RESUMEN
Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4's effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.
Asunto(s)
Heparina/análogos & derivados , Trombocitopenia/tratamiento farmacológico , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Heparina/farmacología , Heparina/uso terapéutico , Humanos , Megacariocitos/citología , Ratones , Recuento de Plaquetas , Factor Plaquetario 4/sangre , Factor Plaquetario 4/efectos de los fármacos , Factor Plaquetario 4/farmacología , Trombocitopenia/inducido químicamente , Trombocitopenia/etiología , TrombopoyesisRESUMEN
Relapses in acute myelogenous leukemia (AML) are a result of quiescent leukemic stem cells (LSCs) in marrow stromal niches, where they resist chemotherapy. LSCs employ CXCL12/CXCR4 to home toward protective marrow niches. Heparin disrupts CXCL12-mediated sequestration of cells in the marrow. CX-01 is a low-anticoagulant heparin derivative. In this pilot study, we combined CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7 + 3) with CX-01. Twelve patients were enrolled (median age, 56 years; 3 women). Three, 5, and 4 patients had good-, intermediate-, and poor-risk disease, respectively. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had morphologic complete remission after 1 induction (CR1). Eight patients were alive at a median follow-up of 24 months (4 patients in CR1). Three patients received an allogeneic stem cell transplant in CR1. Median disease-free survival was 14.8 months. Median overall survival was not attained at the maximum follow-up time of 29.4 months. No CX-01-associated serious adverse events occurred. Median day to an untransfused platelet count of at least 20 × 109/L was 21. CX-01 is well tolerated when combined with intensive therapy for AML and appears associated with enhanced count recovery and treatment efficacy.
Asunto(s)
Antineoplásicos/uso terapéutico , Quimioterapia Combinada/métodos , Heparina/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Adulto , Anciano , Anticoagulantes/uso terapéutico , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Proyectos Piloto , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. METHODS: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. RESULTS: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 µg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. CONCLUSIONS: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.
Asunto(s)
Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Antiinflamatorios no Esteroideos/farmacología , Biopelículas/efectos de los fármacos , Conservadores de la Densidad Ósea/farmacología , Glicosaminoglicanos/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/crecimiento & desarrollo , Aggregatibacter actinomycetemcomitans/metabolismo , Animales , Antiinflamatorios no Esteroideos/síntesis química , Sitios de Unión , Biopelículas/crecimiento & desarrollo , Conservadores de la Densidad Ósea/síntesis química , Línea Celular , Expresión Génica , Glicosaminoglicanos/síntesis química , Células HEK293 , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Periodontitis/prevención & control , Porphyromonas gingivalis/crecimiento & desarrollo , Porphyromonas gingivalis/metabolismo , Unión Proteica , Ligando RANK/genética , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Neutrophil elastase (NE) is a major inflammatory mediator in cystic fibrosis (CF) that is a robust predictor of lung disease progression. NE directly causes airway injury via protease activity, and propagates persistent neutrophilic inflammation by up-regulation of neutrophil chemokine expression. Despite its key role in the pathogenesis of CF lung disease, there are currently no effective antiprotease therapies available to patients with CF. Although heparin is an effective antiprotease and anti-inflammatory agent, its anticoagulant activity prohibits its use in CF, due to risk of pulmonary hemorrhage. In this report, we demonstrate the efficacy of a 2-O, 3-O-desulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity, to inhibit NE activity and to block NE-induced airway inflammation. Using an established murine model of intratracheal NE-induced airway inflammation, we tested the efficacy of intratracheal ODSH to block NE-generated neutrophil chemoattractants and NE-triggered airway neutrophilic inflammation. ODSH inhibited NE-induced keratinocyte-derived chemoattractant and high-mobility group box 1 release in bronchoalveolar lavage. ODSH also blocked NE-stimulated high-mobility group box 1 release from murine macrophages in vitro, and inhibited NE activity in functional assays consistent with prior reports of antiprotease activity. In summary, this report suggests that ODSH is a promising antiprotease and anti-inflammatory agent that may be useful as an airway therapy in CF.
