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BACKGROUND AND PURPOSE: Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056-opioid interaction. EXPERIMENTAL APPROACH: We used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid-induced adverse effects were assessed in rodent models of morphine-induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxone-induced withdrawal), and abuse (conditioned place preference-associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of µ-opioid receptors. KEY RESULTS: CR4056 (ED50 = 4.88 mg·kg-1 ) and morphine (ED50 = 2.07 mg·kg-1 ) synergized in reducing CFA-induced hyperalgesia (ED50 = 0.52 mg·kg-1 ; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg-1 ) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti-hyperalgesia. These opioid-sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on µ-opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference. CONCLUSION AND IMPLICATIONS: We showed selective synergism between CR4056 and morphine as analgesics. Their combination showed an improved safety and abuse liability profile over morphine alone. CR4056 could be developed as an opioid-sparing drug in multimodal analgesia.
Assuntos
Morfina , Roedores , Analgésicos Opioides/toxicidade , Animais , Tolerância a Medicamentos , Imidazóis , Morfina/efeitos adversos , Quinazolinas , RatosRESUMO
BACKGROUND: Prostaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD). METHODS: CR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA. RESULTS: CR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate. CONCLUSIONS: CR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Animais , Antirreumáticos/metabolismo , Artrite Reumatoide/metabolismo , AMP Cíclico/biossíntese , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Células HEK293 , Humanos , Macrófagos/metabolismo , Masculino , Metotrexato/uso terapêutico , Camundongos Endogâmicos DBA , Ensaio Radioligante/métodos , Ratos Endogâmicos Lew , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células THP-1 , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Injury to the ocular surface increases corneal epithelial production of cyclooxygenase (COX)-derived eicosanoids but this increase correlates poorly to the inflammatory sequelae. Moreover, corticosteroids are effective in treatment of this inflammation but NSAIDs are not. The discovery of COX-2 that is differentially affected by common NSAIDs reopened the question of the role of prostaglandins in ocular surface inflammation. We examined the presence and inducibility of COX-2 in the corneal epithelium following hypoxia-induced injury in vivo and in vitro. COX-2, but not COX-1, protein levels markedly increased following hypoxia. Use of the selective COX-2 inhibitor, NS-398, indicated that COX activity in hypoxic corneas or cells is essentially that of COX-2; in control cells, both COX-1 and COX-2 contributed equally to the production of PGE2. COX-2 protein overexpression induced by hypoxia was not associated with a parallel increase in PGE2 accumulation but the enzyme regained full catalytic activity when cells were re-exposed to normoxia in the presence of heme and arachidonic acid. Decreases in the levels of oxygen and heme, essential substrates/cofactors for COX catalytic activity, contributed to a diminished prostanoid production during hypoxia. These results suggest that in hypoxic injury, molecules other than COX-derived prostanoids play a major pro-inflammatory role. Furthermore, this study provides an explanation for the ineffectiveness of classical NSAIDs in the treatment of hypoxia-related ocular surface inflammation.
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Hipóxia Celular/imunologia , Epitélio Corneano/enzimologia , Epitélio Corneano/imunologia , Isoenzimas/imunologia , Isoenzimas/metabolismo , Ceratite/enzimologia , Ceratite/imunologia , Prostaglandina-Endoperóxido Sintases/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Linhagem Celular , Técnicas de Cultura , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/imunologia , Dinoprostona/metabolismo , CoelhosRESUMO
BACKGROUND AND OBJECTIVE: Glucosamine is a safe and common treatment for osteoarthritis. Even so, literature data on the cardiovascular safety of glucosamine are limited. The objective of this paper is to investigate the long-term effects of crystalline glucosamine sulfate (CGS) on key measures of cardiovascular risk in patients with osteoarthritis. METHODS: We analyzed safety data from two long-term (6-month and 3-year, respectively) randomized controlled trials of CGS. Mean changes in blood pressure, lipids, and glucose were calculated for all patients randomized to CGS or placebo in either study and for subgroups with abnormally elevated baseline values. Shift tables were used to analyze transitions from normal to abnormal levels, or vice versa. RESULTS: This analysis on 428 osteoarthritis patients includes data from subjects who had, on average, high normal blood pressure or high cholesterol at baseline. There were no significant changes in mean blood pressure after 6 months on CGS (systolic: -5±15 mmHg; diastolic: -5±10 mmHg) or placebo (systolic: -7±14 mmHg; diastolic: -4±10 mmHg). Subgroup analysis did not show significant effects in subjects with hypertension. Likewise, blood lipids (total/LDL cholesterol) and blood glucose did not change over 3 years and 6 months of treatment, respectively, even in hypercholesterolemic or hyperglycemic subjects. The proportions of patients whose blood pressure or cholesterol levels shifted from normal to abnormal, or vice versa, were comparable in the CGS and placebo groups. CONCLUSIONS: Long-term use of CGS did not affect blood pressure, lipids, or glucose in patients with osteoarthritis. These findings further support the cardiovascular safety of CGS.
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PURPOSE: Gastrointestinal mucositis, commonly associated with diarrhea, is a dose-limiting toxicity of chemotherapy. The new benzamidine derivative CR3294 reduces tissue damage in animal models of intestinal inflammation. Thus, we tested whether CR3294 had the potential to prevent chemotherapy-induced mucositis. METHODS: In tests on isolated cells, reactive oxygen species (ROS) formation and cytokine release were measured by chemiluminescence and immunoassays, respectively. In studies in vivo, BDF1 mice were given oral CR3294 (2.5-20 mg/kg) for 3 days before receiving 5-fluorouracil. Intestinal crypt survival, cell apoptosis and proliferation, and diarrhea score were assessed. Additionally, nude mice bearing tumor xenografts were treated with CR3294 and/or 5-fluorouracil, and tumor growth was monitored. RESULTS: CR3294 significantly inhibited cytokine release from stimulated leukocytes at concentrations similar to the IC(50) (2.9 +/- 0.2 muM) for ROS production by these cells. Consistent with these molecular findings, CR3294 dose-dependently protected the intestinal mucosa against 5-fluorouracil-induced toxicity in a mouse model of mucositis. The number of surviving crypts per cross-section in mice receiving 20 mg/kg CR3294 was 2.8-fold that in vehicle-treated animals (18.1 +/- 1.9 vs. 6.5 +/- 0.9, P < 0.001). Moreover, CR3294 decreased the cumulative diarrhea score by 50%, reduced by nearly 70% the incidence of severe episodes, and increased by 3-fold the number of mice without diarrhea. CR3294 neither affected the growth of tumor xenografts nor protected tumors from the cytotoxic activity of 5-fluorouracil. CONCLUSIONS: This study demonstrates that CR3294 acts on key molecular targets to reduce the signs of mucositis and the occurrence of diarrhea in mice exposed to the chemotherapy drug 5-fluorouracil.
Assuntos
Amidinas/farmacologia , Antimetabólitos Antineoplásicos/toxicidade , Diarreia/prevenção & controle , Fluoruracila/toxicidade , Mucosite/prevenção & controle , Tioureia/análogos & derivados , Amidinas/administração & dosagem , Animais , Antimetabólitos Antineoplásicos/farmacologia , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Diarreia/induzido quimicamente , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Fluoruracila/farmacologia , Concentração Inibidora 50 , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mucosite/induzido quimicamente , Espécies Reativas de Oxigênio , Tioureia/administração & dosagem , Tioureia/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, namely statins, exert pleiotropic actions beyond lipid-lowering effects. Their pharmacological activity on atherosclerotic plaque stability and vascular inflammation appears to be mediated, at least in part, by nitric oxide (NO). With the aim of enhancing the nonlipid-lowering properties of selected statins, we introduced a NO-releasing moiety into the structure of pravastatin (NCX 6550) and fluvastatin (NCX 6553). NO release was evaluated as nitrosylhemoglobin adduct formation by using EPR spectroscopy in rat blood. Both compounds produced a linear time-dependent increase in nitrosylhemoglobin formation, which is consistent with slow NO release kinetics. In PC12 cells, unlike their native statins, both compounds stimulated cGMP formation (NCX 6550, EC(50) = 2.3 +/- 0.2 microM; NCX 6553, EC(50) = 2.7 +/- 0.2 microM). Moreover, NCX 6550 potently inhibited cell proliferation in rat aortic smooth muscle cells (IC(50) = 2.2 +/- 0.3 microM) with a mechanism that involved both the polyamine and HMG-CoA reductase signaling pathways. Hence, mevalonate or putrescine partially reverted the effects of NCX 6550 and their combination was fully effective. In RAW 264.7 murine macrophage cells stimulated with lipopolysaccharide (1 microg/ml), NCX 6550, but not pravastatin, significantly decreased inducible NO synthase and cyclooxygenase-2 protein expression as well as nitrite accumulation. All together, the data show that the previously undescribed NO-releasing statins retain HMG-CoA reductase inhibitory activity and release bioactive NO slowly. Among the additional properties, compared with native statins, the NO-releasing statins show enhanced antiinflammatory effects. Thus, NO-releasing statins represent an interesting class of drugs having potential in the therapy of disorders associated with endothelial dysfunction and vascular inflammation.