RESUMO
Thrombomodulin (TM) is an integral membrane protein expressed on the surface of vascular endothelial cells that suppresses blood coagulation. Recent studies have shown that TM exhibits anti-inflammatory effects by inhibiting leukocyte recruitment. However, the actual modes of action of TM in vivo remain unclear. Here, we describe the pharmacological effects of recombinant human soluble TM (TM alfa) on leukocyte dynamics in living mice using intravital imaging techniques. Under control conditions, neutrophils exhibited three distinct types of adhesion behavior in vessels: 1) "non-adhesion", in which cells flowed without vessel adhesion; 2) "rolling adhesion", in which cells transiently interacted with the endothelium; and 3) "tight binding", in which cells bound strongly to the endothelial cells. Compared to control conditions, local lipopolysaccharide stimulation resulted in an increased frequency of rolling adhesion that was not homogeneously distributed on vessel walls but occurred at specific endothelial sites. Under inflammatory conditions, TM alfa, particularly the D1 domain which is a lectin-like region of TM, significantly decreased the frequency of rolling adhesion, but did not influence the number of tight bindings. This was the first study to demonstrate that TM alfa exerts anti-inflammatory effects by inhibiting rolling adhesion of neutrophils to vascular endothelial cells in living mice.
Assuntos
Anti-Inflamatórios , Adesão Celular , Neutrófilos/fisiologia , Trombomodulina/fisiologia , Animais , Endotélio Vascular/citologia , Masculino , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Proteínas Recombinantes/farmacologiaRESUMO
Thrombomodulin (TM), an endothelial protein with anti-coagulant activity, is composed of 5 domains, D1-D5. Recombinant human soluble TM (TMα) consisting of D1-D3, which is generated in CHO cells, suppresses inflammatory and nociceptive signals by inactivating high mobility group box 1 (HMGB1), one of damage-associated molecular patterns. TMα sequesters HMGB1 with the lectin-like D1 and promotes its degradation by thrombin binding to the EGF-like D2. We prepared TM's D123, D1 and D2 by the protein expression system of yeast, and evaluated their effects on HMGB1 degradation in vitro and on the allodynia caused by HMGB1 in distinct redox forms in mice in vivo. TMα and TM's D123, but not D1, promoted the thrombin-dependent degradation of all-thiol (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), an effect mimicked by TM's D2, though to a lesser extent. Intraplantar administration of TMα and TM's D123, but not D1, D2 or D1 plus D2, strongly prevented the mechanical allodynia caused by intraplantar at-HMGB1, ds-HMGB1 or lipopolysaccharide in mice. Our data suggest that, apart from the role of D3, TMα and TM's D123 require both lectin-like D1 capable of sequestering HMGB1 and EGF-like D2 responsible for thrombin-dependent degradation of HMGB1, in abolishing the allodynia caused by exogenous or endogenous HMGB1.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Lectinas/metabolismo , Trombomodulina/administração & dosagem , Animais , Humanos , Masculino , Camundongos , Domínios Proteicos , Solubilidade , Trombomodulina/química , Resultado do TratamentoRESUMO
We investigated the anxiolytic effects and mechanism of action of a new anxiolytic drug, (R)-piperonyl-1,2,3,4-tetrahydro[1]benzothieno[2,3-c]pyridine-3- carboxamide hydrochloride (AP521). AP521 showed equal or more potent anxiolytic-like effects compared with diazepam, a benzodiazepine receptor agonist, or tandospirone, a partial 5-hydroxytryptamine (5-HT)1A receptor agonist, in three rat anxiety models; the Vogel-type conflict test, elevated plus maze test, and conditioned fear stress test. Although AP521 did not bind to the benzodiazepine receptor, it did bind to 5-HT1A, 5-HT1B, 5-HT1D, 5-HT5A and 5-HT7 receptors, and showed agonist activity for the human 5-HT1A receptor expressed in HEK293 cells. Tandospirone, which can stimulate the presynaptic 5-HT1A receptors in the raphe, tended to decrease extracellular 5-HT concentration in the medial prefrontal cortex (mPFC) in rats. In contrast, AP521 increased extracellular 5-HT concentration. In addition, AP521 enhanced the anti-freezing effect of citalopram, a selective serotonin reuptake inhibitor, in the fear conditioning model in rats and enhanced the citalopram-caused increase of the extracellular 5-HT concentration in the mPFC. These results suggest that AP521 exhibits potent anxiolytic effects by acting as a postsynaptic 5-HT1A receptor agonist and by enhancing serotonergic neural transmission in the mPFC by a novel mechanism of action.
