In a preferred embodiment of the present invention, the standard solutions and the solutions of the samples of defibrotide to be determined are introduced into the respective wells of the microplate. The euglobulin fraction are prepared at the moment of use and it is the dilution media of defibrotide stock solution. Finally, the solution containing the chromogenic substrate is added. The microplate is then placed in the thermostated reader and, after rapid agitation, readings of the system's absorbance are taken at predetermined intervals and for the predetermined period of time. The raw data obtained are then processed, thus determining the unknown activities of the defibrotide samples.

As it shall be appreciated from the following examples, the method according to the present invention allows to obtain liquid defibrotide formulations, 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.

Liquid defibrotide formulations are preferably marketed in form of containers, more preferably vials, containing 200 mg of defibrotide in 2.5 ml of buffered water solution (preferably at a pH from 6.5 to 8.5, more preferably from 7 to 8), to be diluted before use; consequently, when the biological activity is assessed with the method of the present invention, each container presents a defibrotide activity of 5000 to 7000 IU, preferably 5500 to 6500 IU, more preferably 5600 to 6400.

Those and other aspects of the invention will be better illustrated in the following examples which are not, however, to be regarded as limiting the invention.

EXAMPLES

The following materials were used in the examples given here:

Apparatus

Main features:
Microplate reader for UV-Vis absorbance

determination equipped with thermostatic

chamber and absorbance filter at 405 nm.

Detection
Kinetic Absorbance determination at 405 nm

Plate Type
96-well clear for UV-Vis Determination

Chamber
37° C.
Total Absorbance recording
45-60 min

Temperature

time

Absorbance
About 1 × min

recording frequency

Programs & Software

- Microsoft Excel® (Microsoft Corporation, Redmond, Wash., USA)
  
  Substances
- Defibrotide (Gentium)
- Chromogenic substrate S-2251 (Chromogenix Instrumentation Laboratory S.p.A., Milan, Italy)
- Tris(hydroxymethyl) aminomethane (TRIS)-NaCl, (Sigma-Aldrich, Milan, Italy)
- 1N HCl (Carlo Erba reagenti, Milan, Italy)
- IN NaOH (Carlo Erba reagenti, Milan, Italy)
- Bovine Plasma (Tebu Bio Italia, Magenta (MI), Italy)
- Glacial Acetic Acid (Carlo Erba reagenti, Milan, Italy)
  
  Solutions
- TRIS-NaCl (1 L Preparation): Into a 1 L beaker quantitatively transfer 6.06 g of TRIS-HC1 and 2.2 g of NaCl. Dissolve in 500 mL of purified water and adjust the pH to 7.4 with about 40 mL of HC1 1N. Quantitatively transfer the solution into a volumetric flask of 1 L and dilute the solution to volume with purified water. Store the solution into refrigerator (2-8° C.)
- Chromogenic Substrate S2251 (CAS 63589-93-5) 3 mM (15.2 mL Preparation): Dissolve about 25 mg of chromogenic substrate with 15.2 mL of purified water. Store the solution into refrigerator (2-8° C.)
- Bovine euglobulins (10 mL Preparation). In a container with a minimum capacity of 300 mL introduce 240 mL of ice-cooled purified water and under stirring add 10 mL bovine plasma. Adjust the pH to 5.3±0.1 with acetic acid 1%. Allow to settle at 2-8° C. for 1 to 16 hours. Remove the clear supernatant solution by siphoning and collect the precipitate by centrifugation at 2.800 rpm for 5 minutes and 4° C. Suspend the precipitate dispersing mechanically (e.g., by means of a laboratory glass rod) in 5 mL of ice-cooled purified water, shake for about 5 min and collect the precipitate by centrifugation at 2.800 rpm for 5 minutes and 4° C. Disperse the precipitate mechanically into about 10 mL of TRIS-NaCl; to facilitate the dissolution of the precipitate crush the particles of the precipitate with a suitable instrument (es.: laboratory glass rod). Store the obtained suspension at 2-8° C. for not less than 1 hour and not more than 6 hours before its use.
  
  Standard and Sample Defibrotide Solutions
  
  Reference Stock Solution

Into 20 mL volumetric flask quantitatively transfer about 100 mg of defibrotide reference standard accurately weighed. Dissolve the powder with about 10 mL TRIS-NaCl and bring the volume with the same solvent. Dilute 1:4 the obtained solution with TRIS-NaCl in order to obtain a defibrotide RS solution of about 1.25 mg/mL.

