RU2657523C2 - Pharmaceutical aerosol composition of protease inhibitors with ozone-preserving propellant and its preparation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and is a pharmaceutical aerosol formulation of a protease inhibitor with an ozone-preserving propellant, consisting of a homogeneous mixture of an aqueous solution of an active substance from a group of protein and polypeptide protease inhibitors, containing 0.05–250 mg of the protease inhibitor per ml of the solution, and ingredients of the three-component push out system, namely to: glycerol, ethanol and water-insoluble ozone-saving propellant, with a ratio of an aqueous solution and a three-component system of 0.05–5 and 95.0–99.95 volume %, which was obtained by stepwise successive mixing of each of the listed components with a solution of the active substance, and allowing the aerosol generation of the active protease inhibitor to be generated by the propellant's buoyant force when the resulting composition is sprayed from the aerosol device to the valve.

EFFECT: invention allows to create a pharmaceutical aerosol formulation with active substances of protein and polypeptide nature from the group of protease inhibitors that allows preserving the beneficial properties of the propellant system and the biological functions and activity of protein and polypeptide substances.

6 cl, 5 tbl, 9 ex

Description

The invention relates to medicine and is aimed at creating pharmaceutical aerosols with active substances of a protein nature with propellant push systems for the treatment of many diseases in humans.

Known three-component ejection system "Modulite", consisting of a water-insoluble propellant, glycerol and ethanol, which can be mixed with aqueous solutions [Ganderton et al. 2002]. The main medical propellants of the new generation from the group of ozone-saving (free of chlorine-containing fluorocarbons; CFC-free - chlorofluorocarbons free) are 134A (1,1,1,2-tetrafluoroethane) and 227 (1,1,1,2,3,3 , 3-heptafluoropropane). However, for the use of the Modulit system, it has not been established how and in what proportion the listed four components must be combined to create a homogeneous mixture in which the protein protease inhibitor, including aprotinin, is dissolved in the aqueous phase and to prevent the protein or polypeptide protease inhibitor molecule from being denatured at the phase separation and under the influence of propellant and ethanol. Protein molecules are poorly soluble in these fluorocarbon mixtures and quickly denature, losing activity when mixed with the specified surfactants. To achieve a technical result consisting in the creation of aerosols with physiologically active proteins and polypeptides, it is necessary to select a unique ratio of these auxiliary ingredients and the active substance, which allows (1) to preserve the aerosol-generating properties of the multicomponent composition and (2) not to violate the functional properties of the active substance. To solve this twofold problem, we use the method of stepwise mixing of composite ingredients, in which glycerol, ethanol and, at the last stage, a propellant are added to the aqueous solution of the protease inhibitor.

A known method of treating influenza and other respiratory infections with an aerosol of protease inhibitors, mainly aprotinin, prepared from an aqueous solution or its dry substance [RF patent 2054180]. Aprotinin, an inhibitor of a wide spectrum of proteases, which is a naturally occurring low molecular weight polypeptide consisting of 58 amino acids (molecular weight 6 kD) [Trautschold et al. 1983]. However, this patent does not describe how to prepare an aerosol composition containing a protein or polypeptide protease inhibitor, including aprotinin, as an active ingredient, and a water-insoluble ozone-saving propellant as a buoyancy force so that the composition homogeneously mixes and does not denature the protein and polypeptide molecules inhibitor. This problem is solved in the present invention, which describes the preparation procedure and the composition of the aerosol composition, which allows to generate an aerosol of the active protein substance from the group of protease inhibitors.

Known metering aerosol devices, consisting of a container with a special coating resistant to ozone-saving propellant and a metering device (head), releasing a certain amount of aerosol at the touch of a valve. For example, devices of this type are manufactured by Bespack, Ovar 3M Pharmaceuticals and others. An aerosol device for medical use contains a pressurized propellant, active and auxiliary substances. The pharmaceutical aerosol composition is released from the device by spraying the aerosol composition through the outlet by the buoyancy of the propellant. The objective of the invention was the creation of a universal aerosol composition containing an active substance of a polypeptide nature from the group of protease inhibitors and an ejection system with a water-insoluble ozone-saving propellant suitable for use in various metering aerosol devices.

Influenza and other respiratory infections of a viral and bacterial nature cause great harm to human health. An aerosol of zanamivir powder, a neuraminidase inhibitor, for inhalation in influenza patients is proposed for the treatment of influenza [Moscona 2005]. Aerosol zanamivir inhibits the reproduction of the virus in the respiratory tract by inhibiting viral neuraminidase. It is well known that respiratory infections, including influenza infection, are accompanied by a violation of the proteolytic balance in the respiratory tract [Kido et al. 2007]. To correct this violation, it is proposed the use of protease inhibitors, which have a protein nature or polypeptide structure, consisting of amino acids and their derivatives. The most rational therapeutic method of use is direct irrigation of the source of infection in the respiratory tract with the aerosol form of protease inhibitors, among which aprotinin is a typical representative. Such an action of a protease inhibitor, firstly, blocks the multiplication of the virus by inhibiting the protease activation of viral proteins and, secondly, inhibits the pathogenesis of the disease by reducing the level of harmful proteases directly in the outbreak and in the infected body. As a result, unlike zanamivir, the aerosol of protease inhibitors has a binary antiviral and pathogenetic therapeutic effect. The developed aerosol composition based on ozone-saving propellants, mainly 134A (1,1,1,2-tetrafluoroethane), free of chlorine and therefore not oxidizing to polypeptide molecules, for use in aerosol devices generating an aerosol of an active protease protein and polypeptide structure, allows to optimize the individual use of pharmaceutical protein aerosol and to reduce cross-contamination of infection among people.

The developed pharmaceutical aerosol composition of protease inhibitors, mainly aprotinin, an inhibitor of a wide spectrum of proteases, will find wide medical application, since the violation of the proteolytic balance, which requires correction by protease inhibitors, develops in many diseases of humans and animals. In particular, antiprotease aerosol composition can be used for diseases such as respiratory infections, including influenza, keratoconjunctivitis of viral and bacterial etiology, herpetic lesions of the mucous membranes and skin, chronic obstructive bronchopneumonia, asthma and others.

Examples of the invention

Example 1. Obtaining an aerosol composition from an aqueous solution of aprotinin and ozone-saving propellant

To obtain the aerosol composition, composition N1 is shown; to obtain it, one after the other, the individual components are mixed sequentially in the following sequence and ratios: 0.12 ml of an aqueous solution containing 3.64 mg of protein (or 25,000 kallikrein inhibitory units (KIE)) of aprotinin protein , 1.2 ml of a 96% aqueous solution of glycerol are added to it; then, 2.0 ml of 96% ethanol is added to the resulting mixture, and 0.013 ml of peppermint oil is added to the resulting mixture, and at the final stage, 13.5 ml of 134A propellant is added under constant pressure in a sealed container with constant stirring. As an additional example, the aerosol composition N2 is obtained, which was obtained by mixing the components in a larger volume in the sequence shown in the composition N1. The resulting aerosol formulations have the following ratio of ingredients (table 1).

Table 1. The ratio of components in the aerosol composition of water-soluble aprotinin and water-insoluble propellant 134A Components Composition N1 Composition N2 Component Ratio Range Water Soluble Aprotinin: Aprotinin, concentrated aqueous solution 0.12 ml (contains 25,000 KIE or 3.64 mg of aprotinin protein) 0.18 ml (contains 35,000 KIE or 5.45 mg of aprotinin protein) An aqueous solution of 0.05-5 vol.% Composition Water insoluble propellant ejector system: Propellant 134A (1,1,1,2-tetrafluoroethane) CH ₂ FCF ₃ 13.5 ml 20.0 ml Propellant system as a whole 95.0-99.95 vol. % composition, including glycerol 1-7% vol., ethanol 3-13% vol., propellant 80-96% vol. system. Glycerol (96% aqueous solution) 1.2 ml 1,5 ml Ethanol (96% aqueous solution) 2.0 ml 3.3 ml Auxiliary additives: Peppermint Oil 0.013 ml 0.02 ml Additives 0.05-3 vol. % composition

The resulting aerosol composition is poured forcibly under pressure into sealed containers made of an aluminum alloy and equipped with a metered discharge valve having an opening with a diameter of 0.3 mm. Filled cylinders are stored at a temperature of 18-20 degrees. C for 0.5 and 4 years.

Example 2. Polypeptide protease inhibitors for medical purposes.

The example illustrates a list of various polypeptide protease inhibitors of nature that are dissolved in the water-glycerol-alcohol phase of the proposed aerosol composition, prepared according to the dependent paragraph 1 of the formula, for use as an active ingredient in a pharmaceutical aerosol composition.

Table 2. List of medical polypeptide protease inhibitors Inhibitor name access number
SWISS-PROT Aprotinin P00974 Alpha protease inhibitor P01009 Alpha 1-antichymotrypsin P01011 Cystatin A P01040 Cystatin C P01034 Thiostatin P01042 Calpastatin P20810 Alpha 2-Macroglobulin P01023 Alpha 1-microglobulin P02760 Tissue Pathway Factor 1 Inhibitor P10646 Tissue Pathway Factor 2 Inhibitor P48307 Serous protease inhibitor P00995 Elafin P19957 Leupeptin (Acetyl-Leu-Leu-Arg-Aldehyde) Apoptosis Inhibitor BIRC 5 015392 XIAP Apoptosis Inhibitor P98170 Caspase inhibitor c-FLIP 015519 Tissue inhibitor of metal proteases (1-4) X01683 Serpin A1 (alpha 1-antitrypsin) P01009 Serpin A3 (inti-chymotrypsin) P01011 Serpin F2 (alpha 2-antiplasmin) P08697 Serpin G1 (C1 inhibitor) P05155 Leukocyte secretory inhibitor P03973 Protein Inhibitor Bowman-BIRK X68704 Angiotensin-3 P01019 Protein Inhibitor SPINK-1 NM003122

Example 3. The lack of physical denaturation of the aerosol composition

In order to evaluate the miscibility of the ingredients in the obtained aerosol composition samples, the optical properties, i.e. transparency of the solution. To this end, the aerosol composition was released from the balloon by opening the valve and releasing the aerosol into 15 ml tubes (Falcon; Germany). Immediately after release, a portion of the collected solution from a 15-ml tube was transferred to a measurement cuvette (0.5 ml volume) and the optical density of the solution in the visible light flux was determined using an Ultraspec-2 spectrophotometer (Pharmacia, Sweden). The optical density (transparency) of the aerosol solution is compared with the optical density of distilled water, which is taken as zero. Three portions of the aerosol composition are tested: from a full balloon (issues number 10-40), issues 120-150 (half-filled balloon); issues 190-240 (last fraction of the cylinder). In total, the cylinder contained 25 ml of aerosol composition, which made it possible to make about 300 releases with a volume of 85 μl each of one cylinder. The results of the optical density of the aerosol composition of the three fractions are shown in table 3.

Table 3. Optical density of early and late aerosol fractions Aerosol Fractions Shelf life 0 0.5 years 4 years Early 0,0 ± 0,03 0,0 ± 0,03 0,0 ± 0,06 Average 0,0 ± 0,05 0,0 ± 0,04 0,0 ± 0,03 Late 0,0 ± 0,04 0,0 ± 0,02 0,0 ± 0,06

The data in table 3 show that the aerosol solution generated from the early and late fractions of the container has full transparency like distilled water. This result indicates good compatibility of the components and the absence of precipitation of the components of suspended particles in the aerosol composition both in the container and in the generated aerosol.

Example 4. Biophysical stability of aprotinin in aerosol composition

To test the biophysical properties of aprotinin, the method of fractionation of proteins in a polyacrylamide gel in an electric field is used, the so-called electrophoresis of polypeptides in a polyacrylamide gel (PAGE). Testing is carried out, firstly, early and late portions of the aerosol composition of one cylinder and, secondly, from cylinders stored for 0.5 and 4 years at a temperature of 18-22 ° C. For testing, early, middle and late portions of the aerosol composition are obtained by collecting these fractions, as described in section 3.

Equal aliquots (15 μl) are taken from the collected portions of the aerosol composition, which are mixed with 5 μl of a dissociating solution containing 5% sodium dodecyl sulfate (SDS) and 200 μM dithiothreitol (DTT), heated for 10 min at 70 ° C and applied to 3 % polyacrylamide focusing gel prepared on 0.12 M Tris-HCl (pH 6.8) and 0.1% SDS. The focusing gel has a thickness of 1.2 mm and a height of 1 cm. The separation gel had a height of 7 cm and contained 17.5% acrylamide, 0.3% methylenebisacrylamide, 0.4 M Tris-HCl (pH 8.3), 0.1 % SDS. Focusing and separation gels are polymerized using a system of catalysts - ammonium persulfate (0.05%) and tetramethylene diamine (TEMED; 0.2%). The electrode buffer contained trishydroxyaminomethane (0.03 M), glycine (0.2 M), 0.1% SDS and had a pH of 8.3, which was placed in 75 ml in the anode and cathode chambers, electrophoresis buffer system according to the Laemmli method ( 1970). Electrophoretic fractionation is carried out at 70V on a PAG plate 8 cm wide for 2 hours. After electrophoresis, the polypeptides in the gel were stained with Kumasi Blue R-350 (0.1%), which was dissolved in a mixture of water: ethanol: acetic acid in a volume ratio of 5: 5: 1 for 2 hours at room temperature. Unbound paint is washed from the gel in a mixture of water: ethanol: acetic acid with a ratio of 88: 5: 7, respectively. For comparison, a commercial preparation of purified aprotinin isolated from cattle lungs (Sigma, USA) was used as a standard sample of aprotinin.

The figure 1 shows the results of the analysis of samples of the aerosol composition obtained from the container immediately after filling and after storage for 0.5 and 4 years at room temperature (18-22 degrees C). The first, aprotinin from the aerosol composition of the early and late fractions had a typical profile of electrophoretic mobility for a polypeptide with a molecular weight of about 7 kD, and was fully consistent with the electrophoretic characteristics of standard aprotinin. Second, no high molecular weight protein precipitates were found in aprotinin aerosol samples. This macromolecular zone (VMZ), corresponding to mol. mass of 150-250 kDa, is shown in figure 1 in a frame on top of the separation gel. As can be seen in the figure, no appreciable amounts of protein were detected in the precipitate zone in the samples obtained immediately after filling the cylinders and in the samples from cylinders stored for 4 years. These results indicate that aprotinin in the aerosol composition retains its original structural properties and does not form precipitates during storage of the aerosol composition and its subsequent spraying.

After electrophoresis, proteins in the gel are evaluated by gel scanning. To do this, the gel is stained with Kumashi blue and scanned in visible light using a ScanJet 6300 scanner. The quantification of the intensity of protein spots in the scan is determined using a program that allows you to scan the optical density of the sites. Using the obtained intensity values of spots on the electrophoregram, the ratio of the area areas calculated per unit area of the scanned zone for aprotinin and impurity proteins in the high molecular weight region corresponding to molecular weights from 150 to 250 kDa is calculated. The intensity of the main peak of aprotinin (molecular weight of about 7000 daltons) is taken as 100%.

, $$N=\frac{A}{B}\times \mathrm{one\; hundred}$$

,

Where:

A is the optical intensity of the high molecular weight zone (VMZ);

B is the optical intensity of the aprotinin spot;

N is the percentage of high molecular weight impurities.

The amount of protein in the high molecular zone does not exceed 3%.

Example 5. Immunological stability of aprotinin in aerosol composition.

The immunological properties of aprotinin in the aerosol composition are tested by its interaction with specific antibodies using the Western blot method. Three samples are examined: standard aprotinin (Sigma, USA) and aerosol composition from a cylinder stored for 0.5 and 4 years at a temperature of 20-22 degrees Celsius. After electrophoresis on PAG, proteins from the gel are transferred onto a nitrocellulose protran membrane with a pore diameter of 0.45 microns (Shleiher & Schull, Germany) and then the protein adsorbed on the membrane is tested for interaction with aprotinin-specific antibodies. The interaction on the aprotinin membrane with antibodies is identified by enhanced chemiluminescence using a peroxidase conjugate with a secondary antispecies antibody. As a substrate for peroxidase, a commercial preparation by Pierce (USA) is used and its luminescence is recorded on a Kodak X-ray film (USA). First, figure 2 shows that the aprotinin of the standard and of both the studied samples of the aerosol composition interacts well with antibodies against aprotinin, which confirms its immunological stability in the aerosol composition. Second, after 4-year storage of the aerosol composition, aprotinin retains the ability to react with specific antiaprotinin antibodies. This result shows that aprotinin preserves the native immunological structure during storage of the aerosol composition for 4 years.

Example 6. The preservation of the antiprotease activity of aprotinin in an aerosol composition.

The antiprotease activity of the aerosol composition is checked by its ability to inhibit the hydrolytic function of trypsin. To do this, establish a standard amount of a standard trypsin preparation (Sigma; USA), which cleaves the chromogenic substrate L-ZAPA (Z-Arg-pNA; BAC, Switzerland) with the formation of nitroanilide (NA) with a yellow color intensity of about 0.8 units at a wavelength 405 nm (OD ₄₀₅ ). This amount is about 100 ng trypsin with a total reaction volume of 150 μl. Further, this amount of trypsin in a volume of 50 μl is mixed with serial dilutions of aerosol composition samples (volume 50 μl) and incubated for 30 min at 20 ° C to bind aprotinin with trypsin and inhibit it. After that, L-ZAPA substrate (25 μl of a solution with a concentration of 1 mg / ml) was added to the mixture, incubated for 15 min at 20 ° C and the hydrolytic reaction was stopped by adding 25 μl of a 1 M hydrochloric acid solution and the absorbance was measured at 405 nm to determine the residual trypsin activity (shown in figure 3). To calculate the molar ratio of trypsin and aprotinin, at which 50% inhibition of trypsin occurs, the concentrations of trypsin and aprotinin protein in the initial solutions are determined. To determine the concentration of aprotinin protein, the standard Bradford method is used, in which bovine serum albumin (Sigma, USA) and Kumashi blue G-250 (Sigma, USA) are taken as a standard. Given the dilution factor of the test aerosol composition, in which there was a 50% decrease in the optical density of OD _{405 of the} released substrate nitroanilide, the trypsin / aprotinin molar ratio was calculated.

The aprotinin titration curves in aerosol composition immediately after preparation of the cylinders and after 4 year storage are shown in Figure 3. Studies show that 50% trypsin inhibition is observed with the aprotinin starting material at a trypsin / aprotinin molar ratio of 1/1. When testing the aerosol composition, a similar antitrypsin activity of aprotinin is observed and 50% inhibition (shown by an arrow in figure 4) is also recorded at a trypsin / aprotinin molar ratio of 1/1. Thus, this testing shows that aprotinin remains stable in aerosol composition and stably maintains antiprotease activity at the level of the initial aprotinin during storage for 4 years.

Example 7. The preservation of the activity of protease inhibitors in a three-phase water-glycerol-alcohol aerosol mixture

An aqueous solution of protease inhibitors containing either polypeptide aprotinin, leupeptin (oligopeptide: acetyl-Leu-Leu-Arg-aldehyde (Sigma, USA)), or protein alpha 1-antitrypsin (Boehringer, Germany) with a concentration of 1 mg of dry matter per ml of solution , successively mixed with an aqueous solution of 99% glycerol solution (FisherBiotech, USA) and 96% medical grade ethanol. Two ranges of the ratio of ingredients A and B are tested. To increase the homogeneity of the aerosol composition, surfactants can be included, examples B and D. After incubation of the aerosol composition for 1 hour at 20 degrees. C determine antiprotease activity by inhibiting the hydrolytic activity of trypsin. The hydrolytic activity of trypsin is determined by the ability to cleave the chromogenic substrate L-zapa (BACNEM, Switzerland) according to the method described in Example 6. A positive inhibition reaction is the result in which a 75% decrease in the optical signal of free nitroanilide is determined in a sample with a test inhibitor sample and more compared to the control trypsin without an inhibitor. It was found that mixing protein and polypeptide protease inhibitors with a three-phase water-glycerol-alcohol mixture does not inhibit their activity in the aerosol composition.

Note *) The resulting mixture was combined with A134 propellant in a ratio of 4.5% and 95.5% or 20 and 80%, according to claim 2, was stirred at a reduced temperature, incubated for one hour and sprayed at room temperature. The generated aerosol was condensed in vitro at room temperature as described in Example 3, and the antitrypsin activity was determined. The ratio of the aqueous phase and the propellant system in the composition is 0.07% by volume (for a ratio of 4.5-95.5%) and 0.3% (for a ratio of 20-80%), respectively.

Note *) The resulting mixture was combined with A134 propellant in the ratio of 4.5% and 95.5% or 20 and 80%, according to claim 2, and the antitrypsin activity of the generated aerosol was determined. The content of dimethyl sulfoxide and tween-20 in the total aerosol composition is 0.4% by volume (for a ratio of 4.5-95.5%) and 1.8% (for a ratio of 20-80%), respectively.

Example 8. The preservation of the antiviral activity of aprotinin in aerosol composition.

To test the antiviral properties of the aerosol composition, we used the HA0 viral protein cleavage criterion and its aprotinin inhibition during propagation of the human influenza virus A / Puerto Rico / 34 (H1N1) and A / Aichi / 2/68 (H3N2) in chicken embryos. 9-day-old chicken embryos were infected by introducing the virus into the allantoic cavity of the embryo in an amount of about 1000 viral particles. Immediately after infection, the studied aerosol composition was additionally introduced into the allantoic cavity of the embryo. After 24 hours of incubation of the embryos at 37 ° C, allantoic fluid was obtained from the chicken embryos and the amount of the synthesized infectious virus accumulated and the protein composition of the virus were examined.

To prove the antiviral effect of the aerosol composition, the protein composition of the virus synthesized in chicken embryos in the presence of an aerosol composition was studied. Allantoic fluid (AF) samples were subjected to 2-step centrifugation to purify viral particles. To precipitate cell debris, AF samples were centrifuged at 4000 rpm for 30 min and then 3 ml of clarified AF was centrifuged in a Spinko L7-50 ultracentrifuge (SW 55.1 rotor) at 27000 rpm for 2.5 hours in a test tube of 5, 5 ml, at the bottom of which 2 ml of an 18% sucrose solution prepared on phosphate buffer was layered (FB: 10 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ pH 7.2; 2.7 mM KCl; 137 mM NaCl). During centrifugation, the virus passed through a sucrose layer and settled on the bottom of the tube (virus preparation), while the contaminating proteins remained in the supernatant. Viral sediment polypeptides were subjected to PAGE electrophoresis according to the Laemmli method (1970) described in section 3 above and analyzed by Western blot (WB) techniques with antibodies to the HA protein. For this purpose, proteins from the gel were transferred onto a nitrocellulose membrane (Protran; Shleiher & Schull, Germany) in a buffer for semi-dry transfer of proteins (0.05 M Tris; 0.01 M glycine; pH 9.7; 0.01% SDS; 17 5% ethanol). The transfer was carried out at 0.8 mA per cm ² membrane for 1 hour. After transfer, the membrane was saturated overnight in a 3% solution of skim milk of cows and then incubated for 1 hour at 20 ° C in PBS containing 0.5% BSA and guinea pig antibodies against the viral HA protein, and immune complexes formed on the membrane identified using peroxidase conjugate against guinea pig immunoglobulin (Pierce; USA) by enhanced chemiluminescence (ECL) with an ECL substrate (Pierce; USA).

The profile of the HA0 polypeptides (mol. Weight 75 kD) and HA1 (mol. Weight 55 kD) in the preapartes of the virus is shown in Figure 4. It should be borne in mind that the HA0 polypeptide is part of non-infectious virions, while HA1 makes the virions infectious. The transition HA0 → HA1 is carried out by trypsin proteases of the chicken embryo, to which the action of aprotinin is directed. As can be seen in figure 4, in the embryos not treated with an aerosol composition, the virus was detected in active HA1 (about 95%), which indicated the formation of an infectious virus in such embryos. In contrast, in the virus from the embryos treated with the aerosol composition, undigested HA0 prevailed (about 50%), which showed the predominant accumulation of the non-infectious virus in these embryos. These results show that aprotinin in aerosol composition retains its antiprotease function even after 4-year storage and blocks the activation of influenza virus by inhibiting the cleavage of HA0 → HA1.

To assess the effect of the aerosol composition on the reproduction of the virus, the yield of the infectious virus in chicken embryos treated and not treated with the aerosol composition was compared. The aerosol composition was tested, stored for 5 months and 4 years at room temperature (18-22 ° C). The yield of the virus was determined according to the generally accepted method of titration of an infectious virus by the method of infectious foci in a cell culture of MDSK. Each aerosol sample was introduced into 3 embryos and the virus yield was examined independently in each of the embryos. The results are shown in Table 5. As can be seen, both aerosol samples from the cylinders exhibited a pronounced virus inhibitory effect and reduced the accumulation of the infectious virus by a factor of 100 or more. It is important to note that the virus-inhibiting activity of the aerosol composition, stored for 4 years, was equal to that of the initial sample of the aerosol composition before storage. These results prove that the aerosol composition has antiviral activity in inhibiting the reproduction of the infectious virus, and this activity of the composition remains at a high initial level for at least 4 years of storage.

Table 5. Inhibition of influenza virus by aerosol composition Infectious virus titer (if / ml) Embryo Number ^*) Control (without aprotinin) 0.5 years 4 years one. 2.7 × 10 ⁷ 1.3 × 10 ⁵ 2.0 × 10 ⁵ 2. 1.9 × 10 ⁷ 1.5 × 10 ⁵ 1.7 × 10 ⁵ 3. 1.2 × 10 ⁷ 0.7 × 10 ⁵ 3.3 × 10 ⁴ four. 0.9 × 10 ⁸ 4.3 × 10 ⁴ 5.1 × 10 ⁴ 5. 2.9 × 10 ⁷ 1.8 × 10 ⁴ 2.8 × 10 ⁵ Average value 1.92 ± 2.0 × 10 ⁷ 1.14 ± 1.46 × 10 ⁵ 1.47 ± 1.1 × 10 ⁵ ( ^* ) 9-day-old chicken embryos were infected with the influenza virus by introducing about 1000 virions into the allantoic cavity of the embryo and 75 μl of the aerosol composition obtained 0.5 and 4 years after storage at a temperature of 20 degrees was additionally introduced. C. Take 5 embryos per sample composition. After 24 hours of incubation of the embryos at 37 ° C, allantoic fluid (AF) was taken from the chicken embryos and the amount of the synthesized infectious virus was determined by titration of the infectious focuses in the MDSC cell culture. The amount of virus is calculated by the number of viral particles per ml of allantoic fluid (average values ± σ). As a control, embryos were taken that were infected with the influenza virus without the introduction of an aerosol composition.

Example 9. The dispersion profile when spraying an aerosol composition.

The determination is carried out microscopically after the release of the aerosol onto the glass. The aerosol can is shaken and sprayed 1 dose onto a clean, dry, defatted glass slide perpendicular to the spray direction at a distance of 6 cm from the outlet of the spray nozzle. The determination of the value of wet particles is carried out using a microscope at 450 × magnification immediately after spraying the aerosol onto the glass. The particle diameter is determined using a transparent matrix having a marked grid. The calculation is carried out on 10 fields of view (figure 5). The measurement is carried out 3 times from one balloon at different times of aerosol release, 3 balloons are used in the test, the distribution of particles by fractions with a certain range of particle diameters is calculated as a percentage of the total number of particles on glass in the field of view.

Literary sources

1. RF patent 2054180. "A method for the treatment of viral respiratory infections, aerosol for its implementation", priority of August 21, 1991

2. 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.

3. Moscona A. 2005. Oseltamivir resistance - disabling our influenza defenses. N Engl J Med. 353 (25): 2633-6.

4. Trautschold I., Werle E., Zickgraf-Rudel G. 1967. Trasylol. Biochem. Pharmacol 16: 59-72.

5. Kido H, Okumura Y, Yamada H, Le TQ, Yano M. Proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents. Curr Pharm Des. 2007; 13 (4): 405-14. Review PubMed PMID: 17311557.

6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15; 227 (5259): 680-5. PubMed PMID-5432063.

Brief Description of the Figures

Figure 1. Structural stability of aprotinin in aerosol composition. Equivalent amounts of aerosol composition of standard aprotinin and aerosol composition samples stored for 0.5 and 4 years were subjected to electrophoresis in 15% PAG. After electrophoresis, the gels stained Kumashi with blue R350 and the stained gels were photographed in normal light. As marker proteins with a famous mole. weight used a mixture of the company Fermentas (Latvia).

Figure 2. Immunological stability of aprotinin in aerosol composition.

Samples of the aerosol composition stored for 0.5 and 4 years were subjected to electrophoresis in 15% PAG and analyzed by Western blot (WB) technique with antibodies to the aprotinin protein. The amount of protein in the aerosol composition applied to the gel track is indicated in the figure under the tracks. For the registration of proteins that react with antibodies to aprotinin, the enhanced chemiluminescence (ECL) technique with peroxidase substrate by Pierce (USA) was used. Positive protein components are shown in the figure as black zones. As marker proteins with a famous mole. weight (positions on the membrane are shown by arrows) used a mixture of the company Fermentas (Latvia).

Figure 3. Antiprotease activity of the aerosol composition. Different amounts of aerosol composition in a volume of 50 μl obtained after 0.5 and 4 years after storage at 220 ° C were mixed with 50 μl of 0.15 M phosphate buffer (FB) containing 100 ng of trypsin and incubated for 30 min at a temperature of 200 ° C. After that, 25 μl of L-zapa substrate (1 mg / ml) was added to the mixture, incubated for 30 min at 200 ° С, and the reaction was stopped by adding 25 μl of a 1 M hydrochloric acid solution. The residual trypsin activity in the sample was determined by the staining intensity (OD) of the released nitroanilide, which was measured at 405 nm. The ordinates show the average OD405 values for three independent measurements; the abscissa shows the amounts of aprotinin protein in one sample.

Figure 4. Inhibition of proteolysis of the viral protein HA0 → HA1 aerosol composition.

9-day-old chicken embryos are infected with the influenza virus by introducing about 1000 virions into the allantoic cavity of the embryo and additionally introduce 75 μl of the aerosol composition of the early and late fractions. Test two samples of the aerosol composition obtained after 0.5 and 4-year storage in aerosol cans at a temperature of 220 ° C. After 24 hours of incubation of the embryos at 370 ° C, allantoic fluid (AF) was taken from the chicken embryos from which the virus was isolated and the HA0 and HA1 protein profile was tested. Virus polypeptides are subjected to PAGE electrophoresis and tested using Western blot (WB) techniques with antibodies to the HA protein. The figure shows samples obtained from 5 individual embryos. The figure shows quantitative data on the ratio of HA0 / HA1 proteins in the viral preparation, which were obtained by scanning signals on the membrane and calculating peak areas using the TINA program. The total content of HA0 + HA1 is taken as 100%.

Figure 5. Dispersion profile of particles generated from an aerosol composition.

The aerosol composition prepared according to the recipe (1) in table 1 was sprayed from the balloon by opening the valve on the surface of defatted glass located 5.5 cm from the outlet of the balloon. The diameter of the droplets on the glass was determined using a light microscope at a magnification of 400 on a micron grid deposited on the glass. The abscissa axis shows the particle diameter ranges: 1 (0.5-10 microns), 2 (10-50 microns), 3 (50-100 microns), 4 (100-250 microns). The ordinate shows the fractional fraction of a given diameter in% of the total particle content in the aerosol.

Claims (6)

1. The pharmaceutical aerosol composition of a protease inhibitor with an ozone-saving propellant, consisting of a homogeneous mixture of an aqueous solution of the active substance from the group of protein and polypeptide protease inhibitors containing 0.05-250 mg of a protease inhibitor per ml of solution, and the ingredients of a three-component ejection system, namely : glycerol, ethanol and a water-insoluble ozone-saving propellant, with a ratio of an aqueous solution and a three-component system of 0.05-5 and 95.0-99.95 volume%, which was obtained in stepwise sequential cm by combining each of the listed components with a solution of the active substance, and allowing the aerosol of an active protease inhibitor to be generated due to the buoyancy of the propellant when spraying the resulting composition from an aerosol device with a valve. 2. The composition according to p. 1, characterized in that the aqueous solution of the protease inhibitor is gradually mixed with each subsequent component of the ejection system in the sequence glycerol, ethanol and fluorine-containing propellant, mainly 134A, taken in a volume ratio of 1-7: 3-13: 80 -96% of the system volume, respectively. 3. The composition according to p. 1, characterized in that the active substance selected from the group of protease inhibitors is represented by substances of protein and polypeptide nature, such as aprotinin, alpha 2-antiplasmin, alpha1-antitrypsin, antitrypsin, cystatin, mainly aprotinin, or a combination. 4. The composition according to p. 1, characterized in that the active substance is selected from the group of antiprotease oligopeptides consisting of two or more amino acid residues and their modified amino acid analogues. 5. The composition according to p. 1, additionally containing one or more organoleptic additives from the group of vegetable oils, including peppermint oil, aromatic mint, menthol oil in an amount of 0.05-0.9% of the total volume. 6. The composition according to p. 1, additionally containing one or more additives from the group of surfactants, including tween-20, tween-80, span-20, span-80, oleic acid, citric acid, glyceryl monooleate, dimethyl sulfoxide, polyethylene glycol , in an amount of 0.01-3.0% of the total volume.

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