Assuntos
Ansiolíticos/farmacologia , Piridinas/farmacologia , Serotonina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Células Cultivadas , Citalopram/farmacologia , Diazepam/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Isoindóis/farmacologia , Masculino , Piperazinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Ratos , Receptores de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Tiofenos/uso terapêuticoRESUMO
High-mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, facilitates pain signals as well as inflammation. Intraplantar or intraspinal application of HMGB1 elicits hyperalgesia/allodynia in rodents by activating the advanced glycosylation end-product specific receptor (receptor for advanced glycation end-products; RAGE) or Toll-like receptor 4 (TLR4). Endogenous HMGB1 derived from neurons, perineuronal cells or immune cells accumulating in the dorsal root ganglion or sensory nerves participates in somatic and visceral pain consisting of neuropathic and/or inflammatory components. Endothelial thrombomodulin (TM) and recombinant human soluble TM, TMα, markedly increase thrombin-dependent degradation of HMGB1, and systemic administration of TMα prevents and reverses various HMGB1-dependent pathological pain. Low MW compounds that directly inactivate HMGB1 or antagonize HMGB1-targeted receptors would be useful to reduce various forms of intractable pain. Thus, HMGB1 and its receptors are considered to serve as promising targets in developing novel agents to prevent or treat pathological pain. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.
Assuntos
Proteína HMGB1 , Trombomodulina , Humanos , Dor , Receptor para Produtos Finais de Glicação Avançada , TrombinaRESUMO
Oxaliplatin, a platinum-based chemotherapeutic agent, is widely used to treat colorectal cancer, but it induces peripheral neuropathy as a serious dose-limiting side effect. Recently, thrombomodulin alfa, a recombinant human soluble thrombomodulin, was reported to prevent oxaliplatin-induced peripheral neuropathy in a clinical phase 2 study. Here we conducted preclinical pharmacology studies. Rats were given oxaliplatin (6 mg/kg) intravenously to induce mechanical hyperalgesia associated with peripheral neuropathy. Single intravenous administration of thrombomodulin alfa (0.1, 0.3, 1 mg/kg) dose dependently prevented the development of oxaliplatin-induced mechanical hyperalgesia, with no sex difference in the efficacy. The preventative effect of thrombomodulin alfa on mechanical hyperalgesia was attenuated by antithrombin or carboxypeptidase inhibitor. In addition, carboxypeptidase B, a homolog of activated thrombin-activatable fibrinolysis inhibitor (TAFI) and human-derived activated protein C, prevented mechanical hyperalgesia, whereas antithrombin or other anti-coagulants did not. These results suggest that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy through the activation of TAFI and protein C by modulating thrombin activity, but the effects are independent of an anticoagulant effect. On the other hand, thrombomodulin alfa did not affect the anti-cancer activity of oxaliplatin on human colon cancer cell lines or mice transplanted with HCT116 cells. These results indicate that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy without affecting the anti-tumor activity of oxaliplatin. Therefore, thrombomodulin alfa is a promising drug to prevent the symptoms of oxaliplatin-induced peripheral neuropathy.
Assuntos
Analgésicos/uso terapêutico , Antineoplásicos/efeitos adversos , Neoplasias do Colo/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Oxaliplatina/efeitos adversos , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Trombomodulina/uso terapêutico , Analgésicos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carboxipeptidase B2/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Feminino , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuralgia/induzido quimicamente , Neuralgia/metabolismo , Oxaliplatina/uso terapêutico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Proteína C/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Trombomodulina/genética , TatoRESUMO
In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.
Assuntos
Anti-Inflamatórios/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Fenilacetatos/química , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/química , Pirimidinas/química , Tiofenos/química , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Área Sob a Curva , Descoberta de Drogas , Humanos , Concentração Inibidora 50 , Camundongos , Fenilacetatos/farmacocinética , Fenilacetatos/farmacologia , Inibidores de Fosfodiesterase/farmacocinética , Inibidores de Fosfodiesterase/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Tiofenos/farmacocinética , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Vômito/induzido quimicamenteRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) considerably impairs cancer patients' QOL, and may lead to discontinuation of drug treatment of cancer. Currently, there is no effective strategy against CIPN. Therefore, it is an urgent issue to develop clinically available drugs that prevent or treat CIPN. We have shown that high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, plays an essential role in the development of CIPN. Most interestingly, thrombomodulin α, approved as a medicine for treatment of disseminated intravascular coagulation (DIC) in Japan, causes thrombin-dependent degradation of extracellular HMGB1 that is released in response to chemotherapeutics, and prevents CIPN. Thus, we expect that targeting HMGB1 or its receptors would lead to prevention of CIPN in cancer patients in near future.
Assuntos
Antineoplásicos/efeitos adversos , Proteína HMGB1 , Terapia de Alvo Molecular , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Trombomodulina/uso terapêutico , Humanos , Japão , Doenças do Sistema Nervoso Periférico/prevenção & controleRESUMO
High mobility group box 1 (HMGB1), a nuclear protein, once released into the extracellular space under pathological conditions, plays a pronociceptive role in redox-dependent distinct active forms, all-thiol HMGB1 (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), that accelerate nociception through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4), respectively. Thrombomodulin (TM), an endothelial membrane protein, and soluble TM, known as TMα, promote thrombin-mediated activation of protein C and also sequester HMGB1, which might facilitate thrombin degradation of HMGB1. The present study aimed at clarifying the role of thrombin in TMα-induced suppression of peripheral HMGB1-dependent allodynia in mice. Thrombin-induced degradation of at-HMGB1 and ds-HMGB1 was accelerated by TMα in vitro. Intraplantar (i.pl.) injection of bovine thymus-derived HMGB1 in an unknown redox state, at-HMGB1, ds-HMGB1 or lipopolysaccharide (LPS), known to cause HMGB1 secretion, produced long-lasting mechanical allodynia in mice, as assessed by von Frey test. TMα, when preadministered i.pl., prevented the allodynia caused by bovine thymus-derived HMGB1, at-HMGB1, ds-HMGB1 or LPS, in a dose-dependent manner. The TMα-induced suppression of the allodynia following i.pl. at-HMGB1, ds-HMGB1 or LPS was abolished by systemic preadministration of argatroban, a thrombin-inhibiting agent, and accelerated by i.pl. co-administered thrombin. Our data clearly indicate that TMα is capable of promoting the thrombin-induced degradation of both at-HMGB1 and ds-HMGB1, and suppresses the allodynia caused by either HMGB1 in a thrombin-dependent manner. Considering the emerging role of HMGB1 in distinct pathological pain models, the present study suggests the therapeutic usefulness of TMα for treatment of intractable and/or persistent pain.
Assuntos
Proteína HMGB1/metabolismo , Hiperalgesia/metabolismo , Neuroimunomodulação/fisiologia , Trombina/metabolismo , Trombomodulina/metabolismo , Animais , Proteína HMGB1/imunologia , Hiperalgesia/imunologia , Masculino , Camundongos , Trombina/imunologia , Trombomodulina/imunologiaRESUMO
A series of conformationally restricted analogues of milnacipran, a weak NMDA receptor antagonist, were designed by a method based on allylic strain. The conformational analysis study showed that the allylic-strain-based conformational restriction indeed occurred and that the affinity for the NMDA receptor was efficiently improved by the conformational restriction.
