defibrotide and Ischemia

Defibrotide is a polydeoxyribonucleotide extracted from mammalian organs (porcine) and prepared by controlled depolymerization, resulting in a single-stranded deoxyribonucleotide with a mean molecular weight of 15 to 30 kDa. Early studies of experimental pharmacology in different animal species (1981-1986) were focused on an antithrombotic effect in arteries, veins, and the microcirculation, attributed to a dose-dependent activation of fibrinolysis. More recently, a number of new data have raised interest in an "anti-ischemic" action of the substance, as suggested by its evident protective effect in different models of tissue and organ ischemia. Protection from myocardial ischemia was demonstrated in different experimental models, and several ischemic features, such as heart muscle contracture, loss of high-energy substrates, decline in beta-adrenergic receptor sensitivity, and drop in infarct-related blood flow, were successfully prevented. Similar protective effects were observed during liver and kidney ischemia and in different types of experimental shock. The mechanisms involved in the anti-ischemic properties of defibrotide have been investigated in studies of experimental and human pharmacology. The substance has been shown to modulate arachidonic acid metabolism by enhancing the production and release of prostacyclin and prostaglandin E2 from tissues and whole blood, and inhibiting leukotriene B4 generation in leukocytes. Furthermore, an effect of defibrotide on activation of polymorphonuclear leukocytes and their incorporation into thrombi was described. Favorable effects on blood rheology were also reported. In summary, defibrotide deserves attention for therapeutic trials in clinical conditions characterized by organ or tissue ischemia.

Topics: Animals; Antifibrinolytic Agents; Endothelin-1; Epoprostenol; Humans; Ischemia; Polydeoxyribonucleotides; Vasoconstriction

Defibrotide is a single-stranded DNA fraction obtained from mammalian lung and is able to increase prostacyclin production by endothelial cells. It has profibrinolytic and antithrombotic properties, and we have successfully used it as an anti-ischaemic drug in many in-vivo experimental models. In fact, we showed that defibrotide treatment significantly protected rat heart, kidney and liver from ischaemia and postischaemic reperfusion injury. In rats treated with defibrotide, the functionality and metabolic activity of ischaemic organs were significantly protected from impairment as compared to controls treated only with the vehicle of the drug. In the present work we evaluated all our results, together with those of others, in order to hypothesize the mechanism of action of the drug. We postulated that the prominent function of defibrotide is to inhibit platelet and leukocyte adhesion to endothelial cells: this may depend on reduced cell activation possibly following drug interaction with adenosine receptors. Defibrotide could also favour endothelial-cell function through binding with haemoglobin: such binding permits oxygen release and preservation of endothelium-derived relaxing factor. Moreover, prostacyclin production by endothelial cells is responsible for many drug activities and also limits superoxide radical generation.

Topics: Adenine Nucleotides; Animals; Cell Adhesion; Endothelium; Epoprostenol; Heart Transplantation; Ischemia; Kidney; Kidney Transplantation; Leukocytes; Myocardial Reperfusion Injury; NAD; Platelet Adhesiveness; Polydeoxyribonucleotides; Rats; Rats, Wistar; Receptors, Purinergic P1; Reperfusion Injury

1. The purpose of this study was to investigate the protective effects of defibrotide, a single-stranded polydeoxyribonucleotide, on ischaemia-reperfusion injury to the liver using a rat model. 2. Ischaemia of the left and median lobes was created by total inflow occlusion for 30 min followed by 60 min of reperfusion. Hepatic injury was assessed by the release of liver enzymes (alanine transferase, ALT and lactic dehydrogenase, LDH). Hepatic oxidant stress was measured by superoxide production, lipid peroxidation and nitrite/nitrate formation. Leukocyte-endothelium interaction and Kupffer cell mobilization were quantified by measuring hepatic myeloperoxidase (MPO), polymorphonuclear leukocyte adherence to superior mesenteric artery (SMA) and immunostaining of Kupffer cell. 3. Defibrotide treatment resulted in a significant inhibition of postreperfusion superoxide generation, lipid peroxidation, serum ALT activity, serum LDH activity, MPO activity, serum nitrite/nitrate level, leukocyte adherence to SMA, and Kupffer cell mobilization, indicating a significant attenuation of hepatic dysfunction. 4. A significant correlation existed between liver ischaemia/reperfusion and hepatic injury, suggesting that liver ischaemia/reperfusion injury is mediated predominantly by generation of oxygen free radicals and mobilization of Kupffer cells. 5. We conclude that defibrotide significantly protects the liver against liver ischaemia/reperfusion injury by interfering with Kupffer cell mobilization and formation of oxygen free radicals. This study provides strong evidence that defibrotide has important beneficial effects on acute inflammatory tissue injury such as that occurring in the reperfusion of the ischaemic liver.

Topics: Animals; Free Radicals; Ischemia; Kupffer Cells; Liver; Male; Polydeoxyribonucleotides; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Defibrotide (a polydeoxyribonucleotide) and oligotide (an oligodeoxyribonucleotide) obtained from mammalian single-stranded DNA, have been demonstrated to have anti-ischemic activity in some experimental models of ischemia/reperfusion of kidney in rats. We hypothesized that their anti-ischemic activity could be related to an inhibition of leukocyte-endothelial cell adhesion and also the consequent generation of oxygen free radicals by leukocytes. We studied the in vitro adhesion of neutrophils to human umbilical vein endothelial cells under basal conditions and following neutrophil or endothelial cell activation (using 10(-7) fMLP and 500 U/ml TNF-alpha, respectively). Defibrotide and oligotide significantly inhibited neutrophil adhesion to endothelial cells (after only 1 min of drug treatment). When the anti-LFA-1 70H12 F(ab)2 monoclonal antibody was used, the drugs exerted only slight additional inhibition of the adhesion of fMLP-activated neutrophils to endothelium. These results, confirmed in NIH/3T3-ICAM-1-transfected cells, demonstrate that defibrotide and oligotide interfere with leukocyte adhesion to endothelial cells by an LFA-1-dependent mechanism.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Ischemia; Leukocytes; Lymphocyte Function-Associated Antigen-1; Mice; N-Formylmethionine Leucyl-Phenylalanine; Oligodeoxyribonucleotides; Polydeoxyribonucleotides

Defibrotide is a polydeoxyribonucleotide-derived anti-ischemic drug with multiple sites of action involving both plasmatic and cellular targets. This agent has been demonstrated to produce profibrinolytic, cytoprotective, and vaso-facilatory actions. Since monocytes are increased in the mediation of some of the pathophysiologic responses seen in ischemic disorders, the functional properties of these cells were investigated in experimental conditions to evaluate their behavior during resting and stimulated states. Defibrotide was supplemented in these systems to determine its modulatory action. In this investigation Defibrotide was found to decrease the PAI-1 levels and may indicate that this may be the mechanism for its profibrinolytic actions. Defibrotide was also found to reduce the procoagulant activity of monocytes in these experimental settings. Both PAI and procoagulant factors play an important role in the pathophysiology of inflammation, DIC, and ischemia. Defibrotide induced reduction of these two factors represents the mechanism whereby this agent produces its therapeutic action.

Topics: Blood Coagulation Factors; Cytokines; Fibrinolytic Agents; Hemostasis; Humans; Ischemia; Monocytes; Plasminogen Activator Inhibitor 2; Polydeoxyribonucleotides; Thromboxane B2

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

Defibrotide (Df) has been reported to protect various organs from ischemic damage. The aim of this study was to evaluate the effect of Df on renal function and morphology after warm kidney ischemia. Divided into two groups, 14 pigs underwent bilateral renal clamping for 90 min. One group (7 pigs) was treated with Df (32 mg/kg per h) for 6 h, whereas the control group received 5000 IU of heparin. Serum levels of blood urea nitrogen (BUN) and creatinine measured for 6 days were significantly higher in the control group (peak 26.8 +/- 16.7 vs 11.0 +/- 2.9 mmol/l and 501.2 +/- 351.4 vs 230.2 +/- 68.0 mumol/l P < 0.05). Renal biopsy evidenced a lesser extent of tubular and endothelial damage in Df-treated animals. In conclusion, Df provided relevant protection against renal ischemic injury.

Topics: Animals; Fibrinolytic Agents; Ischemia; Kidney; Kidney Function Tests; Polydeoxyribonucleotides; Renal Circulation; Swine

We evaluated whether defibrotide, a single-stranded polydeoxyribonucleotide that enhances prostacyclin (PGI2) release from various isolated organs, could also release PGI2 from the rabbit kidney and prove effective against renal ischemic injury. Isolated perfused kidneys responded to defibrotide (100, 250 and 500 micrograms ml-1 min-1) with a dose-dependent release of immunoreactive 6-keto-PGF1 alpha (4-fold increase at highest dose), which was prevented by indomethacin pre-treatment. In vivo, venous blood withdrawn from heparinized rabbits (and representative of renal outflow) was conveyed over a collagen matrix, onto which platelets adhered and aggregated. Recording the weight increase of the matrix was used as a bioassay to follow the time-course of released PGI2. We observed that renal outflowing blood from defibrotide treated animals (50 mgKg-1 i.v.) displayed lower (P less than 0.05 versus controls) platelet activation, consistent with enhanced PGI2 release from the kidneys. Furthermore, the duration of this effect was longer lasting than that predicted from the known plasma half-life of the drug. After transient (30 min) occlusion of the renal arteries, glomerular filtration rate (GFR) dropped by about 50% (P less than 0.01) during the first reperfusion hour in control animals, with only mild recovery having occurred 4 h later. Defibrotide (16 mgKg-1 bolus + 16 mgKg-1h-1, i.v.) could not antagonize the initial impairment (40% GFR reduction), but allowed full recovery at the end of the observation period (P less than 0.05 vs controls). Indomethacin, instead, caused a dramatic reduction of GFR (70%) during early reperfusion, with no subsequent recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acute Kidney Injury; Animals; Epoprostenol; Fibrinolytic Agents; In Vitro Techniques; Ischemia; Kidney; Male; Perfusion; Platelet Adhesiveness; Platelet Aggregation; Polydeoxyribonucleotides; Rabbits