Sample Stock Solution

Into 20 mL volumetric flask quantitatively transfer about 100 mg of defibrotide sample accurately weighed. Dissolve the powder with about 10 mL TRIS-NaCl and bring the volume with the same solvent. Dilute 1:4 the obtained solution with TRIS-NaCl in order to obtain a defibrotide sample solution of about 1.25 mg/mL.

Reference and Sample Solutions Preparation

- a) Defibrotide 125 μg/mL: dilute 1:10 defibrotide stock solution (Reference and Sample) with TRIS NaCl (corresponding to 50 μg/mL into the plate well). Into an eppendorf tube quantitatively transfer 500 μL of the prepared solution and mix with 500 μL of euglobulin. Close the tube and store in ice-cooled water.
- b) Defibrotide 62.5 μg/mL: dilute 1:2 solution (a) with TRIS NaCl (corresponding to 25 μg/mL into the plate well), Into an eppendorf tube quantitatively transfer 500 μL of the prepared solution and mix with 500 μL of euglobulin. Close the tube and store in ice-cooled water.
- c) Defibrotide 31.25 μg/mL: dilute 1:2 solution (b) with TRIS NaCl (corresponding to 12.5 μg/mL into the plate well). Into an eppendorf tube quantitatively transfer 500 μL of the prepared solution and mix with 500 μL of euglobulin. Close the tube and store in ice-cooled water.
- d) Defibrotide 12.5 μg/mL: dilute 1:2.5 solution (d) with TRIS NaCl (corresponding to 5 μg/mL into the plate well). Into an eppendorf tube quantitatively transfer 500 μL of the prepared solution and mix with 500 μL of euglobulin. Close the tube and store in ice-cooled water.
  
  Blank Solution

Mix 1 volume of euglobulins with 1 volume of TRIS NaCl solution (ex.: 500 μL+500 μL)

Plate Deposition

According to the proposed deposition scheme (see Table 1 below) add in each well of the plate 200 μL of standard or sample or blank solution. Note different deposition scheme can be used according to the availability and configuration of automatic pipettes. However not less than 4 depositions for each reference and sample solution must be used for the assay.

Immediately before incubation of the plate into the microplate reader add in each well 50 μL of Chromogenic substrate.

TABLE 1

1
2
3
4
5
6
7
8
9
10
11
12

A
—
BLK
S1_Ca
S1_Cb
S1_Cc
S1_Cd
—
—
—
—
—
—

B
—
BLK
S2_Ca
S2_Cb
S2_Cc
S2_Cd
—
—
—
—
—
—

C
—
BLK
S3_Ca
S3_Cb
S3_Cc
S3_Cd
—
—
—
—
—
—

D
—
BLK
S4_Ca
S4_Cb
S4_Cc
S4_Cd
—
—
—
—
—
—

E
—
BLK
U1_Ca
U1_Cb
U1_Cc
U1_Cd
—
—
—
—
—
—

F
—
BLK
U2_Ca
U2_Cb
U2_Cc
U2_Cd
—
—
—
—
—
—

G
—
BLK
U3_Ca
U3_Cb
U3_Cc
U3_Cd
—
—
—
—
—
—

H
—
BLK
U4_Ca
U4_Cb
U4_Cc
U4_Cd
—
—
—
—
—
—

S1, S2, S3, S4: Reference Solution deposition 1, deposition 2, deposition 3, deposition 4, U1, U2, U2, U5: Sample solution deposition 1, deposition 2 deposition 3, deposition 4, Ca, Cb, Cc etc.: Defibrotide reference and sample concentration a, b, c etc.

BLK Blank Solution

Calculation and Results

From the kinetic plot “absorbance versus time” of the standard preparations (ex.: S1_Ca, S1_Cb, S1_Cc) identify a suitable linear range (ex.: from 30 to 35 min, see FIG. 3).

Identification of the linear kinetic range (A@405 nm vs time).

Calculate for each preparation of the standard and of the sample the response of the assay (Slope) in the pre-defined time range as follow.

Sample

&

⁢

Standard
⁢

Response

=

A
b

-

A
a

T
b

-

T
a

×
1
⁢
0
⁢
0
⁢
0

Where:

Aa is the Absorbance values at Time Ta (30 min from the plot above)

Ab is the Absorbance value at Time Tb (35 min from the plot above)

Report the obtained value in a tabular format as reported in table 2.

TABLE 2

Concentration
Standard Preparation