Asunto(s)
Antiinflamatorios/farmacología , Proteína HMGB1/metabolismo , Heparina/análogos & derivados , Elastasa de Leucocito/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Neumonía/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Animales , Línea Celular , Quimiotaxis/efectos de los fármacos , Modelos Animales de Enfermedad , Heparina/farmacología , Interleucina-13 , Elastasa de Leucocito/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/fisiopatologíaRESUMEN
Nosocomial pneumonia (NP, or hospital-acquired pneumonia) is associated with infections originating from hospital-borne pathogens. Persistent microbial presence and acute lung injury are common features of these infections, contributing to the high mortality rates and excessive financial burden for these patients. Pseudomonas aeruginosa (PA), a gram-negative opportunistic pathogen, is one of the prominent pathogens associated with NP. PA pneumonia is characterized by excessive secretion of inflammatory cytokines, neutrophil infiltration, and subsequent lung damage. The persistent presence of PA along with overwhelming inflammatory response is suggestive of impairment in innate immunity. High mobility group box 1 (HMGB1), a recently discovered potent pro-inflammatory cytokine, plays an important role in PA lung infections by compromising innate immunity via impairing phagocyte function through toll-like receptors (TLR) TLR2 and TLR4. ODSH (2-O, 3-O-desulfated heparin), a heparin derivative with significant anti-inflammatory properties but minimal anti-coagulatory effects, has been shown to reduce neutrophilic lung injury in the absence of active microbial infections. This study examined the effects of ODSH on PA pneumonia. This study demonstrates that ODSH not only reduced PA-induced lung injury, but also significantly increased bacterial clearance. The ameliorated lung injury, together with the increased bacterial clearance, resulted in marked improvement in the survival of these animals. The resulting attenuation in lung injury and improvement in bacterial clearance were associated with decreased levels of airway HMGB1. Furthermore, binding of HMGB1 to its receptors TLR2 and TLR4 was blunted in the presence of ODSH. These data suggest that ODSH provides a potential novel approach in the adjunctive treatment of PA pneumonia.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Infección Hospitalaria/tratamiento farmacológico , Heparina/análogos & derivados , Pulmón/efectos de los fármacos , Neumonía Neumocócica/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/fisiología , Animales , Carga Bacteriana/efectos de los fármacos , Infección Hospitalaria/inmunología , Modelos Animales de Enfermedad , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Heparina/administración & dosificación , Humanos , Inmunidad Innata/efectos de los fármacos , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/inmunología , Infecciones por Pseudomonas/inmunología , Regulación hacia ArribaRESUMEN
BACKGROUND: Rosacea is a common disfiguring skin disease of primarily Caucasians characterized by central erythema of the face, with telangiectatic blood vessels, papules and pustules, and can produce skin thickening, especially on the nose of men, creating rhinophyma. Rosacea can also produce dry, itchy eyes with irritation of the lids, keratitis and corneal scarring. The cause of rosacea has been proposed as over-production of the cationic cathelicidin peptide LL-37. METHODOLOGY/PRINCIPAL FINDINGS: We tested a new class of non-anticoagulant sulfated anionic polysaccharides, semi-synthetic glycosaminoglycan ethers (SAGEs) on key elements of the pathogenic pathway leading to rosacea. SAGEs were anti-inflammatory at ng/ml, including inhibition of polymorphonuclear leukocyte (PMN) proteases, P-selectin, and interaction of the receptor for advanced glycation end-products (RAGE) with four representative ligands. SAGEs bound LL-37 and inhibited interleukin-8 production induced by LL-37 in cultured human keratinocytes. When mixed with LL-37 before injection, SAGEs prevented the erythema and PMN infiltration produced by direct intradermal injection of LL-37 into mouse skin. Topical application of a 1% (w/w) SAGE emollient to overlying injected skin also reduced erythema and PMN infiltration from intradermal LL-37. CONCLUSIONS: Anionic polysaccharides, exemplified by SAGEs, offer potential as novel mechanism-based therapies for rosacea and by extension other LL-37-mediated and RAGE-ligand driven skin diseases.
Asunto(s)
Catelicidinas/metabolismo , Glicosaminoglicanos/farmacología , Receptores Inmunológicos/antagonistas & inhibidores , Rosácea/tratamiento farmacológico , Rosácea/metabolismo , Sulfatos/química , Administración Tópica , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Aceite de Crotón/efectos adversos , Modelos Animales de Enfermedad , Glicosaminoglicanos/administración & dosificación , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/uso terapéutico , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ligandos , Antígeno de Macrófago-1/metabolismo , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Selectina-P/metabolismo , Péptido Hidrolasas/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Rosácea/inducido químicamenteRESUMEN
While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.
Asunto(s)
Antiinflamatorios/farmacología , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Heparina/análogos & derivados , Neumonía/tratamiento farmacológico , Receptores Inmunológicos/metabolismo , Trombocitopenia/prevención & control , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/efectos adversos , Antiinflamatorios/farmacocinética , Anticoagulantes/administración & dosificación , Anticoagulantes/efectos adversos , Anticoagulantes/farmacocinética , Antitrombina III/metabolismo , Pruebas de Coagulación Sanguínea , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Factor XIIa/metabolismo , Femenino , Productos Finales de Glicación Avanzada/metabolismo , Proteína HMGB1/metabolismo , Heparina/administración & dosificación , Heparina/efectos adversos , Heparina/farmacocinética , Heparina/farmacología , Humanos , Infusiones Intravenosas , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ligandos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Antígeno de Macrófago-1/metabolismo , Masculino , Melanoma Experimental/sangre , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/metabolismo , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Neumonía/sangre , Neumonía/inducido químicamente , Receptor para Productos Finales de Glicación Avanzada , Proteínas Recombinantes/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Trombocitopenia/sangre , Trombocitopenia/inducido químicamente , Células U937RESUMEN
Heparin desulfated at the 2-O and 3-O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na(+)](i) (Na Green) and [Ca(2+)](i) (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37 degrees C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na(+)/Ca(2+) exchange (NCX) activity and Na(+) channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 microg/ml) reduced [Na(+)](i) and [Ca(2+)](i) during PMI. The NCX inhibitor KB-R7943 (10 microM) or the late Na(+) current (I(Na-L)) inhibitor ranolazine (10 microM) reduced [Ca(2+)](i) during PMI and prevented effects of ODSH on Ca(2+) loading. ODSH also reduced the increase in Na(+) loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of I(Na-L). ODSH directly stimulated NCX and reduced I(Na-L). These results suggest that in the intact heart ODSH reduces Na(+) influx during early reperfusion, when I(Na-L) is activated by a burst of reactive oxygen production. This reduces Na(+) overload and thus Ca(2+) influx via NCX. Stimulation of Ca(2+) extrusion via NCX later after reperfusion may also reduce myocyte Ca(2+) loading and decrease infarct size.
Asunto(s)
Calcio/metabolismo , Heparina/farmacología , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Sodio/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Estimulación Cardíaca Artificial , Separación Celular , Circulación Coronaria/efectos de los fármacos , Femenino , Técnicas In Vitro , Masculino , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Peroxidasa/metabolismo , Sustancias Protectoras , Conejos , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , PorcinosRESUMEN
The response of the NMR relaxation times (T(1), CPMG T(2), and Hahn T(2)) to bleomycin-induced lung injury was studied in excised, unperfused rat lungs. NMR, histologic, and biochemical (collagen content measurement) analyses were performed 1, 2, 4, and 8 weeks after intratracheal instillation of saline (control lungs) or 10 U/kg bleomycin sulfate. The control lungs showed no important NMR, water content, histologic, or collagen content changes. The spin-spin relaxation times for the fast and intermediate components of the CPMG decay (T(2f) and T(2i), respectively) increased 1 week after bleomycin injury (acute inflammatory stage) and then progressively decreased during the following 2-8 weeks (i.e., with the development of the chronic, fibrotic stage of the injury). The slow component (T(2s)) showed no significant changes. The response of T(1) and the slow component of the Hahn T(2) was, on the whole, similar to that of CPMG T(2f) and T(2i). T(1) changes were very small. Lung water content increased 1 week after injury. Histologic and biochemical assessment of collagen showed that collagen content was close to control at 1 week, but markedly increased at 2, 4, and 8 weeks. T(1) and T(2) data were directly correlated with lung water content and inversely correlated with collagen content. Our results indicate that NMR relaxation time measurements (particularly T(2)) reflect the structural changes associated with bleomycin injury. The prolonged T(2) relaxation times observed in the acute stage are related to the presence of edema, whereas the subsequent decrease in these values marks the stage of the collagen deposition (fibrotic stage). CPMG-T(2) and Hahn-T(2) measurements can be valuable as a potentially noninvasive method for characterizing bleomycin-induced lung injury and pathologically related lung disorders.