, and decreased cisplatin-induced upregulation of inflammatory components. Moreover, treatment with the B1 receptor antagonist prior to cisplatin administration normalized serum creatinine, blood urea levels, protected from acute tubular necrosis, apoptosis-related genes, and prevented upregulation of pro-inflammatory cytokines. Thus, we propose that kinins have an important role in cisplatin-induced acute kidney injury by impairing immune cells migration to renal tissue during cisplatin nephrotoxicity. KEY MESSAGE: Kinin B1 receptor is upregulated after cisplatin exposure. Kinin B1 receptor deficiency diminishes the nephrotoxicity caused by cisplatin. Kinin B1 receptor deficiency ameliorates the inflammatory response. Kinin B1 receptor deficiency diminishes apoptosis caused by cisplatin. Kinin B1 receptor antagonism ameliorates renal function after cisplatin injection.