Method of definition biological activity of defibrotide

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

 

The technical field to which the invention relates.

This invention relates to a method of determining the biological activity of defibrotide and more particularly to an indirect enzymatic method of determination of biological activity of defibrotide.

The level of technology

Defibrotide (Merck Index, 1996, no. 2915) is a substance of natural origin, which is obtained by extraction from animal organs and which consists of the sodium salt of polyethoxylated having low molecular weight.

Defibrotide was the subject of numerous pharmacological studies, which suggest that it can be used in therapy as an antithrombotic agent (U.S. patent US 3829567).

In addition, defibrotide has been used successfully in the treatment of peripheral arteriopathy, acute renal failure (U.S. patent US 4694134) or acute myocardial ischemia (U.S. patent US 4693995).

Like other biological substances that are produced by extraction, defibrotide is also subject to the limited variability of the composition, which is typical of natural biopolymers. A classic example of this situation is heparin, the variability from batch to batch in relation to chain length, molecular weight, composition, degree sulfotyrosine etc. is well known is on. The consequence of this is that the same quantity by weight of defibrotide might actually be equivalent from the point of view of specific biological activity.

The extraction, isolation and purification may not per se guarantee the absolute reproducibility of this product due to its inherent biopolymer structure.

However, with good control you can reduce this variability: for this purpose, research was conducted standardized industrial processes to highlight defibrotide extraction from bodies such as, for example, the study described in U.S. patent US 4985552.

The product obtained in accordance with the above process, characterized by defining some specific physico-chemical parameters, such as, for example, electrophoretic mobility, the extinction coefficient, the ability to the optical rotation and reversible hyperchromeity. However, these parameters depend mainly on the structure of defibrotide and can not provide information on its biological activity.

To the authors ' knowledge this invention, the only ways that were reported up to the present time, were used to assess the biological activity of defibrotide are Cup test definition lysis of fibrin and thrombosis ograficheskie registration time of lysis euglobulin (Prino G., Mantovani M, Niada R., Coccheri S., Lima, A., Indagini preliminari sull attivita fibrinolitica, Nell animale e Nell'uomo di una nuova sostanza presente in diversi organi animali, Simposio Internazionale: La ricerca scientifica Nell'industria farmaceutica in Italia, Rome, 2-4 October 1975 - II Farmaco, Ed. Prat. (1969), 24, 552-561).

However, these methods have significant experimental complexity, poor reproducibility and accuracy and, in the specific case thromboelastography registration, linearity of response, limited to a very narrow concentration ranges.

Thus, up to this time were not known for reliable, accurate and reproducible methods for determining the biological activity of defibrotide.

The authors of this invention have developed a simple and reliable method for determining the biological activity of defibrotide, which allows you to control samples, obtained by extraction and, hence, allows to standardize medical drugs based on defibrotide.

The method belongs to this invention, allows the detection of specific biological activity of defibrotide in comparison with the standard pattern with high precision, high speed and reproducibility.

Disclosure of inventions

Thus, this invention relates to a method of determining the specific biological activity of the sample defibrotide, predusmatriva usamu stages:

a) bringing into contact of defibrotide, plasmin and substrate specific for plasmin, which by reaction with plasmin provides the medium, and

b) measuring the amount of formed product in a sequential time points.

The method of this invention is indirect in vitro method for determining the activity of defibrotide, which is based on the functional interaction between defibrotide and plasmin.

From literature it is known that plasmin is a proteolytic enzyme in the cascade of coagulation/fibrinolysis, is able to cleave fibrin, fibrinogen and other plasma proteins.

The enzymatic activity of plasmin typically define various standard in vitro tests. One of the most frequently used methods is the determination using spectrophotometry or fluorimetry chromogenic or fluorogenic compounds, which are released through the action of plasmin on different substrates (Haemostasis, (1978), 7, 138-145). Typically use of peptide substrates having the formula A1-A2-A3-X, where A1and a2are amino acids, which are predominantly non-polar And3is lysine or arginine and X represents the measured release of the connection, for example, para-nitroaniline (pNa) or 2-naphtylamine (NA (Haemostasis, (1978), 7, 146-149). In addition to the above-mentioned peptide substrates, success was achieved with the use of other, more simple compounds, such as, for example, p-nitrobenzyl-p-toluensulfonyl-L-arginine (Haemostasis, (1978), 7, 105-108).

In these tests the speed with which the compound X is released into the incubation medium, is proportional to the activity (International Units) of plasmin present in the sample.

Now it was found, and this is the principle of this invention that tests based on the evaluation of plasmin described above, defibrotide increases the rate of release of compound X in proportion to its concentration.

The method applies this invention involves first bringing the sample defibrotide, plasmin and plasmin substrate into contact with each other.

Sample defibrotide used to determine in accordance with this invention are usually obtained by extraction of organs according to known procedures, such as, for example, the procedures described in U.S. patent US 4985552, which was already mentioned.

The usual party, manufactured industrially by defibrotide was chosen as the reference sample (standard) and used to construct the calibration curves in accordance with the method of this invention.

Usually this pic is b provides precise and accurate measurements of defibrotide even in the presence of impurities, such as, for example, RNA, heparin, degraded defibrotide (defibrotide, from which was removed purine or pyrimidine) or ethanol, provided that they are in concentrations, usually less than 10% by weight, such so as not to disrupt the system.

In addition to determining defibrotide this method also allows you to identify other biologically equivalent substances produced from defibrotide, such as, for example, diaminononane defibrated or, more simply, defibrotide, denatured by heating.

This method is sensitive enough to detect concentrations of defibrotide equal to or smaller than 0.1 μg/ml (final concentration in the system definition), and usually gives good correlation to the maximum values of concentrations equal to or higher than 100 mcg/ml

Used plasmin is usually plasmin any mammal, such as, for example, bovine, porcine or human plasmin, and is preferred plasmin person.

However, although plasmin is the preferred enzyme, the use of other equivalent enzyme systems, such as, for example, the precursor of plasmin, such as plasminogen, or enzymes analogous to plasmin, which are chemically related and have similar functionality is also part of this invention.

In the method of the present invention under the substrate for plasmin can be any substrate that is specific for plasmin, which, in this way releases the detected hydrolysis product X.

Depending on the nature of the detectable group X can be used equally successfully alternative detection system, typically known to a person skilled in the field.

Spectrophotometric or fluorometric detection systems are especially preferred, in particular spectrophotometric system.

Commonly used substrates are substrates that are specific for plasmin. Preferred peptides of the formula A1-A2-A3-X, where A1and A2are amino acids, which are predominantly non-polar And3is lysine or arginine and X is a detectable group. Examples of such substrates are Val-Leu-Lys-pNa, Val-Phe-Lys-pNa or Glu-Phe-Lys-pNa, in which the group X, detected using spectrophotometry, is para-nitroaniline (pNa). Other suitable substrates, for example, Val-Gly-Arg-2NA, contain 2-naphtylamine, which is measured by fluorometry. Particularly preferred substrate is a compound H-D-poured-L-leucyl-L-lysine-p-nitroaniline (H-D-Val-Leu-Lys-pNa).

Plasmin and pacificnet substrates, used to determine defibrotide are usually commercially available.

The detection method of the present invention are realized by putting these reagents and sample defibrotide in aqueous solution at a specific pH and polyarnosti.

In particular, the concentration of plasmin can vary typically from 0,0016 to 0.20 M.E. Ter-Minassian/ml, preferably from 0,0064 to 0,050 M.E. Ter-Minassian/ml and even more preferably equal to approximately of 0.0125 M.E. Ter-Minassian/ml

However, with respect to the substrate for plasmin concentrations from 0.3 to 4 mm, preferably 2.5 to 3.5 mm, and most preferably 3 mm is used usually in the case of a chromogenic substrate, whereas a concentration of from 0.05 to 0.15 mm is used in the case fluorogenic substrate.

The detection method of the present invention, like other enzymatic methods, is sensitive to the pH of the environment.

In fact, it cannot be used at extreme pH values, at which the enzymatic system would be inactivated.

It is preferable that the pH of the environment has not been changed at any point in time during the period of measurement, and therefore, the solution is usually sautereau using a buffer system selected from the systems commonly used in the tests determination of plasmin. For example, a suitable buffer may be a phosphate buffer, citrate the first buffer or Tris(hydroxymethyl)aminomethan-cleaners containing hydrochloride (TRIS) buffer. The operation is performed preferably in the presence of Tris.

In this way, it is generally preferable to maintain a pH in the range from approximately 7 to 8, more preferably at approximately 7,4.

In addition, it is preferable to maintain the concentration of the buffer system in the range from 10 to 200 mm, preferably at approximately 50 mm.

The method of this invention for determining defibrotide provides that plasmin, a substrate for plasmin and defibrated mix. In particular, to ensure correct measurements under this method, it is preferable to add the plasmin or specific substrate or both to the buffer solution containing the sample defibrotide, before the beginning of the measurement phase. The substrate for plasmin preferably added last.

An important parameter of this method of determination is the temperature. It is preferable to maintain the same temperature throughout the measurement duration for all measured samples as in the construction of the calibration curves, and during phase measurements. For this purpose it is preferable to use the device with a temperature-controlled, and, when necessary, can be performed several series of measurements, changing the position of the samples in a suitable manner, so that the warranty is to, this system has a maximum temperature homogeneity.

Usually this method definitions used in the temperature range, for example, from 25 to 40°C, preferably from 35 to 39°and even more preferably at 37°C.

According to this invention the measurement of the concentration of the compound X released into the environment under the action of plasmin, begin when all reagents have been added, and continue for a predetermined time and at a predetermined frequency depending on the chemical nature of X and detection systems.

Like other methods of biological determination method of the present invention also provides for phase calibration and phase measurements, which are preferably performed in parallel to reduce, to the extent possible, the occurrence of experimental variability.

Stage calibration includes receiving data of optical density related to samples with known increasing concentrations of defibrotide (standard), statistical processing of these data and the extrapolation of the calibration curves, which Express the correlation between the increase in the rate of the enzymatic reaction of the present invention and the concentration of defibrotide present in the environment. At the stage of measurement due to the correlation obtained at the stage of feces is brownie, it is possible to identify the unknown biological activity of the sample defibrotide based on the values of optical density, measured and processed under the same conditions.

In more detail, the experimental Protocol usually involves the preparation of several samples, both standard and unknown, at various known concentrations of defibrotide. Sample defibrotide prepare a serial dilution of the original solution in accordance with a previously determined dilution factor.

In this way, it is preferable to prepare at least 5 concentrations of standard and 5 concentrations of the test specimen with getting 5 times or more, preferably 10 repetitions for each concentration of the standard and, similarly, for each concentration of the analyzed sample, usually in sequential dilution of 1:2 initial solutions.

Concentration as standard, and analyzed sample defibrotide is usually from 0.1 to 100 μg/ml, preferably from 0.3 to 50 μg/ml, more preferably from 0.5 to 8 μg/ml

The concentration of the analyzed sample are preferably the same order of magnitude as the concentration of the standard.

According to the illustration above measurements for each concentration is preferably carried out at two microplate, where the placement of each the sample, standard and analyzed sample, respectively, at a corresponding concentration on a single tablet is preferably reverse placement on another tablet. According to this diagram for the location of the samples, which is explained in more detail in the experimental part, for each concentration of standard defibrotide and the analyzed sample defibrotide measure at least 5 or preferably 10 values of optical density for each time point.

A series of measurements described above, carried out at preset time points, that is, first of all, at time t0that is , when all components have been added, before the enzymatic reaction of the invention and then through the exact intervals and for a time sufficient to obtain the required data.

Preferably the measurement of optical density continues up to a maximum of 90 minutes with withdrawals readings made every 1-10 minutes. More preferably the removal of the evidence produced at time t0and then within 20 to 50 minutes every 5 minutes. Photometric readings of optical density is carried out at a wavelength that depends on the nature of the detectable group X released during enzymatic hydrolytic reactions. In the particular case when X is a pNA, optical the current density measured at 405 nm.

Readings of optical density of the standard and unknown samples defibrotide, known as source data, usually directly originate from the same device, which ensures the operation of reading; they bring to the table so that the value of optical density is expressed for each time point and for each hole.

Then these raw data was processed using, for example, Microsoft Excel spreadsheets®. This first operation data leads to the calculation of the average optical density and associated standard deviations at each time point and for each series of readings, with each series of readings contains at least 5 and preferably 10 repetitions for each concentration as standard, and analyzed sample defibrotide.

Further statistical processing of data is carried out with the use of programs such as Sigma Plot Computer Program® (SPSS, Chicago, USA), which converts the mathematical relationship that exists between the values of optical density of these samples and the time for each series of the concentrations of defibrotide, to obtain straight lines, the slope of which is proportional to the concentration of defibrotide.

More precisely, in the interval, which has a linearity of response, preferably from 20 to 50 minutes for each of the 5 or predpochtitelno 10 repetitions of the same concentration this program calculates the regression line, which is characterized by the coefficient of the linear regression b, the correlation coefficient r2cutoff on the coordinate axis of the segment.

The straight lines obtained in accordance with this procedure, usually have a good correlation, expressed by high values of r2usually not less than 0.97, preferably r2≥0,99.

The data obtained with the help of this software may be reproduced in tabular digital data or can be presented graphically for each series of concentrations.

As shown in figure 1, by setting aside time on the abscissa and absorbance on the ordinate get straight lines, the slope of which b is proportional to the velocity of enzymatic reactions: by increasing the concentration of defibrotide will increase the rate of hydrolysis and the proportion will increase the value of b. Finally, the magnitude of the slope, calculated as described above, for each series of repetitions of the standard defibrotide and the analyzed sample defibrotide correlated with the decimal logarithm of the concentration of defibrotide to which they relate.

Graphically this correlation gives a sigmoidal curve for the standard sigmoid curve for the analyzed sample (figure 2); the Central part of these sigmoidal curves given two straight lines, which are usually the camping parallel and the distance between which is a function of the difference between the biological activity of the analyzed sample and standard.

In this interval of linearity determine the activity of the unknown sample defibrotide in comparison with the activity of the standard in accordance with the methodology of the biological definition using parallel lines described Finney DJ, Statistical Method in Biological Assay, 2nded. Ch. Griffin, London.

This methodology can be applied when, as in this invention, the biological response is a linear function of the logarithm of the concentration of analyte and when there is parallelism and linearity between the straight lines associated with the standard and, therefore, unknown concentrations.

Preferably the statistical processing of these data, the calculation of the relationship of these activities and, therefore, determination of unknown activity defibrotide done using specialized programs created on the basis of the above methodology.

However, statistical data processing, which, in chemical analysis in General, and in this way, more specifically, allows to minimize the frequency error and experimental variability, is not required in the method of this invention, but merely provides a method of assessing results, which is well known to specialists in this field and which is usually used in this field.

This invention relates also to set is to determine the biological activity of defibrotide in accordance with the method of the present invention, containing at least:

a) a measured quantity of substrate for plasmin, as defined above, and

b) a measured quantity of plasmin.

Preferred kits contain 20-30 mg of substrate specific for plasmin, per unit of plasmin and even more preferably 25 mg of substrate per unit of plasmin.

In accordance with this invention sets containing H-D-Val-Leu-Lys-pNA as a substrate specific for plasmin, and plasmin man, are especially preferred.

The kits of this invention may also contain buffered aqueous solution, preferably a solution, buffered with 50 mm TRIS-HCl at pH 7.4.

Optionally, the kits of this invention also contain a measured quantity of defibrotide (standard) to allow control measurements.

In the preferred embodiment of the present invention the standard solutions and the sample solutions defibrotide, which are to be determined, enter into the appropriate wells of the microplate. The plasmin solution is prepared at the point of use and distribute the wells containing defibrotide, and, finally, add the solution containing the substrate for plasmin. Then, the microplate is placed in a temperature-controlled tablet reader and after rapid mixing shall take readings of the optical density of this system cher the C pre-specified intervals and for a pre-determined period of time. Then the received source data to process, identifying unknown activity samples defibrotide.

These and other aspects of the present invention will be illustrated in more detail in the following examples, which, however, should not be construed as limiting the invention.

Brief description of figures

Figure 1:

The kinetics of release of pNA from the substrate S-2251 under the action of plasmin, which is activated and not activated by defibrotide (concentration of 0-100 μg/ml, 0-20 minutes).

Figure 2:

The dependence of the slope of the straight lines corresponding to the standard and test sample defibrotide, concentration defibrotide.

Figure 3:

The kinetics of release of pNA chromogenic substrate S-2251 under the action of plasmin in the presence of defibrotide (concentration 0.5 μg/ml, 5 times).

Figure 4:

The kinetics of release of pNA from the substrate S-2251 under the action of plasmin in the presence of defibrotide (concentration of 2.0 μg/ml, 5 times).

Figure 5:

The kinetics of release of pNA from the substrate S-2251 under the action of plasmin in the presence of defibrotide (concentration of 8.0 ág/ml, 5 times).

Figure 6:

The dependence of the release of para-nitroaniline concentration defibrotide.

The implementation of the invention

EXAMPLES

In the examples used the following material is s.

Equipment

Detector for microplate having 96 wells, MRX TCII (Dynex Technologies, Chantilly, VA, USA), temperature-controlled and equipped with a program of enzyme kinetics

A microplate having 96 wells with a flat base (Greiner L., Kremunster, Austria, cat. 655101)

- Pipette with continuously adjustable volume Pipetman P200 (30-200 mm) and 8×200 (20-200 ál) and pipette tips 200 ál of certified quality (Gilson, Milan, Italy)

- pH meter MM Radiometer (Analitica De Mori, Milan, Italy)

Program

- Microsoft Excel® Microsoft Corporation, Redmond, WA, USA)

Sigma Plot Computer Program® (SPSS, Chicago, USA)

Substances

- Defibrated (Gentium)

- Plasmin person, 1 unit, P-4895 (Sigma Aldrich, Milan, Italy)

- Chromogenic substrate S-2251, 820332-39 (Chromogenix Instrumentation Laboratory S.p.A., Milan, Italy)

- Tris(hydroxymethyl)aminomethan (TRIS), 255285-9 (Sigma-Aldrich, Milan, Italy)

- 1 n HCl 1090571000 (Merck)

-1 n NaOH 1091411000 (Merck)

Solutions

TRIS-HCl-buffer

to 2.42 g Tris was dissolved in distilled water and dilute to a volume of 100 ml 16 ml of 1 n HCl is added to this solution followed by the addition of an additional quantity of water to obtain a final volume of 400 ml. pH of this last solution is 7,40. If these values are different, the pH adjusted to obtain the desired value by adding 1 n HCl or 1 n NaOH.

The solution plasmin

One unit (1 M.E. Ter-Minassian) plasmin person is dissolved in 4 ml of TRIS-HCl buffer at 0°the. Then, working always on ice, this solution was divided into aliquots of 200 μl, which is stored at -20°in plastic test tubes (10 ml

A solution of chromogenic substrate S-2251

25 mg of S-2251 dissolved in br15.15 ml of distilled water and stored at +4/+8°C.

Standard solutions of defibrotide

Preparation of standard solutions

- (End) concentrations from 0.1 to 100 µg/ml

60 mg defibrotide dissolved in 3 ml of TRIS-HCl buffer and diluted 1:15 TRIS-HCl-buffer solution. Thus obtained solution having a concentration of 1,333 mg/ml, subjected to serial dilutions 1:2 to obtain solutions of defibrotide having a concentration of 666 µg/ml, 333 μg/ml and 166 µg/ml of This solution, which is used as initial solution, further diluted to obtain solutions having concentrations 83,33, 41,67, 33,33, 25, 16,66, 8,33, 5, 2,5, 1,66, 0,83, 0,5 and finally, 0.16 µg/ml.

- (End) concentration from 0.5 to 8 μg/ml

60 mg defibrotide dissolved in 3 ml of TRIS-HCl buffer and diluted 1:1500 TRIS-HCl-buffer solution. Thus obtained solution having a concentration 13,33 mg/ml, subjected to serial dilutions 1:2 to obtain solutions of defibrotide having a concentration of 6.66, 3,33, 1.66, and of 0.83 μg/ml, respectively.

Procedure

Take 150 μl each of the standard solutions defibrotide described above, and is worn in the wells of the microplate.

Then prepare quick solution plasmin added at 0°3.8 ml of TRIS-HCl buffer in a test tube containing 0.2 ml of plasmin person. All gently stirred until dissolution, take 50 ál and contribute to the wells of the microplate, followed by adding 50 μl of S-2251 for each hole.

The microplate placed in MRX TCII reader installed on 37°C, stirred for approximately 10 seconds; reading the optical density is carried out at 405 nm at the initial moment of time t0and then every two minutes in the interval from 10 to 20 minutes in accordance with the program of enzyme kinetics.

Then the experimental data measured for concentrations of defibrotide from 0.1 to 100 µg/ml, process (Excel and Sigma Plot) and represent on the graph (the regression line), as shown as an example in the following figures 1 and 2.

The magnitude of the angular coefficients b (slope) these straight lines corresponding to the standard and test sample defibrotide (figure 1), lay relative concentrations of defibrotide (logarithmic scale) (figure 2).

As you can see from this graph, linear response, which allows the identification of a straight line is observed in the Central part of this curve. In this interval of linearity determine the activity of unknown samples defibrotide in comparison to the situation with the standard in accordance with the above mentioned methodology biological definition using parallel lines, described Finney DJ, Statistical Method in Biological Assay, 2nded. Ch. Griffin, London. In order for this methodology to be applicable, it is important to have, in addition to linearity, parallelism between the straight lines relating to the standard and, accordingly, to defibrotide to be checked.

The test to determine the biological activity of the unknown sample defibrotide, in comparison with standard defibrotide, preferably carried out using concentrations that give the linear part of the sigmoid curve, defined above. In particular, preferred are concentrations of the standard and unknown defibrotide in the range from 0.5 to 8 μg/ml

Placement in the wells of repetitions of various concentrations of defibrotide for the standard and the test sample is as follows.

Standard defibrotideThe analyzed sample
123456789101112
And--------- ---
In-0.58.04.02.01.01.02.04.08.00.5-
-1.00.58.04.02.02.04.08.00.51.0-
D-2.01.00.58.04.04.08.00.51.02.0-
E-4.02.01.00.58.08.00.51.02.04.0-
F-8.04.02.01.00.50.51.02.04.08.0-
G------------
N ------------

Standard solutions of defibrotide placed in columns 2-6, whereas samples defibrotide that must be determined is placed in columns 7-11, at the specified concentrations. On the second tablet of the provisions of these samples are preferably reverse. The outer rows and columns of the microplate are not used for process definition, but it is filled with water to ensure maximum temperature homogeneity throughout the system.

The microplate placed in MRX TC reader installed on 37°C, stirred for approximately 10 seconds; reading the optical density is carried out at 405 nm at the initial moment of time t0and then every 5 minutes during the period from 20 to 50 minutes in accordance with the program of enzyme kinetics.

Then the total optical density process (Excel and Sigma Plot)bring to the table and represent the graph (the regression line).

You can then calculate the ratio of the activity and to determine the activity of the unknown sample defibrotide in comparison with the standard using the same system of calculation, which is described above.

As an example, the following table is Itza (1, 2 and 3) and graphs (figures 3, 4 and 5)related to the sample defibrotide, tested at a concentration of 0.5 to 2.0 and 8.0 µg/ml, respectively.

Additionally, based on the data presented in tables 1, 2 and 3, we calculated the release of para-nitroaniline in μm/min under the action of plasmin on the chromogenic substrate in the presence of defibrotide.

Values of b are given in tables 1, 2 and 3, in other words, the slopes of the kinetic curves represent the change of absorbance per minute, multiplied by 1000 (ΔPU/min × 1000) (usually for the sole purpose to avoid numbers with decimal places for scale for spectrophotometric measurements in figures 3, 4 and 5 have introduced the factor 1000; consequently, the actual spectrophotometric readings absorption get, dividing b by 1000).

From knowledge of the literature of the molar extinction coefficient for para-nitroaniline (8270 M-1cm-1that corresponds 0,00827 PU μm on para-nitroaniline), it follows that 1 μm para-nitroaniline: 0,00827 (Absorption) = X μm para-nitroaniline: Spectrophotometric reading (b/1000).

Thus we have calculated in the previous experiments, the number of released para-nitroaniline in micromol and brought the values obtained in table 4.

The correlation between the concentration of defibrotide and released para-nitroaniline in microns presented on the corresponding semi-log graph (6)

The regression line obtained by correlation analysis for 5 repetitions, described by the following equations:

Repeat 1 para-Nitroaniline in μm/min = 0.7317+0.3820 logX

Repeat 2 para-Nitroaniline in μm/min = 0.6625+0.2907 logX

Repeat 3 para-Nitroaniline in μm/min = 0.6708+0.2741 logX

Repeat 4 couple-Nitroaniline in μm/min = 0.6792+0.2907 logX

Repeat 5 para-Nitroaniline in μm/min = 0.7100+0.3323 logX

In a more General form of the release of a pair of nitroanaline described by the equation:

pNA(μm/min)=a+b logX,

where:

a=0.6908±0.0291 (Average ± Standard deviation)

b=0.3140±0.0437 (Average ± Standard deviation)

X = concentration of defibrotide (µg/ml)

or

a (intercept) can vary from 0.6625 to 0.7317,

b (slope) can vary from 0.2741 to 0.3820.

1. The method of determining the biological activity of defibrotide providing stage:

a) bringing into contact in a reaction medium of defibrotide, plasmin and substrate specific for plasmin, which is due to reaction with plasmin provides the medium, and

b) measuring the amount of the formed product in a sequential time points, where a substrate specific for plasmin, is a compound of the formula A1-A2-A3-X, where A1and a2are nonpolar amino acids, AZ is Lisino is or arginine and X is measured by the product and where the concentration of the substrate for plasmin is from 0.3 to 4 mm, preferably from 2.5 to 3.5 mm, more preferably 3 mm, while

c) determine the rate of release of the measured product X during the enzymatic reaction for each sample defibrotide as standard and testing

d) establish correlation, mathematically and/or graphically, the above speed release with the appropriate concentrations of defibrotide with the receipt of the biological activities of the analyzed samples defibrotide.

2. The method according to claim 1, wherein the plasmin plasmin is a mammal, preferably a human plasmin.

3. The method according to claim 1, in which the measured product X is selected from para-nitroaniline and 2-naphtylamine.

4. The method according to claim 1, in which a substrate for plasmin is H-D-poured-L-leucyl-L-lysine-p-nitroaniline.

5. The method according to claim 1, wherein the plasmin has a concentration of from 0,0064 to 0,050 M.E. Ter-Minassian/ml, and the substrate for plasmin has a concentration of from 2.6 to 3.5 mm.

6. The method according to claim 5, in which the concentration of plasmin equal of 0.0125 M.E. Ter-Minassian/ml, and the concentration of the substrate for plasmin equal to 3 mm.

7. The method according to claim 1, in which the measured product X is measured by spectrophotometry.

8. The method according to claim 1, wherein the reaction medium is an aqueous solution, buffered to pH 7 to 8, preferably to a pH of 7.4.

9. The method according to claim 1, in which the temperature of this system of support is when 35-39° C, preferably at 37°C.



 

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FIELD: medicine; forensic medicine.

SUBSTANCE: relates to methods of differential diagnosis of death due to acute carbon monoxide poisoning in ethyl alcoholic intoxication, and can be applied in expert practice for identification of direct cause of death. Microscopic morphometric test of adrenal glands is taken using semi-quantitative 4-points evaluation system to estimate blood flow in the left adrenal zona glomerulosa, intensity of lipidation in the left adrenal zona glomerulosa, intensity of diffuse lymphoid infiltration in the right adrenal zona glomerulosa, to calculate in mcm2 area of adrenocorticocyte nuclei of the right adrenal zona glomerulosa using formula: Р2=100%-Р1, where P1 is probability of assigning case to control group, P2 is probability of assigning case to those died of carbon monoxide poisoning, A is intensity of blood flow in the left adrenal zona glomerulosa, B is intensity of lipidation in the left adrenal zona glomerulosa, C is intensity of diffuse lymphoid infiltration in the right adrenal zona glomerulosa, D isarea of adrenocorticocyte nuclei of the right adrenal zona glomerulosa in mcm2, and atР2>Р1 death due to acute carbon monoxide poisoning in ethyl alcoholic intoxication is diagnosed.

EFFECT: more high precision and information value of differential diagnostics is provided.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method is based on evaluation of degree of inflammatory response in patients with critical ischemia of lower extremities, with a glance of both classic signs of systemic inflammatory response, and additional clinical and laboratory signs of syndrome of systemic inflammatory response. Analysis of venous blood indices is taken, at the same time body temperature ≥38°С or ≤36°С, tachycardia, tachypnea, leucocytes >12х109/L or <4х109/L are measured in patients in the first, the third and the seventh days of hospitalization and one point is assigned to each index, fever 37.0-38.0°С, leucocytes <9-12x109/L, leucocytic left shift >6%, trophic disturbances, C-reactive protein, increased fibrinogen are assigned by 0.5 point each, pain in rest in shin and foot, edema of shin and foot, hyperemia of shin and foot skin and hyperlactatemia are assigned by 0.2 point each, total points at first, third and seventh days are summed up and at total sum from 5.2 to 8.6 favorable outcome of disease is forecasted, at values from 8.7 to 13.6 complications are forecasted, and at values 13.7 and more, fatal outcome is forecasted.

EFFECT: method allows evaluating precisely degree of syndrome of systemic inflammatory response and optimises therapeutic approach.

1 tbl, 3 ex

FIELD: analytical methods in toxicological chemistry.

SUBSTANCE: invention relates to methods for determining title compound in biological material for use in practice of chemicotoxicological and clinical laboratories. Biological tissue is disintegrated, two times infused with acetone each time for 30 min, resulting extracts are combined, combined extracts are filtered, filtrate is evaporated to give dry residue, the latter is dissolved in chloroform and extracted with 0.1 hydrochloric acid, acid extract is washed with diethyl ether, alkalinized with 10% sodium hydroxide solution to pH 8-10, saturated with sodium chloride, and extracted with ethylacetate. Ethylacetate extract is separated, dehydrated, and evaporated to give residue, which is dissolved in mixture acetonitrile/1 N sulfuric acid (8:2 v/v), and chromatographed on column packed with sorbent "Silasorb C-18" having particle size 30 μm while using acetonitrile/1 N sulfuric acid (8:2 v/v) as mobile phase. Fractions of eluate containing subject substance are combined and optical density of combined eluate is measured at wavelength 286 nm.

EFFECT: reduced analysis time, increased degree of recovery, accuracy, and sensitivity of determination.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves running biological sample through column containing absorbent and carrying out ultraviolet detection on absorption at λ=210 nm. The sample is diluted with eluent. Absorbent with inverted C18 phase is used. Eluent composition is as follows: acetonitryl, sodium salt of octane sulfonic acid, trifluoroacetic acid, distilled acid.

EFFECT: enhanced effectiveness in determining streptotricine complex in biological samples.

2 cl, 8 dwg, 1 tbl

FIELD: veterinary medicine.

SUBSTANCE: for fractionating the proteins of canine gastric juice due to disk electrophoresis technique in polyacrylamide gel after polymerization of macroporous gel it is necessary to stratify on it about 0.9-1.1 ml homogenized gastric juice, as for fractionating the proteins of gastric juice it should be carried out at -40-49°C, moreover, this fractioning takes place in a freezing camber supplied with a temperature controller. The innovation enables to carry out qualitative analysis for estimating the state of protein-formation hepatic function.

EFFECT: higher efficiency.

1 dwg, 1 ex

FIELD: medicine, phthisiology, pulmonology.

SUBSTANCE: one should detect the content of adenosine deaminase (ADA) and cytosis in bronchoalveolar lavage fluid (BALF) to calculate ADA coefficient: CADA=ADA/cytosis. At CADA value being equal or above 0.52±0.13 one should diagnose tuberculous etiology of exudative pleurisy. The innovation enables to carry out early diagnostics of tuberculous etiology of exudative pleurisy and provides the chance to conduct early diagnostics in patients who has got inconsiderable quantity of exudates in pleural cavity.

EFFECT: higher accuracy of diagnostics.

4 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation refers to the methods of cytological diagnostics. Smears obtained out of cervix uteri canal should be dyed with nuclear dye to be then studied upon a computed analyzer. On the image obtained it is necessary to measure the area of the nuclei of normal and tumor cells to establish their average values, moreover, the average value of the area of the nuclei of normal cells should be considered to be the standard. Then one should determine the multiplicity of excessive average value of the nuclei of tumor cells against the standard and at the value of this parameter ranged 1.1-1.4 times - one should diagnose hyperplasia, at 1.5-1.9 times - cervical intraepithelial neoplasia degree I (benign stage of carcinogenesis), at 2.0-2.4 times - cervical intraepithelial neoplasia stage II (boundary stage of carcinogenesis), and at its value being 2.5 and higher - cervix uteri cancer (malignant stage of carcinogenesis). The innovation enables to carry out accurate objective differential diagnostics of carcinogenesis stages in cervix uteri upon any cytological preparations.

EFFECT: higher accuracy and efficiency of diagnostics.

2 ex

FIELD: medicine, surgery.

SUBSTANCE: the present innovation deals with evaluating the intensity of inflammatory reaction in surgical wound due to studying bactericidal activity of wound secreta. One should take 20 mcl recombinant test-strain E.coli that carries genes of lux-operon of marine luminescent bacterium Photobacterium leiognathi, at concentration ranged 107-108 CFU/ml, mix with equal volume of wound secreta (experiment) and 0.85%-sodium chloride solution (control). The both samples should be kept at 37°C for 20 min. One should apply upon a slide and calculate the quantity of luminescent cells/area unit. It is necessary to calculate the coefficient of bactericidity (Cb) by the following formula: where Ac and Ao - average value of the quantity of luminescent objects detected at automatized calculation of 10 vision fields in control (c) and experiment (e), correspondingly. At Cb>60% the intensity of inflammatory reaction should be evaluated to be high, that corresponds to the development of infectious-inflammatory complication in the area of surgical interference. The innovation enables to apply the method in case of any diseases that require surgical therapy, moreover, invasive procedures are excluded, the specificity of the method has been increased.

EFFECT: higher efficiency of evaluation.

3 ex

FIELD: medicine, gastroenterology, pathomorphology.

SUBSTANCE: for predicting the state of antral department of gastric mucosa (GM) in children and teenagers it is necessary to carry out morphometry of leukocytes of stroma and stromal cells in gastrobioptate of gastric antral department. One should determine individual leukocytic-stromal index (ILSI) as the ratio of total quantity of leukocytes against total quantity of stromal cells. At ILSI being below 1.0 one should diagnose intact GM of antral department, at values ranged 1.0 to 1.5 - histological signs of tense GM adaptation and at values above 1.5 - chronic nonatrophic antral gastritis. The innovation enables to increase objectivity and accuracy of diagnostics.

EFFECT: higher efficiency of diagnostics.

3 ex, 2 tbl

The invention relates to immunology

The invention relates to the diagnosis and treatment of diseases such as atherosclerosis and thrombosis

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

The invention relates to medicine, in particular to the creation of a new pharmaceutical compositions containing collagenase microbial origin

The invention relates to biotechnology, medical Microbiology, concerns the detection hydrolytically active enzyme, in particular aspartic protease in a sample or preparation

The invention relates to biotechnology and can be used for the development of anti-NPC therapeutic agents

The invention relates to the field of Bioorganic chemistry, namely to a new 6-(moselhotel-L-prolyl - L-arginyl)aminonaphthalene-1-isobutylamino formula:

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as a substrate for the determination of thrombin

The invention relates to physico-chemical biology and biotechnology, namely, biological chemistry, molecular biology, Bioorganicheskaya chemistry, and can be used by scientific institutions in the study of the properties of proteins and enzymes
The invention relates to Bioorganic chemistry, namely to new tribromide 6-(glycyl-glycyl - L-arginyl - L-arginyl)aminonaphthalene - 1-cyclohexylsulfamate and 6-(benzyloxycarbonylglycine-glycyl - L-arginyl - L-arginyl)aminonaphthalene - 1-cyclohexylsulfamic

The invention relates to the field of Bioorganic chemistry, namely to new connections dibromide 6-(D-leucyl-L-prolyl-L-arginyl), aminonaphthalene-1-pentanitroaniline (1) and its benzyloxycarbonyl (Z) is the derivative (2)
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