Assuntos
Acrilamidas/síntese química , Compostos Alílicos/síntese química , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Acrilamidas/química , Acrilamidas/farmacologia , Compostos Alílicos/química , Compostos Alílicos/farmacologia , Animais , Córtex Cerebral/metabolismo , Cristalografia por Raios X , Técnicas In Vitro , Modelos Moleculares , Conformação Molecular , Ensaio Radioligante , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Two molecules with known growth hormone secretagogue (GHS) agonist activity were used as templates to computationally screen approximately 80000 compounds. A total of 108 candidate compounds were selected, and five of them were found to be active in the low-micromolar range in both cell-based and direct binding assays. These compounds were structurally diverse and significantly differed from known GHS agonists. The most active compound was subjected to SAR evaluation, which slightly increased its potency and identified molecular regions important for specific GHS agonist activity.
Assuntos
Acetamidas/química , Formamidas/química , Hormônio Liberador de Hormônio do Crescimento/agonistas , Acetamidas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Simulação por Computador , Formamidas/farmacologia , Hormônio Liberador de Hormônio do Crescimento/química , Humanos , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
RATIONALE: The prefrontal cortex is implicated in the pathophysiology of depression, and hypoactivity of this brain area has been found in depressed patients. Reduced function of the serotonergic and noradrenergic systems is another feature of depression. OBJECTIVES: The present study was aimed at characterizing neurochemically and behaviorally the serotonin and noradrenaline reuptake inhibitor (SNRI), milnacipran, in the prefrontal cortex in comparison with tricyclic antidepressants and selective serotonin reuptake inhibitors. METHODS: Sodium-dependent monoamine uptake measurement, radioligand binding assays, microdialysis procedure, forced swimming test and conditioned fear stress test were carried out in rats. RESULTS: Milnacipran selectively inhibited sodium-dependent [(3)H]serotonin (5-hydroxytryptamine, 5-HT) and [(3)H]noradrenaline (NA) uptake into the synaptosomes from rat cerebral cortex (IC(50)=28.0 and 29.6 nM, respectively) without any affinities for various neuroreceptors. In the medial prefrontal cortex, milnacipran (10 and 30 mg/kg, PO) caused a dose-related increase in the extracellular levels of 5-HT and NA with similar potency, whereas imipramine (10 and 30 mg/kg, PO) caused a dominant increase in the output of NA compared to 5-HT. Milnacipran (30 and 60 mg/kg, PO) significantly reduced the duration of both the immobility time in the forced swimming test and the freezing time in the conditioned fear stress test in rats, which are animal behavioral models for depression and anxiety, respectively. Imipramine and maprotiline were active in the former test, but not in the latter. Fluoxetine and fluvoxamine on the other hand were more active in the conditioned fear test. CONCLUSION: These findings show that milnacipran acts as a SNRI in vitro and in vivo and may be useful for the treatment of anxiety as well as depression.
Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Comportamento Animal/efeitos dos fármacos , Ciclopropanos/farmacologia , Córtex Pré-Frontal/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Análise de Variância , Animais , Ligação Competitiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Condicionamento Psicológico , Relação Dose-Resposta a Droga , Medo/efeitos dos fármacos , Medo/psicologia , Fluoxetina/farmacologia , Fluvoxamina/farmacologia , Imipramina/farmacologia , Masculino , Maprotilina/farmacologia , Microdiálise , Milnaciprano , Monoaminoxidase/efeitos dos fármacos , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Norepinefrina/farmacocinética , Córtex Pré-Frontal/efeitos dos fármacos , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Serotonina/farmacocinética , Natação , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Thrombomodulin alfa (TM-α, recombinant thrombomodulin) significantly improved disseminated intravascular coagulation (DIC) when compared with heparin therapy in a phase III study. Post-marketing surveillance of TM-α was performed to evaluate the effects and safety in patients with sepsis-induced DIC. METHODS: From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC treated with TM-α were registered. DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. The DIC resolution and survival rates on day 28 after the last TM-α administration, and changes in DIC, systemic inflammatory response syndrome (SIRS), and sequential organ failure assessment (SOFA) scores and coagulation and inflammation markers were evaluated. RESULTS: The most frequent underlying disease was infectious focus-unknown sepsis (29.8%). The mean ± SD values of age, dose, and the duration of TM-α administration were 64.7 ± 20.3 years, 297.3 ± 111.4 U/kg/day, and 5.6 ± 3.4 days, respectively. A total of 1,320 subjects (73.9%) received combined administration with other anticoagulants. Both coagulation and inflammation markers, such as fibrin/fibrinogen degradation products, prothrombin time ratio, thrombin-antithrombin complex, and C-reactive protein, as well as JAAM DIC, SIRS, and SOFA scores, significantly and simultaneously decreased after TM-α administration (p < 0.001). DIC resolution and 28-day survival rates were 44.4% and 66.0%, respectively. The 28-day survival rate decreased significantly according to the duration of DIC before TM-α administration (p < 0.001). Total adverse drug reactions (ADRs), bleeding ADRs, and serious bleeding adverse events occurred in 126 (7.1%), 98 (5.5%), and 121 (6.8%) subjects, respectively. On day 28, after the last TM-α administration available for an antibody test, only one patient was positive for anti-TM-α antibodies (0.11%). CONCLUSION: Our results suggest that TM-α is most effective for treating patients with sepsis-induced DIC when administered within the first 3 days after diagnosis.
RESUMO
(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, 4a), which is a conformationally restricted analogue of antidepressant milnacipran [(+/-)-1], is a new class of potent noncompetitive NMDA receptor antagonists. A series of PPDC analogues modified at the 1-phenyl moiety, that is, the analogue 6 lacking 1-phenyl group, the 1-(fluorophenyl) analogues 4b,c,d, the 1-(methylphenyl) analogues 4e-g and the 1-(naphthyl) analogues 4h,i were synthesized. Analogue 6, lacking the 1-phenyl group, was completely inactive showing that the aromatic moiety is essential for the NMDA receptor binding. Among the analogues synthesized, the 1-o-fluorophenyl and 1-m-fluorophenyl analogues 4b and 4c showed potent affinities for the NMDA receptor [IC(50)=0.16+/-0.001 microM (4b), 0.15+/-0.02 microM (4c)], which were improved to some extent compared to those of the parent compound PPDC (IC(50)=0.20+/-0.02 microM). On the other hand, compounds 4b and 4c showed none of the 5-HT-uptake inhibitory effect, while PPDC turned out to be a weak 5-HT-uptake inhibitor.
Assuntos
Ciclopropanos/síntese química , Ciclopropanos/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Antidepressivos/síntese química , Antidepressivos/farmacologia , Córtex Cerebral/química , Hidrocarbonetos Aromáticos , Concentração Inibidora 50 , Conformação Molecular , Ensaio Radioligante , Ratos , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, ), which is a conformationally restricted analogue of the antidepressant milnacipran [(+/-)-1], represents a new class of potent NMDA receptor antagonists. A series of PPDC analogues modified at the carbamoyl moiety were synthesized. Among these, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-dipropylcyclopropanecarboxamide (4d) was identified as the most potent NMDA receptor antagonist in this series and clearly reduced the MMDA receptor mediated potentiation of rat hippocampal slices, a model of long-term potentiation (LTP). The three-dimensional structure of 4d was also analyzed in detail to clarify the receptor-binding conformation.