Hepatic protection, antithrombotic, anti-inflammatory, profibrinolytic, anti-ischemic
Defibrotide does not have a single defined molecular target but exerts its effects through multiple mechanisms involving the vascular endothelium. It binds to and protects endothelial cells from damage, inhibits platelet aggregation, and promotes fibrinolysis through modulation of tissue plasminogen activator and thrombomodulin. The compound also exhibits anti-inflammatory effects by reducing cytokine release and leukocyte adhesion to endothelial cells, and protects against ischemia-reperfusion injury.

By agonism at adenosine A1 and A2 receptors, defibrotide sodium limits the release of inflammatory mediators and decreases the production of intracellular adhesion molecule-1, which in turn has anti-inflammatory effects[1]. By raising prostaglandin I2 and E2 synthesis and suppressing plasminogen activator inhibitor type 1 activity, defibrotide sodium prevents platelet aggregation and promotes fibrinolysis[1]. Moreover, defibrotide sodium promotes the synthesis of tissue factor pathway inhibitor while decreasing tissue factor production, which reduces the activation of the extrinsic coagulation cascade and fibrin deposition[1]. Tumor necrosis factor α-mediated endothelial cell damage as well as fludarabine-induced activation and death are prevented by defibrotide sodium[1].
In vitro, defibrotide has been shown to protect endothelial cells from damage induced by various insults, including chemotherapy agents, oxidative stress, and inflammatory cytokines. It inhibits platelet aggregation and promotes fibrinolysis through modulation of tissue plasminogen activator and thrombomodulin. The compound also reduces the expression of adhesion molecules on endothelial cells, thereby decreasing leukocyte adhesion and transmigration. These activities contribute to its antithrombotic, anti-inflammatory, and endothelial-protective properties.

When intraperitoneally (ic) administered before intravenous (ic) thrombin (100 μg/kg), Defibrotide sodium (64 mg/kg bolus plus 64 mg/kg/h for 1 h) dramatically lowers the capacity of thrombin to generate cranial thromboembolism in rabbits[2]. Intravenous (iv) defibrotide (64 mg/kg bolus plus 64 mg/kg/h for 1 h) administered intravenously (iv) prior to intravenous (iv) thrombin administration in rabbits greatly reduces the reversible accumulation of radiolabelled platelets into the thoracic circulation[2]. Pretreatment with defibrotide (150-175 mg/kg, IV) considerably reduces the majority of animals that die after receiving an intravenous injection of human thrombin (1250 μg/kg) in mice[2].
In vivo, defibrotide has demonstrated efficacy in preclinical models of venous occlusive disease, ischemia-reperfusion injury, and inflammatory conditions. Clinical studies have shown that defibrotide improves survival in patients with severe hepatic VOD/SOS following HSCT. The compound has been shown to restore blood flow in hepatic sinusoids, reduce liver enzyme elevations, and improve multiorgan function. It has also been studied for its potential in preventing VOD in high-risk patients.

In vitro enzyme/receptor binding assays for defibrotide are challenging due to its nature as a mixture of oligonucleotides. However, binding studies with endothelial cells have been conducted using radiolabeled defibrotide to demonstrate its association with cell surface receptors. These studies have identified binding sites on endothelial cells that mediate its protective effects. The compound's interactions with tissue plasminogen activator and thrombomodulin have also been characterized using standard enzyme activity assays.

In vitro cellular studies are conducted using endothelial cell cultures (e.g., human umbilical vein endothelial cells, HUVECs). Cells are treated with defibrotide at various concentrations (typically μg/mL to mg/mL ranges) and exposed to damaging agents such as chemotherapy drugs, oxidative stress, or inflammatory cytokines. Endpoints include cell viability, apoptosis, expression of adhesion molecules, and release of inflammatory mediators. Endothelial barrier function is assessed by measuring permeability or transendothelial electrical resistance.

Defibrotide can be dissolved in saline for in vivo studies [2]

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1. Administration of bovine thrombin (100 u kg-1) into the carotid artery of rabbits induces a sustained accumulation of 111 Indium-labelled platelets within the cranial vasculature over the subsequent 3 h.
2. Intracarotid (i.c.) administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.c. thrombin (100 u kg-1) significantly reduces the ability of thrombin to induce cranial thromboembolism in rabbits.
3. Intravenous (i.v.) administration of thrombin (20 u kg-1) in rabbits induces a reversible accumulation of radiolabelled platelets into the thoracic circulation which is significantly reduced by i.v. administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.v. thrombin. In contrast, platelet accumulation in response to adenosine diphosphate (ADP; 20 micrograms kg-1, i.v.) or platelet activating factor (PAF; 50 ng kg-1, i.v.) is not significantly affected by this treatment.
4. Intravenous administration of the nitric oxide (NO)-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg kg-1) potentiates platelet accumulation induced by low dose thrombin (10 u kg-1, i.v.) within the pulmonary vasculature of rabbits. The potentiated response is significantly abrogated following pretreatment with defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h, i.v.).
5. Intravenous injection of human thrombin (1250 u kg-1) to mice induces death within the majority of animals which is significantly reduced by pretreatment with defibrotide (150-175 mg kg-1, i.v.). In contrast, death induced by i.v. collagen (1.25 mg kg-1) plus adrenaline (75 microg kg-1) is not significantly affected by defibrotide pretreatment.
6. The inhibitory effect of defibrotide in mice is abolished following concomitant treatment with the inhibitor of fribrinolysis, tranexamic acid (100 mg kg-1, i.v.), but is unaffected following treatment with the cyclo-oxygenase inhibitor, aspirin (300 mg kg-1, i.p.).
7. The protective effect of defibrotide against thrombin-induced thromboembolism in the mouse is potentiated by recombinant tissue-plasminogen activator (rt-PA; 1 mg kg-1, i.v.) or unfractionated heparin (10 u kg-1, i.v.) administration.
8. The results suggest that defibrotide may possess antithrombotic activity on thrombin-induced thromboembolism which, at least in the mouse, may be partially mediated via induction of the fibrinolytic pathway[2].
In vivo animal experiments are performed in rodent models of VOD, ischemia-reperfusion injury, and inflammatory conditions. Defibrotide is administered via intravenous injection at various doses. Endpoints include survival, liver function tests, histopathological examination of liver tissue, and measurement of inflammatory markers. In VOD models, the compound has been shown to reduce sinusoidal obstruction and improve liver function. Pharmacodynamic studies evaluate its effects on coagulation, fibrinolysis, and endothelial function.

Absorption, Distribution and Excretion
The bioavailability after oral administration is 58-70%, while the bioavailability after parenteral administration (intravenous and intramuscular injection) is 100%. Biological Half-Life t1/2-α = minutes (10-20 minutes in rats); t1/2-β = several hours
Pharmacokinetic properties of defibrotide include a molecular weight range of 13,000-20,000 Da for the oligonucleotide mixture. The compound is administered intravenously and has a half-life of several hours. It is metabolized primarily in the liver and excreted via the kidneys. The compound is soluble in water and saline (e.g., >5 mg/mL). Storage as powder at -20°C is recommended for long-term stability. Clinical dosing is typically based on body weight.

Hepatotoxicity
Diffluride treatment was not associated with elevated serum transaminases or clinically significant liver injury other than the SOS characteristics associated with its treatment. In a trial of diffluride for SOS prevention in 356 children who underwent hematopoietic stem cell transplantation (HCT), the incidence of serious adverse events such as bleeding, gastrointestinal upset, and liver injury was similar between children treated with diffluride and those who did not. Probability Score: E (Unlikely to be a cause of clinically significant acute liver injury).
Toxicological data for defibrotide from clinical trials indicate that it is generally well-tolerated. The most common adverse events include hypotension, diarrhea, nausea, and vomiting. Hemorrhagic complications are rare but have been reported. No significant systemic anticoagulant effects are associated with defibrotide use. The compound is contraindicated in patients with hypersensitivity to defibrotide or any of its components. Use in pregnancy and lactation should be considered only if the potential benefit justifies the potential risk.

[1]. Defibrotide: An Oligonucleotide for Sinusoidal Obstruction Syndrome. Ann Pharmacother. 2018 Feb;52(2):166-174.

[2]. The effect of defibrotide on thromboembolism in the pulmonary vasculature of mice and rabbits and in the cerebral vasculature of rabbits. Br J Pharmacol. 1993 Dec;110(4):1565-71.

Difubrotide is a sodium salt of a mixture of single-stranded oligodeoxynucleotides derived from porcine mucosal DNA. It has been shown to possess antithrombotic, anti-inflammatory, and anti-ischemic properties (but no significant systemic anticoagulant activity). It is marketed in several countries under the trade names Dasovas (FM), Noravid, and Prociclide. In the United States, it was approved in March 2016 under the trade name Defitelio. Difubrotide is a complex mixture of single-stranded oligodeoxynucleotides derived from porcine intestinal mucosa with antithrombotic and fibrinolytic activities, used to treat severe sinusoidal obstruction syndrome (SOS) following hematopoietic stem cell transplantation (HCT). Difubrotide has been used to treat patients with severe liver injury, and no worsening of serum transaminase elevations or clinically significant specific liver injury cases were observed during treatment. Difubrotide is a mixture of single-stranded oligodeoxynucleotides derived from porcine intestinal mucosa with antithrombotic, thrombolytic, and fibrinolytic activities. Following administration, although its exact mechanism of action is not fully elucidated, difrubitide induces the release of prostaglandin I2 (PGI2), E2 (PGE2), and prostacyclin, and reduces the expression of adhesion molecules on endothelial cells. This relaxes vascular smooth muscle and prevents platelets from adhering to each other and to endothelial cells, thereby protecting the vascular endothelium. Difrubitide increases tissue-type plasminogen activator (t-PA) activity and decreases plasminogen activator inhibitor-1 activity. This product enhances plasmin activity, prevents thrombus formation, and dissolves thrombi.
See also: Difrubitide sodium (note moved to).

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Indications
Indications for the treatment of severe hepatic venous occlusion (VOD), also known as sinusoidal obstruction syndrome (SOS), following hematopoietic stem cell transplantation (HSCT) with renal or pulmonary dysfunction.
FDA Label

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Mechanism of Action
This product appears to prevent thrombosis and help dissolve thrombi by increasing the levels of prostaglandin I2, E2, and prostacyclin, altering platelet activity, enhancing the function of tissue plasminogen activator (TPA), and decreasing the activity of TPA inhibitors. Prostaglandin I2 relaxes vascular smooth muscle and prevents platelets from adhering to each other. Prostaglandin E2 also inhibits platelet aggregation at certain concentrations. Furthermore, recent studies have shown that this drug also has additional anti-inflammatory and anti-ischemic effects. However, it is currently unclear whether these effects can be applied clinically (e.g., for the treatment of ischemic stroke).
Defibrotide sodium is also known as Defitelio (brand name), Dasovas, Noravid, and Prociclide. It has the UNII identifier L7CHH2B2J0. The compound is supplied as a powder and should be stored under recommended conditions. It is indicated for the treatment of severe hepatic veno-occlusive disease (VOD/SOS) with renal or pulmonary dysfunction following hematopoietic stem-cell transplantation. The recommended dose is 6.25 mg/kg every 6 hours administered as a 2-hour intravenous infusion.

13-20 (KDa)

83712-60-1

83712-60-1(sodium);1118915-78-8 (free acid);1402907-09-8 (disodium);1118915-79-9 (monosodium);

135565962

Powder form

4

7

5

31

773

0

Defibrotide;1118915-78-8; UNII-E1942E7K32; STA-1474; UNII-E1942E7K32; 5-Hydroxy-2-isopropyl-4-(4-(1-methyl-1H-indol-5-yl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl dihydrogen phosphate; E1942E7K32; Defibrotide sodium

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Soluble in H2O or saline (e.g. >5 mg/mL)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)