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 27.5 to 32.5 IU/mg and, more preferably, from 28 to 32 IU/mg.

1 - 20 . (canceled) 
     
     
         21 . A liquid defibrotide formulation having a biological activity of about 25 to about 35 IU/mg, and a concentration of at least 80 mg/mL, wherein the defibrotide biological activity can be determined at concentrations of 1000 μg/mL or more, wherein the biological activity is determined by a method comprising the steps of:
 a) bringing into contact defibrotide, mammalian euglobulin 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 biological activity of the defibrotide. 
 
     
     
         22 . The liquid defibrotide formulation of  claim 21 , wherein the formulation has a biological activity of about 27.5 to about 32.5 IU/mg. 
     
     
         23 . The liquid defibrotide formulation of  claim 22 , wherein the formulation has a biological activity of about 28 to about 32 IU/mg. 
     
     
         24 . The liquid defibrotide formulation of  claim 21 , wherein the formulation is a water solution. 
     
     
         25 . The liquid defibrotide formulation of  claim 24 , wherein the formulation has a pH of from about 6.5 to about 8.5. 
     
     
         26 . The liquid defibrotide formulation of  claim 25 , wherein the formulation has a pH of from about 7 to about 8. 
     
     
         27 . The liquid defibrotide formulation of  claim 21 , wherein the euglobulin is human, rabbit or bovine euglobulin. 
     
     
         28 . The liquid defibrotide formulation of  claim 21 , wherein plasmin which reacts with the substrate specific for plasmin is released by the plasminogen contained in euglobulin. 
     
     
         29 . The liquid defibrotide formulation of  claim 21 , wherein the substrate specific for the plasmin is a chromogenic substrate. 
     
     
         30 . The liquid defibrotide formulation of  claim 21 , wherein the substrate specific for the plasmin is a compound of formula A1-A2-A3-X in which A1 and A2 are non-polar amino acids, A 3  is lysine or arginine and X is the measurable product. 
     
     
         31 . The liquid defibrotide formulation of  claim 30 , wherein the measurable product X is selected from the group consisting of para-nitroaniline and 2-naphthylamine. 
     
     
         32 . The liquid defibrotide formulation of  claim 30 , wherein the substrate specific for plasmin is H-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline. 
     
     
         33 . The liquid defibrotide formulation of claim  130 , wherein the measurable product X is measured by spectrophotometry or spectrofluorimetry. 
     
     
         34 . The liquid defibrotide formulation of  claim 21 , wherein the mammalian euglobulin is 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 about 2.5 to about 3.5 mM. 
     
     
         35 . The liquid defibrotide formulation of  claim 21 , wherein said method is carried out in a reaction medium which is an aqueous solution buffered to a pH of from about 7 to about 8. 
     
     
         36 . The liquid defibrotide formulation of  claim 21 , wherein the temperature is maintained at from about 35 to about 39° C. 
     
     
         37 . The liquid defibrotide formulation of  claim 21 , wherein the concentration of the substrate specific for plasmin is from about 0.3 to about 4. 
     
     
         38 . The liquid defibrotide formulation of  claim 21 , wherein the method comprises the steps of: c) determining the rate of release of the measurable product during the course of the enzymatic reaction of both a standard sample and a test sample; d) correlating the rate of release with the corresponding defibrotide concentration to obtain the biological activity of the test sample of defibrotide. 
     
     
         39 . The liquid defibrotide formulation of  claim 37 , wherein the concentration of the substrate specific for plasmin is about 3 mM.