TWI615473B - Euglobulin-based method for determining the biological activity of defibrotide - Google Patents

Abstract

The invention discloses a method for determining the biological activity of a defibrotide polynucleotide, comprising the steps of: a) contacting a mammalian euglobulin and a matrix specific for plasmin to defibrotide polynucleoside An acid, the matrix provides a measurable product by interaction with the plasmin; and b) measuring the amount of product produced in a continuous period of time to determine the biological activity of the defibrotide polynucleotide. The present invention also discloses a liquid defibrotide polynucleotide formulation which is preferably an aqueous solution having defined biological activity, and particularly having a defibrotide activity of from 25 to 35 IU/mg, preferably from 27.5 to 32.5 IU/mg, and more preferably 28 to 32 IU/mg.

Description

Eulogous globulin-based method for determining the biological activity of defibrotide polynucleotides

The present invention relates to a method for determining the biological activity of a defibrotide polynucleotide, and more particularly to an indirect enzyme method for determining the biological activity of a defibrotide polynucleotide.

The defibrotide polynucleotide (Merck Index, 1996, no. 2915) is a naturally occurring substance which is extracted from an animal organ and which is composed of a low molecular weight polydeoxyribonucleotide sodium salt. Defibrotide has become the subject of many medical studies, which have been speculated to be useful as an anticoagulant (U.S. Patent No. 3,829,567).

In addition, defibrotide polynucleotides have also been used to treat peripheral arterial vascular disease, acute renal insufficiency (U.S. Patent No. 4,694,134) or acute myocardial ischemia (U.S. Patent No. 4,693,995).

Currently, defibrotide polynucleotides are being used in clinical trials for the treatment and prevention of venous obstructive disease (VOD).

Like other extracted biological substances, defibrin The variability of the composition of polynucleotides is limited, which is typical of typical natural biopolymers. A classic example of this condition is heparin, which is known to vary in chain length, molecular weight, composition, degree of thiophenolation, and the like, depending on the batch. The result is that the same weight of defibrotide polynucleotide may actually have a non-equal specific biological activity.

Due to its inherent biopolymer nature, processes for extraction, separation and purification do not ensure absolute reproducibility of the product.

However, if the control is good, this variability may be reduced. To this end, research has been conducted to obtain a standardized industrial process for the separation of defibrotide polynucleotides from organ extraction, such as those described in U.S. Patent No. 4,985,552.

The products obtained by the above processes are characterized by measuring specific physicochemical parameters such as electrophoretic mobility, extinction coefficient, optical rotation, and mass average relative molecular mass. However, these parameters essentially depend on the structure of the defibrotide polynucleotide and do not provide information on its biological activity.

As far as we know, the only methods reported for assessing the biological activity of defibrotide polynucleotides are fibrin plate assay and thromboelastographic recording (Prino G., Mantovani). M., Niada R., Coccheri S., Butti A., Indagini preliminari sull ' attività fibrinolitica, nell ' animale e nell ' uomo and di una nuova sostanza diversi organi animali, Simposio Internazionale: La ricerca scientifica nell'industria farmaceutica , Orthodox, Rome, October 2-4, 1975 - Il Farmaco, Ed. Prat.) (1969), 24, 552-561), and the plasmin-based method disclosed in U.S. Patent No. 7,338,777. The above are all incorporated into the prior art of this case.

However, in the above methods, the characteristics of thromboelastographic recording are considerable experimental complexity, insufficient reproducibility and precision, and, in some specific examples, coagulation elasticity. The linearity of the reaction recorded by the schema is limited to a very tight concentration range.

With regard to plasmin-based methods, the activity of plasmin is usually determined by various standards in in vitro tests. One of the most commonly used methods is the determination of chromogenic or fluorescent compounds by spectrophotometry or spectroscopic spectroscopy, which are released by the action of plasmin on a suitable substrate (Haemostasis, (1978). ), 7, 138-145). A peptide matrix having the formula A ₁ -A ₂ -A ₃ -X is generally used, wherein A ₁ and A ₂ are predominantly non-polar amino acids, A ₃ is a peracid or arginine, and X represents The released compound is measured, for example, para-nitroaniline (pNa) or 2-naphthylamine (NA) (Haemostasis, (1978), 7, 146-149). In addition to the above peptide matrix, other simpler compounds have been successfully used, such as p-nitrobenzyl-p-toluenesulphonyl-L-arginine (Haemosta) Sis, (1978), 7, 105-108).

The rate at which the compound X is released into the medium is proportional to the plasmin activity (units/mg) present in the sample. The method disclosed in U.S. Patent No. 7,338,777 is based on the aforementioned fibrin In the lysozyme evaluation test, defibrotide increases the release rate of Compound X proportionally to its concentration.

However, this method is performed in TRIS buffer and does not require any other plasma/serum activator/inhibitor. Therefore, the program does not reflect physiological conditions or properly stimulate the mechanism of action of defibrotide in vivo.

Thus, to date, no effective, accurate, and reproducible methods have been described and are effective in measuring the biological activity of defibrotide polynucleotides, while accurately reflecting the mechanism of action of the product in complex biological systems (in vivo). .

We have established simple and reliable methods for determining the biological activity of defibrotated polynucleotides such that the extracted samples are controlled and thus standardized for defibrotide-based drug preparation.

The method of the present invention can determine the specific biological activity of a defibrotide polynucleotide, and is highly accurate and correct compared to a reference standard.

The present invention relates to a specific biological activity for determining a defibrotide polynucleotide sample, the method comprising the steps of: a) contacting a euglobulin and a matrix specific for plasmin to defibrotide polynucleoside An acid that provides a measurable product by interaction with the plasmin; and b) measures the amount of product formed over a continuous period of time.

The invention is an indirect in vitro method for determining The biological activity of fibrin polynucleotides is based on the functional interaction between defibrotide polynucleotides and euglobulin.

Euglobulin is a fraction of serum globulin that is insoluble in distilled water but soluble in saline solution.

Euglobulin contains fibrinogen, PAI-1, tissue plasminogen activator (tPA), plasminogen, and a small amount of α2-antiplasmin (alpha 2-antiplasmin) And factor VIII.

The inventors of the present invention unexpectedly discovered that the defibrotide polynucleotide catalyzes the hydrolysis of plasminogen to plasmin. Thus, when the defibrotide polynucleotide is cultured with a euglobulin and a matrix specific for plasmin, wherein the matrix is, for example, Haemostasis, (1978), 7, 138-149, the formula A ₁ -A ₂ -A ₃ -X peptide, which is incorporated herein by reference, the rate of release of the compound X to the medium is proportional to the concentration of the defibrotide.

In other words, defibrotide catalyzes the hydrolysis of plasminogen contained in euglobulin to plasmin; plasmin is reacted with a matrix specific for plasmin. To provide a measurable product, wherein the substrate is preferably a chromogenic substrate.

The method of the present invention further comprises the step c) determining the release rate of the measurable product during the enzyme reaction between the standard sample of the defibrotide polynucleotide and the test sample; d) mathematically and/or graphically Indicates the association between the release rate and the corresponding defibrotide concentration to obtain a test sample of the defibrotide polynucleotide. Biological activity.

The sample of the defibrotide polynucleotide used in the present invention is generally extracted from an organ according to a known process, and the process is described, for example, in U.S. Patent No. 4,985,552, which is incorporated herein by reference. ,.

According to the present invention, a batch of normal industrially produced defibrotide polynucleotides is selected as a reference sample (standard) and used to prepare a calibration curve.

In general, the methods of the present invention provide accurate and accurate measurement of defibrotide polynucleotides, even in the presence of contaminants, such as RNA, heparin, degraded defibrotide polynucleotides (purine and pyrimidine removed) Defibrotide) or ethanol, usually less than 10% by weight, does not damage the system.

In addition to the determination of defibrotide polynucleotides, the methods of the invention may also measure other organism-derived matrices derived from defibrotated polynucleotides, such as deaminated defibrotide polynucleotides, or more simply a defibrotide polynucleotide that is denatured/degraded by physicochemical conditions.

The sensitivity of the method of the present invention is sufficient to detect defibrotide at a concentration (final concentration in the assay system) of less than or equal to 2.5 μg/ml, and generally exhibits a good correlation up to a maximum concentration value of greater than or equal to 1000 μg/ml.

The euglobulin used is usually any mammalian euglobulin moiety, such as bovine, porcine, rabbit or human euglobulin, preferably using human and bovine euglobulin.

However, although the euglobulin moiety is the selected enzyme system, other equivalent enzyme systems are used, such as diluted plasma and serum (diluted up to 1:10 in buffer), artificially formed or isolated plasminogen It is within the scope of the invention that tPA, uPA, PAI-1 & 2 are similar to α2-antiplasmin and chemically and biologically related enzyme systems having similar functions.

