defibrotide and Disease-Models--Animal

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapy for the treatment of high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. Acute graft-versus-host disease (aGvHD) remains the most frequent cause of non-relapse mortality following allo-HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug successfully employed for prophylaxis and treatment of veno-occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD-associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte-endothelial interactions by down-regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches.

Topics: Acute Disease; Animals; Biomarkers; Biopsy; Cell Communication; Cell Line; Chemotaxis, Leukocyte; Cytokines; Disease Models, Animal; Endothelium; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Inflammation Mediators; Leukocytes; Mice; Polydeoxyribonucleotides; Tissue Donors; Transplantation, Homologous

In this study, we aimed to investigate the additive effect of mesenchymal stem cells (MSC) and defibrotide (DFT) in a rat model of femoral arterial thrombosis.. Thirty Sprague Dawley rats were included. An arterial thrombosis model by ferric chloride (FeCl3) was developed in the left femoral artery. The rats were equally assigned to 5 groups: Group 1-Sham-operated (without arterial injury); Group 2-Phosphate buffered saline (PBS) injected; Group 3-MSC; Group 4-DFT; Group 5-MSC + DFT. All had two intraperitoneal injections of 0.5 ml: the 1st injection was 4 h after the procedure and the 2nd one 48 h after the 1st injection. The rats were sacrificed 7 days after the 2nd injection.. Although the use of human bone marrow-derived (hBM) hBM-MSC or DFT alone enabled partial resolution of the thrombus, combining them resulted in near-complete resolution. Neovascularization was two-fold better in hBM-MSC + DFT treated rats (11.6 ± 2.4 channels) compared with the hBM-MSC (3.8 ± 2.7 channels) and DFT groups (5.5 ± 1.8 channels) (P < 0.0001 and P= 0.002, respectively).. The combined use of hBM-MSC and DFT in a rat model of arterial thrombosis showed additive effect resulting in near-complete resolution of the thrombus.. El objetivo de este estudio consistió en investigar el efecto aditivo de las células madre mesenquimales (MSC, por sus siglas en inglés) y del defibrótido (DFT) en un modelo de trombosis arterial femoral en ratas.. Se incluyeron 30 ratas Sprague Dawley. Se desarrolló un modelo de trombosis arterial mediante cloruro de hierro (FeCl3) en la arteria femoral izquierda. Las ratas se asignaron equitativamente en cinco grupos: grupo 1, intervención quirúrgica simulada (sin lesión arterial); grupo 2, inyección de solución salina tamponada con fosfato (PBS); grupo 3, MSC; grupo 4, DFT; grupo 5, MSC + DFT. Todas las ratas recibieron dos inyecciones intraperitoneales de 0,5 ml: la primera se administró 4 horas después del procedimiento y la segunda 48 horas después de la primera. Se sacrificó a las ratas siete días después de la segunda inyección.. Aunque el uso por separado de MSC derivadas de médula ósea humana (hBM-MSC) o de DFT permitió una resolución parcial del trombo, la combinación de ambos tuvo como resultado la resolución casi completa. La neovascularización fue doblemente mejor en las ratas tratadas con hBM-MSC + DFT (11,6 ± 2,4 canales) en comparación con los grupos asignados por separado a hBM-MSC (3,8 ± 2,7 canales) y DFT (5,5 ± 1,8 canales) (P < 0,0001 y P= 0,002, respectivamente).. El uso combinado de hBM-MSC y DFT en un modelo de trombosis arterial en ratas mostró que el efecto aditivo tuvo como resultado la resolución casi completa del trombo.

Topics: Animals; Combined Modality Therapy; Disease Models, Animal; Fibrinolytic Agents; Male; Mesenchymal Stem Cell Transplantation; Polydeoxyribonucleotides; Rats; Rats, Sprague-Dawley; Thrombosis

The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria.. DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival.. Therapeutic use of DF in malaria is proposed.

Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Antimalarials; Blood Coagulation; Cells, Cultured; Complement Activation; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Female; Glycosylphosphatidylinositols; Hemoglobins; Humans; Inflammation Mediators; Malaria, Cerebral; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide; Plasmodium berghei; Plasmodium falciparum; Platelet Aggregation; Polydeoxyribonucleotides; Receptors, Purinergic P1; Severity of Illness Index; Thromboplastin; Time Factors

Adenosine has many actions potentially useful as adjunct to a cardioplegia. Defibrotide was recently shown to have protective effects during cardiac arrest. The aim of this study was to compare these 2 substances to delineate their profile of action in the setting of cardioplegic arrest.. A Langendorff model for isolated rat hearts was employed: 3 groups of 8 hearts each were used, respectively with plain St. Thomas cardioplegia as control (group C), and the same solution added with adenosine (group A) or defibrotide (group D). The hearts had a baseline perfusion for 30 minutes with Krebs-Henseleit solution at 37 degrees C, cardioplegia administration for 3 minutes, then 30 minutes of ischemia without any perfusion and finally 30 minutes of reperfusion with Krebs-Henseleit solution at 37 degrees C.. The time to attain heart arrest was 20% shorter in group A, but this difference did not reach statistical significance (A: 13.6+/-1.5; D: 16.8+/-2.7; C: 17.3+/-2.2 s). The heart rate during reperfusion in group A was almost identical to baseline, while in both group C and D it was significantly lower (A: 101%, D: 93.4%, C: 82.4%, p<0.01).A and D decreased significantly the release of creatine phospokinase compared to group C (p=0.006). Lactate dehydrogenase release was lower in both treatment groups, although statistical significance was not reached. Peak positive dP/dT decreased more in controls during reperfusion (A: -23+/-6%, D: -17+/-5%, C: -31+/-5%, p=ns). Negative dP/dT was significantly worse in controls compared to both treatments (A: -19+/-6%, D: -12+/-5%, C: -34+/-7%, p=0.035).. Both adenosine and defibrotide have protective effects in an isolated model of cardioplegic arrest. Adenosine is significantly more active on heart rate while defibrotide is more active on contractily. Further studies are justified in order to test the combination of these 2 drugs.

Topics: Adenosine; Animals; Cardioplegic Solutions; Coronary Circulation; Disease Models, Animal; Drug Combinations; Fibrinolytic Agents; Heart Arrest, Induced; Heart Rate; In Vitro Techniques; Male; Myocardial Ischemia; Polydeoxyribonucleotides; Rats; Rats, Wistar; Recovery of Function; Vasodilator Agents

1. Two polydeoxyribonucleotides, produced by the controlled hydrolysis of DNA of mammalian lung (defibrotide and its lower molecular weight fraction, P.O. 085 DV), were studied for their ability to modify the release of nitrite and the coronary flow in perfusates collected from isolated, normally perfused hearts of guinea-pigs and from hearts subjected to regional ischaemia and reperfusion. 2. In guinea-pig normally perfused hearts, both defibrotide (DFT) and its fraction, P.O. 085 DV, increase the amount of nitrite appearing in perfusates in a concentration-dependent fashion. At the highest concentration studied (10(-6) M), P.O. 085 DV was more effective than DFT. A concomitant increase in the coronary flow was observed. 3. The increase in nitrite in perfusates and the increase in coronary flow induced by both DFT and P.O. 085 DV were significantly reduced by NG-monomethyl-L-arginine (L-NMMA, 10(-4) M), an inhibitor of nitric oxide synthase (NOS). 4. The endothelium-dependent vasodilator, acetylcholine (ACh), enhances the formation of nitrite and the coronary flow. Both the increase in coronary flow and in the formation of nitrite were significantly reduced by L-NMMA (10(-4) M). 5. In guinea-pig hearts subjected to ischaemia and reperfusion, the effect of both compounds in increasing the amount of nitrite in perfusates was more evident and more pronounced with P.O. 085 DV. 6. Reperfusion-induced arrhythmias were significantly reduced by both compounds to the extent of complete protection afforded by compound P.O. 085 DV. 7. The cardioprotective and antiarrhythmic effects of DFT and P.O. 085 DV are discussed.

