Method for determining the biological activity of defibrotide
Method for determining the biological activity of defibrotide by a) bringing into contact defibrotide, plasmin 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.
1. A method for determining the biological activity of defibrotide, which comprises the steps of:
a) bringing into contact defibrotide, plasmin 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.
2. The method according to claim 1 , wherein the plasmin is a mammalian plasmin.
3. The method according to claim 2 , wherein the plasmin is human plasmin.
4. The method according to claim 1 , wherein the substrate specific for the plasmin is a compound of formula
A 1 -A 2 -A 3 -X
in which A 1 and A 2 are non-polar amino acids, A 3 is lysine or arginine and X is the measurable product.
5. The method according to claim 4 , wherein the measurable product X is selected from the group consisting of para-nitroaniline and 2-naphthylamine.
6. The method according to claim 4 , wherein the substrate for the plasmin is H-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline.
7. The method according to claim 4 , wherein the measurable product X is measured by spectrophotometry.
8. The method according to claim 1 , wherein the plasmin has a concentration of from 0.0064 to 0.050 I.U./ml and the substrate for the plasmin has a concentration of from 2.5 to 3.5 mM.
9. The method according to claim 8 , wherein the concentration of plasmin is 0.0125 I.U./ml and the concentration of the substrate for the plasmin is 3 mM.
10. The method according to claim 1 , wherein the reaction is carried out in a reaction medium which is an aqueous solution buffered to a pH of from 7 to 8.
11. The method according to claim 10 , wherein the reaction medium is an aqueous solution buffered to a pH of 7.4.
12. The method according to claim 1 , wherein the temperature is maintained at from 35 to 39° C.
13. The method according to claim 12 , wherein the temperature is maintained at 37° C.
14. The method according to claim 1 , wherein the concentration of the substrate for the plasmin is from 0.3 to 4 mM.
15. The method according to claim 14 , wherein the concentration of the substrate for the plasmin is from 2.5 to 3.5 mM.
16. The method according to claim 14 , wherein the concentration of the substrate for the plasmin is 3 mM.
17. A method for determining the biological activity of defibrotide according to claim 1 , which method comprises the steps of:
a) 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;
b) correlating, mathematically and/or graphically, the rate of release with the corresponding defibrotide concentration to obtain the biological activity of the test sample of defibrotide.