RU2750933C1 - Method for testing the biological activity of aerosol preparations - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine. A method for testing the biological activity of aerosol preparations is disclosed, where aerosol preparations are medical or technical, in which an aerosol generated under the action of a coercive ejection force is released into an open container and incubated for 0.01 minutes or more to condense the liquid phase of the aerosol cloud and vaporize the volatile components of the aerosol composition, determine the quantitative yield of the aerosol composition, the concentration of active substances, their biological activity, at the same time, to prevent aggregation and denaturation of the studied active substances, 10 µl or more of deionized water or a buffer solution with a pH range from 2.0 to 13.0 is added to the container.EFFECT: invention makes it possible to test the structure and activity of pharmaceutical and other biologically active substances of aerosol composition after its spraying, that is, in the resulting aerosol cloud, which allows evaluating the effect of the aerosol process on the properties of substances in the aerosol cloud.3 cl, 10 dwg, 2 tbl, 10 ex

Description

The present invention relates to medicine. The invention discloses a method that allows you to study aerosol preparations of active substances for medical use, after their aerosol spraying using systems with forced buoyancy force in devices of propellant, mechanical, ultrasonic membrane types. The developed method is based on the condensation of the liquid water-containing phase of the generated aerosol cloud in an open container, followed by testing the biological activity of pharmaceutical and other active substances in the initial aerosol composition in the device for generating aerosol.

Known aerosol systems in which the propellant, which is under pressure in a device with a valve [1, 2], and aerosol devices (nebulizers) with mechanical or ultrasonic excitation and a membrane nebulizer, such as tabletop nebulizers of ejector or ultrasonic types and manual inhalers of mechanical type "respimat" or manual inhaler of membrane type Micro-Air "Omron" [3]. When the valve is opened or when aerosol excitation is started, the aerosol composition is ejected and sprayed, the so-called aerosol generation.

A known method for assessing the physical properties of the generated aerosol, such as the particle size of the generated aerosol or the stability of aerosol particles at different distances from the spray device after the release of the aerosol cloud [4]. Such measurements of dispersion or distance stability of aerosol are carried out either automatically by scattering the laser beam by a cloud of aerosol on a laser particle detector (a number of companies: Horiba, Malvern, Shimadzu, Sympatec Helos), operating on the principle of laser diffraction and an optical controller [4, 5], or with the help of a light or electron microscope when studying aerosol drops that have fallen on glass or a substrate, placed at different distances from the aerosol device (nebulizer) [6]. However, these methods do not allow assessing the structural and biological safety of pharmaceutical and other biologically active substances included in the aerosol composition after being sprayed in the resulting aerosol cloud.

We have developed a simple and convenient method that makes it possible to study the structural stability and biological activity of pharmaceutical, disinfecting and other biologically active substances in the resulting aerosol cloud generated from an aerosol composition in a device with a propellant system or other types of devices with forced aerosolization. The essence of the method is the condensation of the liquid water-containing phase of the aerosol cloud into an open container, which allows collecting the resulting condensate and natural evaporation of the propellant and other volatile substances of the aerosol composition. The developed method is applicable both for aqueous aerosols (so-called wet aerosols) and propellant compositions with micronized active substances (so-called dry aerosols). After collecting the condensate, the concentration, structure and biological activity of the contained pharmaceutical and other biologically active substances are determined and recalculated to the original aerosol composition. For more effective dissolution and prevention of aggregation and denaturation of the studied active substances in the resulting condensate, an additional amount of deionized water or a buffer solution with a pH of 2.0-12.0 containing surfactants from the group of surfactants or organic solvents, such as tween-20, tween-80, span-20, span-80, sodium dodecyl sulfate, nonidet-P40, triton X-100, beta-octylglucoside, dimethyl sulfoxide, formamide, acetonitrile and others.

The advantage of the developed method is that it allows testing the structure and activity of pharmaceutical and other biologically active substances of aerosol composition after aerosol spraying (aerosol generation), i.e. in the resulting aerosol cloud. Thus, the method makes it possible to assess the effect of the aerosolization process on the properties of biologically active substances and their structural and functional stability in an aerosol cloud. The condensate obtained according to the presented method, devoid of gaseous components of the aerosol composition, makes it possible to determine the biological activity of pharmaceutical, disinfectants and other substances by direct methods of assessing the corresponding enzymatic activities or assessing various physiological activities, such as antiviral, antimicrobial or metabolic activities in cell cultures, organ cultures, nutrient media, animals, etc.

The developed method is of particular value for testing aerosol formulations containing labile substances, such as protein, oligopeptide or multicomponent substances, which can be inactivated during phase transitions from a liquid to a gaseous phase during aerosolization. The creation of protein-containing aerosol compositions using genetic engineering methods is a rapidly developing trend in modern medical aerosol biotechnology and other areas of industrial and household appliances. This stage in aerosol biotechnology poses new problems, one of which is solved in the present invention, which increases the relevance of the invention.

For the implementation of the task, the following method and approaches for its solution are proposed, namely:

• A method for testing the biological activity of aerosol preparations, in which aerosol preparations are medical or technical, characterized in that the aerosol generated by the forced buoyancy force is released into an open container and incubated for 0.01 min or more to condense the liquid phase of the aerosol cloud and evaporation of volatile components of the aerosol composition, determine the quantitative yield of the aerosol composition, the concentration of active substances, their biological activity, while in order to prevent aggregation and denaturation of the active substances under study, 10 μl or more of deionized water or a buffer solution with a pH range of 2.0 to 13.0.

• For more effective dissolution and prevention of aggregation and denaturation of the studied active substances in the resulting condensate, add 10 μl or more of deionized water or a buffer solution with a pH in the range of 2.0-13.0, containing 0.01 - 99% surfactants to the container substances and co-solvents, mainly tween-20, tween-80, span-20, span-80, sodium dodecyl sulfate, nonidet-P40, triton X-100, beta-octylglucoside, dimethyl sulfoxide, formamide, acetonitrile.

Description of figures

Fig. 1. Electrophoretic profile of aprotinin after aerosol spray and protein markers

The lanes show the profile of aprotinin from the formulation after storage for 1 year (lane 3) and 7 years (4) in an aerosol formulation in a valve device at room temperature) and standard aprotinin (Sigma; lane 2). Lane 1 - Protein molecular weight markers. The squares mark the zones of the polyacrylamide gel corresponding to the native aprotinin with mm. 6kD and high molecular weight zone with mm. 60-250 kD (VMZ) for quantitative scanning. The high molecular weight zone contains less than 5% of the mass of proteins in the aprotinin zone.

Fig. 2. Titration of Sigma standard aprotinin

A dose of about 0.3 CIU of aprotinin (shown by the dotted line) caused 50% inhibition of trypsin under the reaction conditions.

Fig. 3. Titration of the test condensate of aerosol aprotinin.

Fig. 4. Testing the antiviral activity of aprotinin in the generated aerosol using the developed method using cell culture

Cell cultures stained with antiviral antibodies were photographed under a light microscope at a magnification of × 80. Wells 1, 2, 3 - show cell samples incubated without condensation (1), with 30 μl of condensate obtained by nebulization from the device, stored with an aerosol formulation for 1 year (2) and with 30 μl of condensate obtained by nebulization from an aerosol device, stored with aerosol composition for 7 years, respectively.

Fig. 5. Testing the anti-trypsin activity of the test condensate of aerosol leupeptin.

Fig. 6. Testing of condensate of aerosol aprotinin.

Fig. 7. Determination of the content of ribavirin in the aerosol cloud

The absorbance of control solutions and samples of aerosol ribavirin condensate was measured at 270 nm. Columns: 1 - sample of 30% glycerin in deionized water; 2 - sample of 75% glycerin solution; 3 - sample of 30 μl of aerosol ribavirin condensate in 500 μl of deionized water; 4 - sample of 70 μl of aerosol ribavirin condensate in 500 μl of deionized water.

Fig. 8. Electrophoretic profile of aprotinin after aerosol spraying and condensation at pH 11.5 in the presence of DMSO

The lanes show the profile of aprotinin from the formulation after storage for 1 year (lane 2) and 7 years (3) in an aerosol formulation in a valve device at room temperature) and the original aprotinin (Sigma; lane 1). On the left, the positions of marker proteins with known molecular weights are shown.

Fig. 9. Testing of condensate of aerosol aprotinin after spraying "in a soft cloud"

Columns: 1 - buffer control sample without trypsin and without condensation (zero control); 2 - sample with trypsin without adding condensate (100% control); 3 - sample containing trypsin and 10 μl of the investigated aerosol condensate with aprotinin.

Fig. 10. Testing the condensate of aerosol favipiravir after spraying "in a soft cloud"

The optical density of control solutions and condensate samples of aerosol favipiravir was measured at 360 nm in 0.5 ml cuvettes. Columns: 1 - deionized water sample; 2 - sample of the control drug without favipiravir; 3 - sample of 25 μl of aerosol favipiravir condensate in 500 μl of deionized water; 4 - sample of 60 μl of aerosol favipiravir condensate in 500 μl of deionized water.

Examples of implementation of the invention

Example 1. Evaluation of the total water-containing liquid fraction of the generated aerosol cloud

The developed method makes it possible to estimate the total content of the active liquid aerosol fraction after spraying the propellant aerosol composition. For this purpose, the aerosol composition is released into the container using the developed method, and after evaporation of the volatile components of the composition, the weight and volumetric amount of the formed condensate is determined. In parallel, the amount of the sprayed composition is determined by weighing the aerosol can (or other device with a valve) before and after generating the aerosol. The results of the obtained measurements are presented in table 1.

1) An aerosol formulation was prepared in a device weighing 297.165 g with a metered valve allowing the release of about 55 μl of the aerosol formulation per press.

2) An aerosol composition was prepared according to the following recipe per dose (release): A134 propellant (0.047 ml); ethanol (0.0075 ml), glycerol (0.0035 ml), aprotinin (0.0005 ml; 95 KIE).

3) Three series of tests were carried out, in each test 10 releases were made into a 10 ml conical tube. After the aerosol was released, the condensate from the walls was collected by centrifugation at 1500 rpm for 2 minutes. The resulting condensate was incubated for 30 min with constant stirring to evaporate volatile components and examined as indicated in the table. The standard deviation shows the average spread of honey by the values according to the recipe and in the experimental determination, expressed in% of the value according to the recipe for the preparation of the aerosol composition.

Example 2. Evaluation of the native structure of an active pharmaceutical substance in a generated aerosol

To study the preservation of the native properties of the active substance of a protein nature, an aerosol composition was used prepared with an aqueous solution of a natural protease inhibitor (aprotinin) isolated from cow lungs, mixed with propellant 134A, glycerin and alcohol in the ratio of substances: propellant A134 (0.047 ml); ethanol (0.0075 ml), glycerol (0.0035 ml), aprotinin (0.0005 ml; 95 KIE). The contents of the can was discharged into a 10 ml conical tube through 25 outlets and the condensate was collected at the bottom of the tube by centrifugation at 1500 rpm for 2 minutes. Then the condensate was incubated for 30 min at a temperature of 20 ° C with the lid open, with constant shaking (to evaporate the residues of the propellant and alcohol), then the volume of the condensate was brought to 350 μL with a solution of 0.02 M Tris-HCl (pH 7.5) ... The structure of aprotinin is determined by the mobility in the gel in high-resolution polyacrylamide gel electrophoresis (PAG) containing sodium dodecyl sulfate (SDS) in the Tris-glycine buffer system of Laemmli (1970). For analysis, take 2, 5 and 10 μl of the test solution, add 13, 10 and 5 μl of deionized water to the samples, respectively, add 6 μl of dissociating buffer solution containing 10% sodium dodecyl sulfate and 5% mercaptoethanol to completely unwind the aprotinin molecule and breaking all disulfide bonds. The obtained samples are heated at a temperature of 75 ° C for 10 min for better dissociation of aprotinin molecules. Samples are applied to wells of 3.5% focusing polyacrylamide gel prepared over 12% polyacrylamide separating gel, and electrophoresis is carried out in an electric field at 50V on the gel for 3 hours.

After electrophoresis, the gel plate is fixed for 1 hour in an aqueous solution containing 50% ethanol and 10% acetic acid. After fixation, the gel is stained for 2 hours in 0.02% Kumasi blue G-250 solution prepared in a solution containing 40% ethanol and 10% acetic acid. Figure 1 shows the test results.

The result of the analysis in PAG shows that after spraying, aprotinin retains its native properties and has a mobility corresponding to the protein with m.w. 6kD, does not have degradation products and high molecular weight aggregation products.

Example 3. Evaluation of the specific antiprotease activity of aprotinin in the generated aerosol

To test the preservation of the specific enzymatic properties of the active substance of a protein nature, an aerosol composition prepared with an aqueous solution of a natural protease inhibitor (aprotinin) isolated from cow lungs was used. The preparation of the propellant aerosol composition of aprotinin and the production of the condensate by the method according to the invention is given in Example 2 above. The specific activity of the protease inhibitor (aprotinin) was analyzed by inhibiting the cleavage of the chromogenic substrate by the proteolytic enzyme, trypsin. The method is based on inhibition of trypsin-catalyzed cleavage of a chromogenic substrate, N-a-benzoyl-DL-arganine-4-nitroanilide (BAPNA), which acquires a color upon cleavage. The difference in the activity of trypsin and trypsin inactivated by aprotinin is determined by the intensity of color staining, which is recorded at a wavelength of 405 nm. One unit of protease inhibitor (NIT) inhibits the activity of two protease units of trypsin by 50%. It is known that one NIT corresponds to 1330 KIE (Kallikrein Inhibitory Unit). 1 KIE is a unit of protease inhibitor activity that inhibits 2 kallikrein units of protease by 50% under optimal conditions). Crystalline trypsin (Sigma), N-a-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) (Sigma) were used, aprotinin (MERK) with a specific activity of 4500-7000 KIE / mg (according to the certificate) was used as a standard ...

At the first stage, two-fold dilutions of standard trypsin are prepared in a buffer containing 25 mM Tris-HCl (pH 7.8) and 250 mM sodium chloride, and 50 μl of each dilution are added to the wells of a 96-well plate. 2-fold dilutions of trypsin are prepared starting at a concentration of 160 μg / ml and ending with a concentration of 1.25 μg / ml. Then, water, the chromogenic substrate BAPNA (0.8 mg / ml is prepared by diluting the stock solution in water) are successively added and incubated for an hour at 35 ° C. The reaction is stopped by the addition of 35% acetic acid and the optical density (OD) is determined at 405 nm on a microplate spectrophotometer (BioRad Model 550). On the basis of the obtained values, trypsin dilution is determined, inducing OD405 (optimal in the range of 0.5-1.5), which is used in titration of aprotinin in the investigated aerosol condensate. According to testing data, this dose of 50% inhibition was 0.3 μg of trypsin per sample.

To construct a calibration curve for aprotinin, two-fold dilutions of a standard aprotinin solution are prepared, starting with a concentration of 50 μg / ml and ending with 0.25 μg / ml. In the same way, prepare two-fold dilutions of the test condensate in a volume of 50 μl, starting with a dilution of 1/20, by two-fold dilutions. Calibration curves for determining the specific anti-trypsin activity of the standard and test aprotinin preparations are shown in Figures 2 and 3. The protocol for testing antiprotease activity with trypsin is shown in Table 2.

The optical density for the test sample of aprotinin shall be 50% of the optical density of the trypsin sample. The calculation of the total activity of aprotinin X (in KIE) contained in the condensate in terms of the original aerosol composition is carried out according to the formula

A _st is the activity of standard aprotinin at the point of 50% inhibition on the calibration curve (KIE), taking into account the specific activity according to the certificate (shown in Fig. 2);

V _o - the total volume of the original test solution (condensate) of aprotinin in 10 outlets (table 1);

K _p is the dilution factor of the test solution of aprotinin at the point of 50% inhibition of trypsin on the curve of the test solution (shown in Fig. 3);

V _pp - the volume of the test solution of aprotinin in the sample (0.05 ml);

K _p is a correction factor due to partial loss of the drug when collecting aerosol condensate from the can (value is 1.2).

The test solution caused 50% inhibition of trypsin at a dilution of about 1/750 under the reaction conditions (shown by the dashed line in Fig. 3). According to the formula for calculating the activity of aprotinin in the preparation:

Taking into account the obtained values Ast = 0.3 KIE and Kp ≈ 750 (shown in Figures 2 and 3), the total activity of aprotinin in the condensate of aerosol aprotinin in the amount of 20 releases was 1720 KIE, which was about 90% of the calculated value according to the recipe for preparing the composition ...

Example 4. Evaluation of the antiviral activity of aprotinin in the generated aerosol by the developed method using cell culture

To test the antiviral activity of a protein substance, an aerosol composition prepared with an aqueous solution of a natural protease inhibitor (aprotinin) isolated from cow lungs was used. The preparation of a propellant aerosol composition of aprotinin and the production of a condensate by the method according to the invention is given above in Example 2. The specific antiviral activity of the obtained aerosol condensate of a protease inhibitor (aprotinin) was tested by the method of focus reduction of the human influenza type A virus, strain A / Aichi / 2/68 (H3N2), which he formed when multiplying in cell culture from human lungs (line Calu-3).

Calu-3 cells were grown in 24-well plates in DMEM medium containing antibiotics and 10% fetal bovine serum. Then, after the formation of the cell monolayer, a serum-free DMEM medium containing the influenza virus A / Aichi / 2/68 was added at the rate of about 100 focus forming units per cell well, and incubated for 1 hour to infect the cells with the virus. Then the medium was removed and a new portion of the DMEM medium (1.3 ml per well) was added, containing various amounts of the investigated aerosol condensate of aprotinin, and incubated for 30 hours at 37 ° C in a CO2 incubator to form viral foci in cell culture.

The cell culture was fixed with a 4% paraformaldehyde solution and viral foci were stained with specific antiviral antibodies (anti-H3 subtype) and peroxidase conjugates, using True Blue as a chromogenic substrate. The results are shown in Figure 4.

As shown in Figure 4, in the control cell samples incubated in a medium without condensation, the formation of distinct viral foci with a diameter of 1.5 to 4.0 mm was observed. In samples containing 50 μl of condensate, foci were smaller with a diameter of 0.3-1.0 mm and their number was more than 5 times lower than in the control sample. The accumulation of the infectious virus in the cellular environment decreased in the presence of condensate by more than 100 compared with the control. This result indicated that aprotinin, after being sprayed from an aerosol device stored with an aerosol composition for 1 and 7 years, retained its biological antiviral activity.

Example 5. Evaluation of the antiprotease activity of leupeptin in the generated aerosol

Leupeptin is a tripeptide (acetyl-Leu-Leu-Arg-aldehyde) with antiprosthetic activity against a wide range of serine proteases. An aerosol composition was prepared with an aqueous solution of leupeptin mixed with propellant 134A, glycerin and alcohol in the ratio of substances: propellant A134 (13 ml); ethanol (1.35 ml), glycerol (2.5 ml), leupeptin (1.5 mg; 0.8 ml of an aqueous solution). The suspension was prepared in a device with a metering valve (60 μl per outlet) with a 0.3 mm spray hole. The specific activity of leupeptin was tested by inhibiting the cleavage of the chromogenic substrate by the proteolytic enzyme trypsin. The method is based on inhibition of trypsin-catalyzed cleavage of a chromogenic substrate, N-a-benzoyl-DL-arginine-4-nitroanilide (BAPNA), which acquires color upon cleavage. The difference in the activity of trypsin and trypsin inactivated by leupeptin is determined by the intensity of color staining, which is recorded at a wavelength of 405 nm.

The contents of the aerosol can was discharged into the container through 25 outlets and the condensate was collected at the bottom of the tube by centrifugation at 1500 rpm for 2 minutes. Then the condensate was incubated for 30 min at a temperature of 20 ° C with constant shaking (to evaporate the residues of the propellant and alcohol), then the volume of the condensate was brought to 200 μL with a solution of 0.02 M Tris-HCl (pH 7.5). To construct a calibration curve for the activity of antiprotease leupetin, two-fold dilutions of the test condensate in a volume of 50 μl were prepared, starting from a dilution of 1/40, by two-fold dilutions in 0.02 M Tris-HCl (pH 7.5). Each well was filled with 50 μl of buffer containing 0.3 μg of trypsin. This amount of trypsin is determined based on the values obtained for dilutions of trypsin, inducing OP405 (in the optimal range of 0.5-1.5). Further, according to the protocol (table 2), water, the chromogenic substrate BAPNA are sequentially added and incubated for an hour at 35 ° C. The reaction is stopped by the addition of 35% acetic acid and the optical density (OD) is determined at 405 nm on a BioRad Model 550 spectrophotometer. The results are shown in Fig. five.

50% inhibition of trypsin was observed at a leupeptin content of about 0.003-0.0017 μg. Thus, after spraying, leupeptin retained its enzymatic activity close to the initial level in the initial aerosol composition.

Example 6. Evaluation of the anti-protease activity of aprotinin in the generated aerosol when collected in the presence of surfactants

When testing a number of pharmaceutical substances, their aggregation is possible during the condensation of an aerosol preparation. For this purpose, the addition of surface active compounds will be required in the condensate collection tank. For this purpose, 150 μl of a buffer containing 0.03 M Tris-HCl (pH 7.5), 50% dimethyl sulfoxide, 20% tween-20 was introduced into a 10 ml test tube. The aerosol composition of aprotinin was prepared according to the prescription in section 2. The contents of the aerosol can was discharged into the prepared container through 25 outlets and the condensate was collected at the bottom of the test tube by centrifugation at 1500 rpm for 2 minutes. Then the condensate was incubated for 30 min at a temperature of 20 ° C with constant shaking (to evaporate the residues of the propellant and alcohol), then the volume of the condensate was brought to 350 μL with a solution of 0.02 M Tris-HCl (pH 7.5).

To construct the calibration curve of aprotinin, two-fold dilutions of the investigated condensate in a volume of 50 μl were prepared, starting from a dilution of 1/40, by two-fold dilutions in 0.02 M Tris-HCl (pH 7.5). Each well was filled with 50 μl of buffer containing 0.3 μg of trypsin. This amount of trypsin is determined based on the values obtained for dilutions of trypsin, inducing OP405 (in the optimal range of 0.5-1.5). According to the test data, such a dose of 50% inhibition under the conditions of the reaction protocol was a dose of 0.3 μg of trypsin per sample. Further, according to the protocol (table 2), water, the chromogenic substrate BAPNA are sequentially added and incubated for an hour at 35 ° C. The reaction is stopped by the addition of 35% acetic acid and the optical density (OD) is determined at 405 nm on a BioRad Model 550 spectrophotometer. The results are shown in Fig. 6.

The test solution caused 50% inhibition of trypsin at a dilution of about 1/800 under the reaction conditions (arrowed). According to the formula for calculating the activity of aprotinin in the preparation, as described in Section 2, the total activity of aprotinin in the condensate of aerosolized aprotinin in the amount of 40 releases was 3640 KIE, which was about 93% of the calculated value according to the recipe for preparing the composition.

Example 7. Evaluation of the content of ribavirin in the generated propellant aerosol

Ribavirin (1 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -1H-1,2,4-triazole-3-carboxamide) has antiviral activity against a wide range of viruses. An aerosol composition was prepared with an aqueous solution of ribavirin mixed with propellant 134A, glycerin and alcohol in the ratio of substances: propellant A134 (13 ml); ethanol (1.35 ml), glycerin (2.5 ml), ribavirin (10.0 mg; 0.8 ml aqueous solution). An aerosol formulation was prepared in a device with a metering valve (60 μL per outlet) with a 0.3 mm spray hole. The content of ribavirin in aerosol condensate by the intensity of absorption in the ultraviolet range at a wavelength of 270 nm. The zero reference standard was an aqueous solution of 75% glycerol and deionized water.

The contents of the aerosol can was discharged into the container through 25 outlets and the condensate was collected at the bottom of the tube by centrifugation at 1500 rpm for 2 minutes. Then the condensate was incubated for 30 min at a temperature of 20 ° C with constant shaking (to evaporate the residues of the propellant and alcohol), then the volume of the condensate was brought to 200 μL with deionized water. To construct a quantitative calibration curve, solutions were prepared with different concentrations of ribavirin in the range of 0.005-5.0 mg per ml. Optical density (OD) was measured on an ULTRASPEC II (LKB) spectrophotometer at 270 nm in 0.5 ml cuvettes. The results are shown in Fig. 7.

Based on a comparison with the calibration curve obtained for marker solutions of ribavirin, it was found that the concentration of ribavirin in the condensate was about 91% of its content obtained by calculation according to the recipe for preparing the aerosol composition and the sprayed volume in one release.

Example 8. Evaluation of the structure of aprotinin in the generated aerosol when collected in the presence of surfactants at pH 11.5

To study the structure of a proteinaceous substance, an aerosol composition was used prepared with an aqueous solution of a natural protease inhibitor (aprotinin) mixed with a 134A propellant, glycerin and alcohol in a substance ratio: A134 propellant (0.047 ml); ethanol (0.0075 ml), glycerol (0.0035 ml), aprotinin (0.0005 ml; 95 KIE). The contents of the can in a volume of 40 aerosol portions were released into a 10 ml conical tube containing 200 μl of 0.04 M Tris-HCl (pH 11.5) and 50% DMSO, the formed condensate was collected at the bottom of the tube by centrifugation at 1500 rpm for 2 minutes. Then the condensate was incubated for 30 min at 37 ° C with constant shaking (to evaporate the residues of the propellant and alcohol). The structure of aprotinin was tested for mobility in a gel in high-resolution polyacrylamide gel electrophoresis (PAG) containing sodium dodecyl sulfate (SDS) in a Laemmli Tris-glycine buffer system (1970) [7]. For analysis, 10 μL of the test condensate was applied to the gel, into which 6 μL of a dissociating buffer solution containing 10% sodium dodecyl sulfate and 5% mercaptoethanol was added to completely dissociate aprotinin molecules and break all disulfide bonds. The samples obtained were heated at a temperature of 75 ° C for 10 min for better dissociation of aprotinin molecules. Samples were applied to wells of 3.5% focusing polyacrylamide gel, located over a 12% separating gel, and electrophoresis was performed in an electric field at 50V on the gel for 3 hours.

After electrophoresis, the gel plate was fixed in an aqueous solution containing 50% ethanol and 10% acetic acid. After fixation, the gel was stained for 2 hours in 0.02% Kumasi blue G-250 solution prepared in a solution containing 40% ethanol and 10% acetic acid. Figure 8 shows the test results.

The results of the analysis in PAG show that after aerosol spraying and condensation in a solution with pH 11.5 containing 50% DMSO, aprotinin retained its native properties and had the mobility of standard aprotinin, while no degradation by-products and high-molecular aggregation products were observed.

Example 9. Evaluation of the antiprotease activity of aprotinin in the generated "soft cloud" aerosol ("Respimat" system)

The Respimat device was filled through a cartridge with an aqueous solution containing 7000 KIE / ml aprotinin, 3% glycerol, 3% ethanol, according to the instructions for the Respimat device (Boehringer Inhelheim, Germany). The resulting aerosol preparation was released into a 50 ml test tube in the amount of 30 starts and collected by centrifugation at 1500 rpm for 2 min, the aerosol condensate was incubated for 15 min at a temperature of 25 ° C with periodic shaking (to evaporate ethanol residues) and added 150 μl of 0.02 M Tris-HCl solution (pH 7.5). To test the antiprotease activity of the resulting condensate, two-fold dilutions were prepared in a volume of 50 μl, starting with a dilution of 1/10. Each well was filled with 50 μl of buffer containing 0.3 μg of trypsin. Then, according to the protocol (table 2), water and the chromogenic substrate BAPNA were sequentially added and incubated for an hour at 35 ° C. The reaction was stopped by the addition of 35% acetic acid and the optical density (OD) was determined at 405 nm on a BioRad Model 550 spectrophotometer. The results are shown in Fig. nine.

As can be seen in the figure, aprotinin in aerosol condensate retained its antiprotease activity and inhibited trypsin activity by 96%.

Example 10. Evaluation of the content of favipiravir in the generated "soft cloud" aerosol ("Respimat" system)

The Respimat device was filled through a cartridge with an aqueous solution containing 3 mg / ml favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide), 3% glycerol, 3% ethanol, 0.5% DMSO, according to the instructions for the Respimat device (Boehringer Inhelheim , Germany). The resulting aerosol composition was released into a 50 ml test tube in the amount of 25 runs and collected by centrifugation. The aerosol condensate was incubated for 20 min at a temperature of 25 ° C with periodic shaking (to evaporate the residual ethanol), and 150 μl of 0.02 M Tris-HCl (pH 7.5) was added. To construct a quantitative calibration curve, solutions were prepared with different concentrations of favipiravir in the range of 0.005-15.0 mg / ml. Optical density (OD) was measured on an ULTRASPEC II (LKB) spectrophotometer at a maximum absorbance of 360 nm. The results are shown in Fig. 10.

Based on a comparison with the calibration curve obtained for marker solutions of favipiravir, it was found that the concentration of ribavirin in the condensate was about 94% of its content obtained by calculation based on the recipe for preparing the aerosol composition and the sprayed volume in one release.

Literature sources

1. Ganderton D, Lewis D, Davies R, Meakin B, Brambilla G, Church T. 2002. Modulite: a means of designing the aerosols generated by pressurized metered dose inhalers. Respir Med. 96 Suppl D: S3-8.

2. Meshcheryakova H.H. 2006. Modulite - a new technology in metered-dose aerosol inhalers. Inhalation therapy. Atmosphere. Pulmonology and Allergology. No. 2, 40-42.

3. Terekhova E.P. 2014. Inhalation devices used to treat bronchial asthma. Practical Pulmonology. No. 4, pp. 45-52.

4. Black D.L., McQuay M.Q., M. P. Bonin. 1996. Laser-based techniques for particle-size measurement: A review of sizing methods and their industrial applications. Progress in Energy and Combustion Science. Vol. 22 (3), pages 267-306.

5. Davies, N.M. and M.R. Feddah, 2003. A novel method for assessing dissolution of aerosol inhaler products. Int. J. Pharm. 255: 175-187.

6. Dolovich, M., 1991. Measurement of particle size characteristics of metered dose inhaler (MDI) aerosols. J. Aerosol Med. 4: 251-263.

7. Laemmli U. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227, 680-685 (1970). https://doi.org/10.1038/227680a0

Claims (3)

1. A method for testing the biological activity of aerosol preparations, where aerosol preparations are medical or technical, characterized in that the aerosol generated by the forced buoyancy force is released into an open container and incubated for 0.01 min or more to condense the liquid phase of the aerosol cloud and evaporation of volatile components of the aerosol composition, determine the quantitative yield of the aerosol composition, the concentration of active substances, their biological activity, while to prevent aggregation and denaturation of the active substances under study, 10 μl or more of deionized water or a buffer solution with a pH range of 2.0 up to 13.0. 2. The method according to claim 1, characterized in that 10 μl or more of a buffer solution with a pH in the range of 2.0-13.0, containing 0.01-99% of surfactants and co-solvents, preferably tween 20, tween-80, span-20, span-80, sodium dodecyl sulfate, nonidet-P40, triton X-100, beta-octylglucoside, dimethyl sulfoxide, formamide, acetonitrile. 3. The method according to claim 1, characterized in that 10 μl or more of deionized water containing 0.01-99% of surfactants, mainly Tween-20, Tween-80, Span-20, is introduced into the container after the aerosol is injected. span-80, sodium dodecyl sulfate, nonidet-P40, triton X-100, beta-octylglucoside, dimethyl sulfoxide, formamide, acetonitrile.

Images