Topics: Acetylcholine; Animals; Arginine; Arrhythmias, Cardiac; Chemical Fractionation; Coronary Circulation; Disease Models, Animal; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibrinolytic Agents; Guinea Pigs; Male; Molecular Weight; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Polydeoxyribonucleotides

Oral Defibrotide decreased leukocyte and platelet counts, raised by cholesterol diet, and the area (%) of aorta endothelial surface involved in atherosclerosis. Frequency of intimal thickening in blood vessels of kidneys and hearts and in cardiac valves was reduced by oral Defibrotide by 47%, 29% and 17%. It is suggested that oral Defibrotide reduced the involvement of the aorta in the atherosclerotic process by acting on leukocytes and platelets, to both reduce their number and deactivate them.

Topics: Administration, Oral; Animals; Arteriosclerosis; Blood Cell Count; Blood Platelets; Diet, Atherogenic; Disease Models, Animal; Leukocytes; Lipids; Male; Polydeoxyribonucleotides; Rabbits; Random Allocation

The authors previously demonstrated the protective activity of defibrotide (a profibrinolytic and antithrombotic drug) on endothelial cells. In the present work they examine the efficacy of defibrotide in protecting rat kidney from ischaemic/reperfusion injury by studying the modifications of intrarenal adenine nucleotide levels. Right renal ischaemia of 60 min and reperfusion of 30 min were induced in adult male Wistar rats. Defibrotide was administered as a bolus through a catheter inserted into the left femoral vein 5 min before the beginning of ischaemia at the dose of 32 mg/kg and continuously infused during ischaemia/reperfusion through the same vein at the final dose of 32 mg/kg in 5 ml of saline at the rate of 3 ml/h. Rats treated with vehicle of the drug were used as controls. At the end of postischaemic reperfusion, the ischaemic and left kidneys were rapidly removed and frozen in liquid nitrogen. Tissue extracts were prepared, and their ATP, ADP, AMP, cAMP, NAD+, and NADH contents were determined by using luminescence methods. In controls, ATP intrarenal levels were significantly higher in the left kidney than in the ischaemic organ of the same rat (3405 +/- 320 vs. 378 +/- 36 nmol/g fresh tissue and mean +/- s.e.m. of 10 experiments). Defibrotide treatment significantly protected ischaemic kidneys from the drop in ATP intrarenal content (1465 +/- 147 vs. 3124 +/- 303 nmol/g fresh tissue measured in the left kidney).

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Disease Models, Animal; Fibrinolytic Agents; Ischemia; Kidney; Male; Polydeoxyribonucleotides; Rats; Rats, Wistar; Reperfusion Injury

Several attempts have been undertaken to reduce the severity of ischemic myocardial injury by exogenous administration of eicosanoids and by modification of endogenous eicosanoid production. The present study investigates whether defibrotide, a compound that stimulates endogenous prostacyclin (PGI2), has a beneficial effect in experimental ischemic myocardial injury. Anesthetized, open-chest minipigs were subjected to 1 h of coronary artery occlusion, followed by 3 h of reperfusion. Defibrotide (32 mg/kg x h) or its vehicle were infused i.v. throughout the experiment. Defibrotide increased cardiac PGI2 formation 3- to 4-fold greater than control (P < .05). Thromboxane levels remained unchanged. Irreversible ischemic injury, as identified by negative tetrazolium staining, amounted to 44 +/- 6% of the area at risk in pigs receiving vehicle but was reduced to 23 +/- 4% by defibrotide (P < .05). This reduced tissue injury in defibrotide-treated pigs was associated with improved functional recovery (left ventricular pressure, + dP/dtmax), during early reperfusion. Recovery did not occur in vehicle-treated pigs. Collagen (2 micrograms/ml)-induced platelet aggregation ex vivo was increased in vehicle-treated pigs during ischemia and reperfusion, but not in animals treated with defibrotide. Polymorphonuclear neutrophil leukocyte accumulation in the ischemic border zone was reduced from 59 +/- 17 cells/mm2 in vehicle-treated pigs to 17 +/- 9 cells/mm2 by defibrotide (P < .05). Pretreatment of the animals with indomethacin (3 mg/kg) prevented the reduction of infarct size and polymorphonuclear neutrophil leukocyte infiltration by defibrotide. Indomethacin increased infarct size in vehicle- and defibrotide-treated pigs by 71 and 59%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Blood Platelets; Cyclooxygenase Inhibitors; Disease Models, Animal; Epoprostenol; Female; Granulocytes; Indomethacin; Leukocyte Count; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Neutrophils; Platelet Count; Polydeoxyribonucleotides; Prostaglandins; Stimulation, Chemical; Swine; Swine, Miniature; Thromboxane A2; Ventricular Function, Left