In the method of the invention, the matrix of plasmin is understood to be any matrix specific for plasmin, under which conditions the detectable hydrolysate X is released.

Depending on the nature of the detectable functional group X, alternative detection systems well known to those skilled in the art can also be employed. Detection systems for spectrophotometric or fluorescent measurements are particularly advantageous, especially for spectrophotometric systems.

The commonly used matrix is a matrix specific for plasminogen-plasmin analysis. Preferably, a peptide of the formula A ₁ -A ₂ -A ₃ -X is used, wherein A ₁ and A ₂ are mainly non-polar amino acids, A ₃ is a peracid or arginine, and X represents measurable Functional group. These matrices are, for example, Val-Leu-Lys-pNa, Val-Phe-Lys-pNa or pyroGlu-Phe-Lys-pNa, wherein the functional group X is p-nitroaniline (pNA) detectable by a spectrophotometer. Other suitable matrices, such as Val-Gly-Arg-2NA, comprise 2-naphthylamine, which can be detected by fluorometry. A particularly preferred substrate is HD-Amidino-L-Acetyl-L-Iso-P-Nitroaniline (HD-Val-Leu-Lys-pNA) Compound.

The particular matrix used to determine the defibrotide activity of the euglobulin moiety is generally commercially available.

The assay of the invention consists in placing a defibrotide polynucleotide sample in a euglobulin solution of a specific pH and molar concentration.

In particular, the euglobulin portion of mammalian plasma is reconstituted by dissolving and diluting the euglobulin in a saline buffer to produce the original volume of plasma (eg, euglobulin obtained from 10 ml of plasma) Partly, it was dissolved in a salt buffer solution and reformed to 10 ml with a pH between 7 and 8).

However, the chromogenic substrate is usually used at a concentration of 0.3 to 4 mM, preferably 2.5 to 3.5 mM, and more preferably 3 mM, and the fluorescent substrate is used at a concentration of 0.05 to 0.15 mM.

As with other enzyme methods, the assay of the invention is sensitive to the pH of the medium.

In fact, it is usually not used for extreme pH values because the enzyme system is deactivated.

Preferably, the pH of the medium does not change at any time during the measurement, and therefore, the euglobulin fraction is reconditioned with a buffer system typically used for biological testing. A suitable buffer system can be, for example, a phosphate buffer, a citrate buffer or a tris(hydroxymethyl)aminomethane hydrochloride and sodium chloride (TRIS-NaCl) buffer. Preferably, the euglobulin moiety is remodulated by TRIS-NaCl.

In the process of the present invention, it is generally preferred to maintain the pH of the medium in the range of from about 7 to 8, more preferably from about 7.4 to 7.6.

Furthermore, it is preferred to maintain the concentration of the buffer system in the range of 10 to 200 mM, more preferably about 50 mM. More specifically, the concentration of TRIS-NaCl should be 50 mM TRIS and 150 mM chlorination sodium.

In the method of the invention for determining the biological activity of a defibrotide polynucleotide, the defibrotide polynucleotide sample solution is directly diluted in the euglobulin portion, followed by the addition of a chromogenic or fluorescent matrix. In particular, in order to be able to measure, it is preferred to first dilute/dissolve the defibrotide polynucleotide in TRIS-NaCl buffer to obtain a mother stock solution of both the sample and the standard. The sample and the standard were diluted from the parent stock to a defined volume of euglobulin fraction by serial dilution until assayed for defibrotide concentrations ranging from about 1 to 1000 [mu]g/mL.

An important parameter of the assay method of the invention is temperature. With regard to establishing the reference curve and during the measurement phase, it is preferred to maintain the same temperature throughout the measurement process and for all samples to be measured. In order to achieve this, it is preferred to use a temperature control device, which can perform array measurement as needed, and appropriately change the position of the sample to ensure maximum thermal homogeneity of the system.

In general, the temperature range used in the measurement method is, for example, 25 to 40 ° C, preferably 35 to 39 ° C, more preferably 37 ° C.

According to the present invention, when all the reagents have been added, the concentration of the compound X released in the medium is measured by the action of the defibrotide polynucleotide, and the measurement is continued for a predetermined period of time at a predetermined frequency as X chemical nature and function of the detection system.

Similar to other bioassay methods, the method of the present invention also provides a calibration phase and a measurement phase, preferably performed on the same microplate to minimize the occurrence of experimental variables.

The calibration phase includes increasing concentrations of known defibrotide polynucleotides (standards), obtaining absorbance data associated with the samples, statistical reprocessing of those data, and inference of calibration curves indicating the enzyme reaction rate of the present invention. The increase is related to the concentration of the defibrotide polynucleotide present in the euglobulin portion. In the measurement phase, since this correlation is obtained during the calibration phase, the unknown biological activity of the defibrotide polynucleotide sample can be determined based on the measured absorbance values under the same conditions.

In particular, the experimental procedures typically provide standards and unknowns for the preparation of several samples at various known concentrations of defibrotide polynucleotides. The mother solution is serially diluted according to a predetermined dilution factor to prepare the defibrotide polynucleotide sample

In the method of the present invention, it is preferred to prepare at least 5 concentrations of the standard and 5 concentrations of the sample to be tested, and prepare at least 4 repetitions for each concentration of the standard and likewise for each concentration of the test sample. It is usually a 1:2 dilution of the parent solution.

The standard and test sample concentration of the defibrotide polynucleotide is usually 0.1 to 1000 μg/ml.

The concentration of the test sample is preferably the same as the concentration of the standard.

According to the above description, the measurement of each concentration is preferably carried out on a microplate, wherein the positions of each of the standards and the samples are preferably alternated at respective concentrations. Based on this sample configuration, it is further illustrated in the experimental section that at least 4 absorbance values are measured each time for each concentration of defibrotide polynucleotide standard and test sample.

The above measurement is carried out at a predetermined time, that is, first at time t ₀ , that is, all the ingredients have been added and before the start of the enzyme reaction of the present invention, and then at a precise interval for a sufficiently long time to obtain the required The data.

Preferably, the absorbance measurement is continued for up to 90 minutes and reading is performed every 1-10 minutes. It is more advantageous to read every minute. The spectral absorbance reading is performed at a wavelength depending on the nature of the detectable functional group X released during the enzymatic hydrolysis reaction. In the case where X is a p-NA, the absorbance at 405 nm was tested.

The absorbance readings of the standard and the unknown defibrotide polynucleotide sample are raw data, which is typically derived directly from the same device that provides the read operation, which is tabulated in this manner to indicate each time point and The absorbance of the well.

The raw data is then processed using, for example, Spread Sheet-Microsoft Excel®. This first processing operation results in a calculation of the calculated average absorbance and associated standard deviation for each set of time points and for each set of readings, each set comprising at least 4 of each of the standard and test sample defibrotide. repeat.

For bioanalytical determinations, statistical processing of data is further performed with commercially available software, such as the Statistical Method in Biological Assay published by Finney DJ in Ch. Griffin, London and relevant Pharmacopoeias, Second Edition. Said.

More precisely, according to the invention, a flat as defined by statistical analysis in European Pharmacopoeia General text 5.3 can be used. The line analysis mode, the slope ration mode, and the four parameter logic mode were performed for bioanalytical determination of defibrotated polynucleotides.

As shown in Figure 1, a line is obtained by placing the time on the abscissa and the absorbance in the ordinate, and the slope "b" is proportional to the rate of enzyme reaction: by increasing the concentration of defibrotide The rate of hydrolysis increases proportionally to the value of "b". Finally, each set of repeats of the standard defibrotide and the test sample defibrotide was calculated as described above, and the correlation between the slope value and the concentration of its associated defibrotide was obtained. A suitable mathematical transformation of the abscissa (ie, the logarithm of the defibrotide concentration) can be used instead of the actual value.

Graphically, the correlation causes the standard to be S-shaped and the test sample to be S-shaped (Fig. 2); the central portion of the S-shape has two straight lines, which are generally parallel, and the distance is the test sample and the standard A function between poor biological activity. This interval was used for the potency assay using the parallel line pattern described by Finney D J in the statistical method published in Griffin, London, 2nd edition.

For a more specific assay, a four-parameter logistic curve mode is used, and in this example, the entire sigmoidal curve of the sample and the standard is used to calculate the relative biological potential of the sample.

In a preferred embodiment of the invention, a sample solution of the standard solution and the defibrotide polynucleotide to be tested is placed in each well of the microplate. The euglobulin is prepared when it is to be used and is a dilution medium for the defibrotide polynucleotide storage solution. Finally, a solution containing a chromogenic substrate is added. Then, place the microdisk in the thermostat reader to And after the rapid shock, the reading of the absorbance of the system is performed during the predetermined interval and the predetermined time. The resulting raw data is then processed, thus determining the unknown activity of the defibrotide polynucleotide sample.

As can be understood from the following examples, a liquid defibrotide polynucleotide formulation, preferably an aqueous solution, having a defined biological activity, and particularly having a defibrotide polynucleotide activity of 25 to 35 IU, can be obtained according to the method of the present invention. The /mg is preferably 27.5 to 32.5 IU/mg, and more preferably 28 to 32 IU/mg.

The liquid defibrotide polynucleotide formulation is preferably sold in the form of a container, preferably a vial containing 200 mg of defibrotide in a 2.5 ml buffered aqueous solution (preferably pH 6.5 to 8.5, More preferably 7 to 8), diluted before use; as a result, when the biological activity is evaluated by the method of the present invention, each container exhibits defibrotide activity of 5,000 to 7,000 IU, preferably 5,500 to 6,500 IU, more Good for 5600 to 6400 IU.

The above and other aspects of the invention are as described in the following examples, however the following examples do not limit the invention.

Example

The following materials were used in the examples of this case:

Device

Program and software

o Microsoft Excel® (Microsoft Corporation, Redmond, Wash., USA)

substance

○ Defibrinated polynucleotide (Gentium)

○ chromogenic substrate S-2251 (Chromogenix Instrumentation Laboratory S.p.A., Milan, Italy)

○ Tris(hydroxymethyl)aminomethane hydrochloride (TRIS)-sodium chloride (TRIS-NaCl), (Sigma-Aldrich, Milan, Italy)

○1N HCl (Carlo Erba reagenti, Milan, Italy)

○1N NaOH (Carlo Erba reagenti, Milan, Italy)

○Bovine plasma (Tebu Bio Italia, Magenta (MI), Italy)

○ Glacial acetic acid (Carlo Erba reagenti, Milan, Italy)

Solution

○ TRIS-NaCl (Preparation 1L): 6.06 g of TRIS-HCl and 2.2 g of NaCl were quantitatively placed in a 1 L beaker and dissolved in 500 mL of pure water was used and the pH was adjusted to 7.4 with approximately 40 mL of 1 N HCl. The solution was dosed into a 1 L flask and the solution was diluted with pure water. This solution was stored in a refrigerator (2-8 ° C).

○ Sesogenic substrate S2251 (CAS 63589-93-5) 3 mM (preparation 15.2 mL): About 25 mg of a chromogenic substrate was dissolved in 15.2 mL of pure water. This solution was stored in a refrigerator (2-8 ° C).

○ Bovine euglobulin (preparation 10 mL). In a container having a minimum capacity of 300 mL, 240 mL of ice pure water was added, and 10 mL of bovine plasma was added with stirring. The pH was adjusted to 5.3 ± 0.1 with 1% acetic acid. Allow to stand at 2-8 ° C for 1 to 16 hours. The supernatant solution was removed with a siphon and centrifuged at 2.800 rpm for 5 minutes at 4 ° C to collect the precipitate. The precipitate was suspended by mechanical dispersion (for example, using an experimental glass rod), shaken in 5 mL of ice-purified water, shaken for about 5 minutes, and centrifuged at 2.800 rpm for 5 minutes at 4 ° C to collect a precipitate. The precipitate was mechanically dispersed in about 10 mL of TRIS-NaCl; to facilitate dispersion of the precipitate, the precipitated particles were destroyed by a suitable instrument (eg, an experimental glass rod). The resulting suspension is stored at 2-8 ° C and stored for not less than 1 hour and no more than 6 hours prior to use.

Standards and samples for defibrinated polynucleotide solutions

Reference storage solution

Approximately 100 mg of defibrotide polynucleotide reference standard was dosed into a 20 mL flask. The powder was dissolved in about 10 mL of TRIS-NaCl and made up to volume with the same solvent. The resulting solution is TRIS-NaCl was diluted 1:4 to give about 1.25 mg/mL of defibrotide RS solution.

Sample storage solution

Approximately 100 mg of the defibrotide polynucleotide sample was dosed into a 20 mL flask. The powder was dissolved in about 10 mL of TRIS-NaCl and made up to volume with the same solvent. The resulting solution was diluted 1:4 with TRIS-NaCl to give a defibrotide polynucleotide sample solution of about 1.25 mg/mL.

Preparation of reference products and sample solutions

a) 125 ug/mL defibrotide polynucleotide: The defibrotide polynucleotide storage solution (reference and sample) was diluted 1:10 with TRIS NaCl (equivalent to 50 ug/mL in the wells). A 500 uL preparation solution was quantitatively placed in a microcentrifuge tube, and the solution was mixed with 500 uL of euglobulin. The centrifuge tube was closed and stored in ice water.

b) 62.5 ug/mL defibrotide polynucleotide: The solution (a) was diluted 1:2 with TRIS NaCl (corresponding to 25 ug/mL in the wells). A 500 uL preparation solution was quantitatively placed in a microcentrifuge tube, and the solution was mixed with 500 uL of euglobulin. The centrifuge tube was closed and stored in ice water.

c) 31.25 ug/mL defibrotide polynucleotide: The solution (b) was diluted 1:2 with TRIS NaCl (corresponding to 12.5 ug/mL in the wells). A 500 uL preparation solution was quantitatively placed in a microcentrifuge tube, and the solution was mixed with 500 uL of euglobulin. The centrifuge tube was closed and stored in ice water.

d) 12.5 ug/mL defibrotide polynucleotide: used TRIS NaCl diluted the solution (d) at 1:2.5 (5 ug/mL relative to the wells). A 500 uL preparation solution was quantitatively placed in a microcentrifuge tube, and the solution was mixed with 500 uL of euglobulin. The centrifuge tube was closed and stored in ice water.

Blank solution

Mix 1 volume of euglobulin with 1 volume of TRIS NaCl solution (eg: 500 uL + 500 uL).

Disk storage

Add 200 uL of standard or sample or blank solution to each well of the microplate according to the proposed storage protocol (see Table 1 below). Different storage options can be used depending on the availability and architecture of the automated pipette. However, for each reference and sample solution, no less than four stores must be used for analysis.

Immediately before placing the disk into the microdisc reader, 50 uL of chromogenic substrate was quickly added to each well.

S1, S2, S3, S4: Reference solution storage 1, storage 2, storage 3, storage 4

U1, U2, U2, U5: sample solution storage 1, storage 2, storage 3, storage 4

Ca, Cb, Cc, etc.: defibrotide polynucleotide reference product and sample concentration a, b, c, etc.

BLK: blank solution

Calculation and results

From the power diagram, the "absorbance value and time" of the standard preparation (for example: S1_Ca, S1_Cb, S1_Cc) identifies the appropriate linear range (for example, 30 to 35 minutes, see Figure 3).

Identification of the linear dynamic range (absorbance value at 405 nm versus time)

The analytical reaction (slope) of each preparation of the standard and the sample over a predetermined time range is calculated as follows.

among them:

Aa is the absorbance at time Ta (30 points from the above figure)

Ab is the absorbance at time Tb (35 points from the above figure)

The values obtained are listed in Table 2.

The reaction of the test substance and the reaction of the standard are plotted against the logarithm of the concentration, and the activity of the test substance is calculated using the parallel line mode defined in Chapter 5.3.2 of the current version of Ph. Eur.

References and samples should be used in no less than 3 consecutive serial dilutions (eg defibrotide concentration 5 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL or 5 μg/mL, 12.5 μg/mL) , 25μg/mL or 12.5μg/mL, 25μg/mL, 50μg/mL

Analysis of variation

Analysis of the variation was performed according to Section 5.3.2.3 of the current version of Ph. Eur. and version 2 of the statistical method in Finney DJ (1964) Bioanalysis.

Validity test

1) Linear regression conditions are important, ie the probability of calculation is less than 0.05. If the threshold is not met, it is impossible to calculate 95% C.I.

2) Non-parallel conditions are not important, ie the probability of calculation is less than 0.05.

3) The condition of the nonlinearity is not important, that is, the probability of calculation is less than 0.05.

Acceptance condition

4) The estimated potency is not less than 90% and no more than 111% of the stated effect.

5) The confidence limit for estimated efficacy (P = 0.95) is not less than 80% and is not greater than 125% of the stated effect.

Calculation

among them:

R: sample results from parallel line mode analysis

The claimed effect: the claimed effect of the standard (dry matter UI / mg)

Standard concentration: concentration of standard (dry matter mg/mL)

Sample concentration: sample concentration (dry matter mg/mL)

Analysis for defibrotide polynucleotide formulations

The above disclosed assays have been used to determine the biological activity of a liquid formulation containing 200 mg of defibrotide polynucleotide (80 mg per ml) in 2.5 ml and having a qualified basis for the composition shown in Table 3.

The results are shown in Table 4.

Figure 1, by defibrotide polynucleotide (concentration A globulin portion of a mammal that is activated or non-activated at 0-50 [mu]g/ml, 0-100 minutes) is a graph illustrating the release kinetics of pNA from matrix S-2251.

Figure 2 is a graph showing the rise of the standard for the defibrotide polynucleotide and the test sample.

Fig. 3 is a graph showing the "absorbance versus time" of a standard preparation (for example, S1_Ca, S1_Cb, S1_Cc), which confirms a suitable linear range (for example, from 30 to 35 minutes).

Claims (24)

A method for determining the biological activity of a defibrotide polynucleotide, comprising the steps of: a) contacting a mammalian euglobulin and a matrix specific for plasmin to a defibrotide, The matrix provides a measurable product by action with the plasminogen hydrolyzed from plasminogen contained in the euglobulin; and b) measuring the product formed during continuous time The amount by which the biological activity of the defibrotide polynucleotide is determined. The method of claim 1, wherein the euglobulin is a mammalian euglobulin. The method of claim 1, wherein the euglobulin is a euglobulin of human, rabbit or bovine. The method of claim 1, wherein the plasminogen which acts on a matrix specific for plasmin is released from plasminogen contained in the euglobulin. The method of claim 1, wherein the matrix is a chromogenic substrate specific for plasmin. The method of claim 1, wherein the plasmin-specific compound is a compound of the formula A ₁ -A ₂ -A ₃ -X, wherein A ₁ and A _{2 are} non-polar amino acids, A ₃ It is an amine acid or arginine, and X is the measurable product. The method of claim 6, wherein the measurable product X is selected from the group consisting of p-nitroaniline and 2-naphthylamine. The method of claim 4, wherein the matrix specific for plasmin is H-D-amidino-L-alkamine-L-isoamine-p-nitroaniline. The method of claim 6, wherein the measurable product X is measured by spectrophotometry or spectroscopic spectroscopy. The method of claim 2, wherein the mammalian euglobulin is reconditioned to the same volume as the original plasma, or diluted 1:10 in a buffer, and the concentration of the chromogenic substrate is 2.5 to 3.5 mM. . The method of claim 10, wherein the concentration of the chromogenic substrate is 3 mM. The method of claim 1, wherein the method is carried out in a reaction medium which is buffered to an aqueous solution having a pH of 7 to 8. The method of claim 12, wherein the reaction medium is buffered to an aqueous solution of pH 7.4. The method of claim 1, wherein the temperature is maintained at 35 to 39 °C. The method of claim 14, wherein the temperature is maintained at 37 °C. The method of claim 1, wherein the concentration of the matrix specific for plasmin is from 0.3 to 4 mM. The method of claim 16, wherein the concentration of the matrix specific for plasmin is from 2.5 to 3.5 mM. The method of claim 17, wherein the concentration of the matrix specific for plasmin is 3 mM. The method of claim 1, wherein the method comprises the steps of: c) determining a rate of release of the measurable product during an enzyme reaction between the standard sample and the test sample; d) mathematically and/or graphically Indicates the release rate and the corresponding defibrotide concentration Linked to obtain the biological activity of the test sample of the defibrotide polynucleotide. The method of claim 1, wherein the defibrotide polynucleotide is a liquid defibrotide polynucleotide formulation having a defibrotide polynucleotide having a biological activity of 25 to 35 IU/mg. The method of claim 20, wherein the liquid defibrotide polynucleotide formulation has a defibrotide polynucleotide having a biological activity of from 27.5 to 32.5 IU/mg. The method of claim 21, wherein the liquid defibrotide polynucleotide formulation has a defibrotide polynucleotide having a biological activity of 28 to 32 IU/mg. The method of claim 20, wherein the liquid defibrotide polynucleotide formulation is buffered at a pH of 6.5 to 8.5. The method of claim 23, wherein the liquid defibrotide polynucleotide formulation is buffered at a pH of 7 to 8.