Claims for Patent: 11,236,328

Summary for Patent: 11,236,328

Title:	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.
Inventor(s):	Ignoni; Terenzio (Solbiate, IT), Kumar; Vijay (Casnate, IT), Islam; Khalid (Massagno, IT)
Assignee:	GENTIUM S.R.L. (Villa Guardia, IT)
Application Number:	17/459,169
Patent Claims:	1. A method of treating Veno-Occlusive Disease (VOD) comprising administering to a patient in need thereof 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, 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 potency of the defibrotide. 2. The method of claim 1, wherein the formulation has a potency of 27.5 to 32.5 IU/mg. 3. The method of claim 2, wherein the formulation has a potency of 28 to 32 IU/mg. 4. The method of claim 1, wherein the formulation is a water solution. 5. The method of claim 4, wherein the formulation has a pH of from 6.5 to 8.5. 6. The method of claim 5, wherein the formulation has a pH of from 7 to 8. 7. The method of claim 1, wherein the euglobulin is human, rabbit or bovine euglobulin. 8. The method of claim 1, wherein plasmin which reacts with the substrate specific for plasmin is released by plasminogen contained in the mammalian euglobulin. 9. The method of claim 1, wherein the substrate specific for the plasmin is a chromogenic substrate. 10. The method of claim 1, 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, A3 is lysine or arginine and X is the measurable product. 11. The method of claim 10, wherein the measurable product X is selected from the group consisting of para-nitroaniline and 2-naphthylamine. 12. The method of claim 10, wherein the substrate specific for plasmin is H-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline. 13. The method of claim 10, wherein the measurable product X is measured by spectrophotometry or spectrofluorimetry. 14. The method of claim 1, wherein the mammalian euglobulin 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 method of claim 1, wherein said method of determining the defibrotide potency is carried out in a reaction medium which is an aqueous solution buffered to a pH of from 7 to 8. 16. The method of claim 1, wherein the method of determining the defibrotide potency is maintained at a temperature of from 35 to 39.degree. C. 17. The method of claim 1, wherein the substrate specific for plasmin has a concentration of from 0.3 to 4 mM. 18. The method of claim 1, wherein the method of determining the defibrotide potency 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 method of claim 17, wherein the concentration of the substrate specific for plasmin is 3 mM.