The effect of defibrotide treatment in protecting liver metabolism from ischemic damage was studied. The drug was administered to male Wistar rats as a bolus (30 mg/kg body weight) at the beginning of 60 min ischemia and then continuously during 60 min of postischemic reperfusion at a dose of 30 mg/kg body weight. This dose was previously identified as useful to protect against myocardial ischemia induced in the cat. ATP and ADP intrahepatic levels were significantly higher in drug-treated rats than in untreated animals. The liver cytoplasmic NAD+/NADH ratio in defibrotide-treated rats was no different from that observed in sham-operated rats. The mitochondrial NAD+/NADH ratio in the liver was also improved by defibrotide treatment. Our data suggest that defibrotide may exert protective activity on hepatocytes useful for inducing a rapid restoration of their metabolism. Such a restoration is possibly related to improvement of microcirculation through an increase in prostaglandin I2 production or oxygen delivery due to drug administration.

Topics: Adenine Nucleotides; Animals; Cytoplasm; Fibrinolytic Agents; Ischemia; Liver; Male; Mitochondria, Liver; NAD; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains

Topics: Adenosine Triphosphate; Animals; Fibrinolytic Agents; Ischemia; Kidney; Liver; Male; Myocardial Reperfusion Injury; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains; Reperfusion Injury

Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, has been demonstrated to exert profibrinolytic and antithrombotic activity through stimulation of vascular prostacyclin (PGI2) production. We studied the effect of defibrotide administration in protecting liver and heart from ischaemic and postischaemic reperfusion damage. Defibrotide was administered as an i.v. bolus (30 mg/kg) at the beginning of liver ischaemia and at the same dose continuously during 60 min of postischaemic reperfusion. ATP levels were significantly improved in livers of defibrotide-treated rats as compared to those obtained in livers of rats treated with vehicle of the drug. Intrahepatic cytoplasmic and mitochondrial NAD+/NADH ratios were higher in defibrotide-treated than in vehicle-treated animals. The hearts, isolated from rats according to the transplantation procedure, were subjected to different times of warm + cold ischaemia. During ischaemia, the hearts were perfused continuously with 60 mg/kg of defibrotide or vehicle of the drug. The loss of creatine phosphokinase and lactate dehydrogenase activities due to an increased ischaemia time was limited in defibrotide-perfused hearts. Intracardiac ATP and ADP levels were significantly higher in defibrotide-treated organs than in controls. Our results demonstrate the efficacy of defibrotide in protecting liver and heart from ischaemia.

Topics: Animals; Coronary Disease; Cytoplasm; Epoprostenol; Ischemia; Liver; Male; Mitochondria, Liver; Myocardium; Nucleotides; Polydeoxyribonucleotides; Rats; Rats, Inbred Strains; Reperfusion Injury

Eight patients with severe lower limb ischaemia, aged 65-80, received defibrotide intravenously for periods from 5 to 21 days (mean 13 days). All patients had intractable rest pain. Five had ischaemic ulcers and 3 had minor gangrene. Five had previous arterial surgery and 6 lumbar sympathetic ganglion injections. Pretreatment ankle pressure indices ranged from 0 to 0.5 (mean 0.19). Rest pain, sleep disturbance and analgesic requirement were assessed on a nominal scale. Rest pain improved in 4 and sleeping pattern in 2 patients. One patient showed a diminution in analgesic requirement. Pressure indices improved in 5 patients. Amputation was performed in 4 patients. Adverse reactions included vomiting and diarrhoea (2), thrombophlebitis at infusion site (3) and generalized skin reaction (1). All patients had 'end-stage' peripheral vascular disease but some showed symptomatic benefit. Further evaluation of defibrotide is indicated.

Topics: Aged; Amputation, Surgical; Blood Pressure; Diarrhea; Female; Fibrinolytic Agents; Humans; Ischemia; Leg; Male; Pain; Pain Management; Polydeoxyribonucleotides; Sleep; Vomiting