Defibrotide (DEF), a compound previously found to stimulate vascular prostacyclin (PGI2) formation, has been investigated in an experimental model of septic shock. Anesthetized pigs were subjected to i.v. infusion of lipid A (1.5 mg/kg per hr for 4 hr). DEF (50 mg/kg per hr) or vehicle were infused i.v. throughout the experiments, starting 1 hr prior to lipid A. Two out of 7 pigs receiving vehicle survived lipid A infusion for 4 hr, whereas 6 out of 7 DEF treated animals survived this period (P less than 0.05). DEF delayed the shock-induced depression of platelet count and preserved platelet secretory function (collagen-induced ATP-secretion). DEF increased plasma PGI2 by 45% (P less than 0.05) during lipid A infusion and tended to reduce thromboxane levels. DEF did not change eicosanoid formation in sham-shock pigs (n = 4 per group). In vivo treatment with DEF significantly increased the stimulatory effect of bradykinin (1 microM) and arachidonic acid (100 microM) on PGI2 formation ex vivo of mesenteric and iliac artery segments. The improvement of survival in lipid A-induced shock by DEF may be related to an enhancement of vascular PGI2 generation, potentially due to a reduction of shock-induced platelet activation and microcirculatory dysfunction.

Topics: Animals; Arachidonic Acid; Blood Platelets; Bradykinin; Disease Models, Animal; Drug Synergism; Epoprostenol; Lipid A; Platelet Count; Polydeoxyribonucleotides; Shock; Survival Rate; Swine; Thromboxane A2

The age of the thrombus is probably a very important determinant of the outcome of thrombolysis. The clinical potential for rapidly dissolving thrombi by thrombolytic therapy is considerable because restoration of the blood flow can rescue the jeopardized district served by the occluded vessel such as for myocardial infarction, deep vein thrombosis, arterial thrombosis, pulmonary embolism, and occlusion of retinal vessels. Defibrotide was effective against 3-, 7-, or 10-day-old thrombi; its ED50s were 32, 65, or 118 mg/kg-1 hour-1, respectively, suggesting that the age of the thrombus could play a role in the outcome of thrombolysis. A similar pattern was also shown for urokinase.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Epoprostenol; Fibrinolytic Agents; Heparin; Male; Polydeoxyribonucleotides; Rabbits; Thrombophlebitis; Ticlopidine; Time Factors; Urokinase-Type Plasminogen Activator

A method of inducing microthrombi in small rat mesenteric vessels (approximately 20 to 30 micron) has been developed to study platelet reactions and to investigate antithrombotic drugs. For microscopy, a high-power water immersion system, based on a Leitz Orthoplan microscope, was used. In sodium pentabarbital anesthetized male Wistar rats and in fawn-hooded bleeder rats vascular lesions were produced with a Coherent CR-2 ion laser (argon laser). The laser induced vascular damage, without intravascular heat precipitates, which led to the adhesion, transformation, and aggregation of platelets at the damaged vessel wall. Fibrin threads were rarely formed in these early platelet thrombi. Thrombus formation was estimated by the number of laser injuries required to induce a defined thrombus. The first steps of thrombus formation, in particular the adhesion and reversible aggregation, were significantly reduced in this model in fawn-hooded bleeder rats. A number of different drugs modified thrombus formation in this model, for instance, ASA, defibrotide, unfractionated or low molecular heparins. It is unlikely that a single mechanism is responsible for the observed antithrombotic effects of each of these drugs. The inhibition of platelet adhesion by changes of membrane glycoproteins, interference with the early activation of plasma proteins, effects on prostaglandin metabolism, inhibition of activators stemming from damaged endothelial cells, and other effects have to be considered.

Topics: Animals; Aspirin; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelium, Vascular; Fibrinolytic Agents; Heparin; Heparin, Low-Molecular-Weight; Lasers; Male; Microscopy, Electron; Microscopy, Interference; Platelet Function Tests; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains