Euglobulin-based method for determining the biological activity of defibrotide

Abstract

It is disclosed a method for determining the biological activity of defibrotide, which comprises the steps of: a) bringing into contact defibrotide, mammalian euglobulin and a substrate specific for the plasmin which, by reaction with the plasmin, provides a measurable product; and b) measuring the amount of product formed at successive times, to thereby determine the biological activity of the defibrotide. Liquid defibrotide formulations are also disclosed, preferably water solutions, having a defined biological activity and, in particular, having an activity of 25 to 35 IU/mg of defibrotide, preferably from 27 to 32 IU/mg and, more preferably, from 28 to 32 IU/mg.

1 . A defibrotide formulation consisting of defibrotide, sodium citrate, and water for injection, having a potency of 25 to 35 IU/mg, and a concentration of at least 80 mg/mL, wherein the defibrotide potency is determined by a method comprising the steps of :
 a) bringing into contact defibrotide, an enzymatic system, and a substrate specific for plasmin which, by reaction with plasmin, provides a measurable product; and   b) measuring the amount of product formed at successive times, to thereby determine the potency of the defibrotide.   
     
     
         2 . The defibrotide formulation of  claim 1 , wherein the formulation has a potency of 27.5 to 32.5 IU/mg. 
     
     
         3 . The defibrotide formulation of  claim 2 , wherein the formulation has a potency of 28 to 32 IU/mg. 
     
     
         4 . The defibrotide formulation of  claim 1 , wherein the formulation is a water solution. 
     
     
         5 . The defibrotide formulation of  claim 4 , wherein the formulation has a pH of from 6.5 to 8.5. 
     
     
         6 . The defibrotide formulation of  claim 5 , wherein the formulation has a pH of from 7 to 8. 
     
     
         7 . The defibrotide formulation of  claim 1 , wherein the enzymatic system is selected from euglobulin fraction, diluted plasma and serum, artificially created plasminogen, and isolated plasminogen. 
     
     
         8 . The defibrotide formulation of  claim 1 , wherein plasmin which reacts with the substrate specific for plasmin is released by the enzymatic system. 
     
     
         9 . The defibrotide formulation of  claim 1 , wherein the substrate specific for the plasmin is a chromogenic substrate. 
     
     
         10 . The defibrotide formulation of  claim 1 , wherein the substrate specific for the plasmin is a compound of formula A1-A 2 -A 3 -X in which A1 and A 2  are non-polar amino acids, A 3  is lysine or arginine and X is the measurable product. 
     
     
         11 . The defibrotide formulation of  claim 10 , wherein the measurable product X is selected from the group consisting of para-nitroaniline and 2-naphthylamine. 
     
     
         12 . The defibrotide formulation of  claim 10 , wherein the substrate specific for plasmin is H-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline. 
     
     
         13 . The defibrotide formulation of  claim 10 , wherein the measurable product X is measured by spectrophotometry or spectrofluorimetry. 
     
     
         14 . The defibrotide formulation of  claim 1 , wherein the enzymatic system is obtained from a volume of plasma and reconstituted to the same volume of the originating plasma or diluted up to 1:10 with suitable buffer and the substrate specific for the plasmin is a chromogenic/fluorogenic substrate having a concentration of from 2.5 to 3.5 mM. 
     
     
         15 . The defibrotide formulation of  claim 1 , wherein said method is carried out in a reaction medium which is an aqueous solution buffered to a pH of from 7 to 8. 
     
     
         16 . The defibrotide formulation of  claim 1 , wherein the method is maintained at a temperature of from 35 to 39° C. 
     
     
         17 . The defibrotide formulation of  claim 1 , wherein the substrate specific for plasmin has a concentration of from 0.3 to 4 mM. 
     
     
         18 . The defibrotide formulation of  claim 1 , wherein the method comprises the steps of: c) determining the rate of release of the measurable product during the course of the reaction of both a standard sample and a test sample of defibrotide; d) correlating the rate of release with the corresponding defibrotide concentration to obtain the potency of the test sample of defibrotide. 
     
     
         19 . The defibrotide formulation of  claim 17 , wherein the concentration of the substrate specific for plasmin is 3 mM.