Glycosaminoglycans in nerve injury: 1. Low doses of glycosaminoglycans promote neurite formation.

This study has shown that glycosaminoglycans added to the culture medium may affect neurite formation in SH-SY5Y neuroblastoma cells. The most effective glycosaminoglycans are heparin and COS 8, a preparation with low anticoagulant activity. Promotion of neuritogenesis was remarkable at concentrations as low as 10(-8) and 10(-10). When added at 10(-4) M both agents are inhibitory. Chondroitin-4 sulfate, dermatan sulfate, and heparan sulfate were also effective, the doses required were, however, as high as 10(-4) M for promoting and 10(-4) M for inhibiting neuritogenesis. Thereby low doses of glycosaminoglycans promote, while higher doses inhibit neurite formation. The effects were observed when neuritogenesis was promoted in neuroblastoma cultures either by deprivation of serum or by addition of retinoic acid, in the former case neuritogenesis occurred within 48 hr; in the latter, in 14 days. PC12 pheochromocytoma cells neuritogenesis was triggered by adding NGF to the culture medium. We have also observed that glycosaminoglycan supplementation to the culture medium lowered the quantity of NGF required to form neurites by PC12 cells. Glycosaminoglycans at the dose of 10(-8) M allow the formation of PC12 neurites even in presence of 1 ng/ml NGF, a dose that normally is ineffective.

Glycosaminoglycans in nerve injury: II. Effects on transganglionic degeneration and on the expression of neurotrophic factors.

Injury to the sciatic nerve leads to the transganglionic degeneration of sensory axons and to the induction of neurotrophins and p75 nerve growth factor receptor synthesis by the denervated Schwann cells. Sciatic nerve axotomy caused a marked loss of substance P and of met-enkephalin in the lumbar cord. Substance P immunostaining and pre-proenkephalin mRNA expression were reduced in the dorsal horn layers I and II ipsilaterally to the lesion. Treating rats with low doses (0.25 mg/kg) of heparin or COS 8, a natural glycosaminoglycan mixture with low anticoagulant activity, the peptide loss was prevented and the content increased of about 50% above control values. The effects of COS 8 treatment were also evident on Schwann cells. COS 8 augmented the increase of nerve growth factor, brain-derived neurotrophic factor, and NT-3 mRNA expression in the distal stump of the axotomized sciatic nerve. Therefore, it can be concluded that glycosaminoglycans neuroprotective effects on lesioned sensory axons might have been mediated by the dramatic promotion of neurotrophin synthesis. Although the in vitro studies (Lesma et al.: J Neurosci Res, 1996) suggested also a likely direct effect as extracellular matrix components that is not mediated by trophic factors.

Defibrotide, a single-stranded polydeoxyribonucleotide acting as an adenosine receptor agonist.

The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.

Pharmacokinetics, absorption, distribution and disposition of [125I]-defibrotide following intravenous or oral administration in the rat.

Defibrotide (D) was labelled with 125I. The radiolabelled compound ([125I]-Defibrotide ([125I]-D)) retained the same profibrinolytic activity as the parent drug. Following single intravenous administration of [125I]-D the half lives of radioactivity associated with D components in plasma were 9.45 min and 11.27 h for alpha and beta phases respectively. Following single oral administration of [125I]-D the half life of radioactivity associated with D components in plasma was 12.83 h for the elimination phase. Bioavailability was apparently 58%. The areas under plasma total radioactivity versus time curves were dose-dependent following both intravenous and oral administration. No significant accumulation of total radioactivity in plasma was observed following multiple oral administration of [125I]-D. Following single intravenous administration of [125I]-D a larger proportion of administered radioactivity was excreted via urine than faeces while following single oral administration excretion via urine and faeces accounted for similar proportions of administered radioactivity. Following both single and oral administration the levels of total radioactivity in tissues and organs examined were generally highest in highly perfused organs and were very high in the thyroid despite pretreatment with non-radiolabelled potassium iodide. Radioactivity was also found to be associated with the aorta wall.

High-performance liquid chromatography determination of polydeoxyribonucleotides in plasma: its application to the determination of defibrotide's pharmacokinetics in the rabbit.

We describe an HPLC method for the determination of whole polydeoxyribonucleotides in animal plasma. This method was compared to a colorimetric method, which evaluates the sugar moiety of polydeoxyribonucleotides, and to an agarose gel electrophoresis method, which evaluates the whole polydeoxyribonucleotides as does the HPLC method, and was found to give results very close to those obtained with these two other methods. A pharmacokinetic study of the antithrombotic, profibrinolytic, polydeoxyribonucleotidic drug defibrotide was carried out by evaluating the plasma drug levels by these three methods. The pharmacokinetic parameters calculated from the data are very similar.

Defibrotide has antiischemic activity in perfused rabbit hearts, preventing tissue Ca++ overloading.

Defibrotide (D), a polydeoxyribonucleotide obtained from mammalian lungs, reduced the ischemic contracture due to low perfusion (0.2 ml/min) of the isovolumic left heart of the rabbit and abolished the irregularity of the rhythm of the heart, thereby restoring the cardiomechanical activity upon reperfusion (20 ml/min). D stimulated the release of PG-like material. Indomethacin infusion completely prevented both the antiischemic activity of D and its ability to increase the generation of prostaglandins in the rabbit heart. Measurement by atomic absorption spectroscopy of calcium content in ischemic heart tissue and its mitochondrial fraction indicated that the ischemic procedure significantly increased tissue calcium content in both. D, Prostacyclin (PGI2) and Nifedipine protected the heart from ischemic ventricular contracture and prevented accumulation of calcium in the heart. The effect of D on preventing Ca++ overload was completely abolished by indomethacin infusion. The results indicate that the beneficial effects of Defibrotide in experimental ischemia are primarily due to a release of Prostaglandin E2 (PGE2) and PGI2, which in turn may inhibit the detrimental effects of calcium overload in myocytes and mitochondria.

Enhancement by defibrotide of prostanoid neosynthesis from arachidonic acid: an adenosine receptor-mediated effect?

The antithrombotic and anti-ischemic drug defibrotide is known to enhance PGI2 and prostaglandin E2 release and to interact competitively and agonistically with adenosine A1 and A2 receptors. To elucidate a possible molecular mechanism of action of this drug, we measured prostanoid neosynthesis from exogenous AA in isolated, intraluminally perfused RbA (5 to 10 preparations/group). Defibrotide (30 micrograms/ml) almost doubled the conversion rate of AA (15 micrograms) into PGI2-la (quantitated by a bioassay system): 56.8 +/- 5.3 versus 32.9 +/- 7.8 ng, compared with controls. The adenosine receptor antagonist 8-PT, at concentrations of 0.625, 1, and 1.6 micrograms/ml, dose-dependently and up to 100% inhibited the effect of defibrotide; however, a higher concentration of this latter (100 micrograms/ml) shifted to the right the 8-PT inhibition curve. A second AA bolus given into RbA caused homologous desensitization: 43 and 62% reduction of AA conversion after two versus one bolus was observed in controls receiving 15 and 30 micrograms AA. This phenomenon was inhibited by 30 micrograms/ml defibrotide (32% reduction only at two boluses, instead of 62% displayed by comparable controls), but this effect was abolished by 1.6 micrograms/ml 8-PT; once again, 100 micrograms/ml defibrotide overcame the antagonism by 8-PT. We conclude that defibrotide enhances prostanoid neosynthesis from AA and reduces homologous desensitization of the cyclooxygenase pathway through a mechanism possibly linked to stimulation of adenosine receptors.

Absorption through the peritoneum of the macromolecular profibrinolytic drug defibrotide in the rabbit.

Defibrotide is the sodium salt of a polydeoxyribonucleotide extracted from mammalian lungs by controlled depolymerization. This macromolecular compound (mean molecular weight 20,000 d) has profibrinolytic activity. It also is antithrombotic after both intravenous and oral administration. The purpose of this study was to see whether defibrotide could activate the fibrinolytic system after intraperitoneal administration, because it has been observed that in ascitic patients treated intraperitoneally with defibrotide, no occlusion of the peritoneal-jugular catheter valve draining the ascitic fluid (according to LeVeen et al.) occurred. The profibrinolytic activity of defibrotide and its plasma levels were dose dependent, with a statistically significant correlation between the fibrinolytic activities and the AUC of the plasma levels of defibrotide. No rebound effect was observed during the duration of the experiments (24 hours). We hypothesize that the fibrinolytic activity elicited by defibrotide prevented the deposition of fibrin in the catheter valve, enabling normal drainage of the ascitic fluid. The lack of rebound makes defibrotide a safe drug. The mechanism by which this macromolecular compound is absorbed is still unknown.

Effects of defibrotide on prostacyclin release from isolated rabbit kidneys and protection from post-ischemic acute renal failure in vivo.

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)

Defibrotide affects the anticoagulant and lipase-releasing activities of heparin.

Defibrotide (DEF), an antithrombotic drug with no anticoagulant activity, given concomitantly with heparin potentiates its effects on thrombin time and lipase-releasing activity. Unlike heparin, DEF has hardly any lipase-releasing activity of its own. Most likely DEF interferes with pharmacokinetics of heparin. It is clear from the data reported here that when used in combination with DEF heparin should be administered at low doses and the coagulation parameters carefully followed.

Thrombolytic activity of defibrotide against old venous thrombi.

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.

Profibrinolytic activities of chemically modified heparins with very low anticoagulant activities.

Many evidences indicate that heparin is an activator of fibrinolysis, but the most important side effect of heparin is bleeding which is a problem particularly in the high risk patient. Here we describe how chemical modifications, associated with separation techniques, can sharply reduce the anticoagulant activities of a heparin while its ability to stimulate fibrinolysis is retained.

Defibrotide decreases cholesterol amount in hypercholesterolemic rabbit aorta, with no modification of plasma or lipoprotein cholesterol.

Defibrotide, (D) an antithrombotic agent, when administered i.v. to cholesterol-fed rabbits decreased cholesterol in the aorta without changing total plasma cholesterol, triglyceride or phospholipid, nor the cholesterol, triglyceride, phospholipid and protein of plasma lipoproteins. Platelet aggregation was decreased in rabbits treated with D. There were fewer vascular lesions in the hearts and kidneys of animals treated with D than in animals fed cholesterol and treated with placebo. These data suggest that the antithrombotic activity of D and its ability to reduce platelet sensitivity could help to reduce the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations.