GB2511011A - Aerosol pharmaceutical composition of protease inhibitors and production thereof - Google Patents

Abstract

The invention relates to medicine and describes a medicinal composition of water-soluble protein or polypeptide compounds from the group of proteolytic enzyme inhibitors for the creation of an aerosol with the aid of a water-insoluble, ozone-saving propellant in aerosol devices provided with a metering valve. The claimed aerosol composition can be used for treating diseases that are accompanied by a proteolytic imbalance, such as respiratory infections, including influenza, viral or bacterial keratoconjunctivitis, herpetic damage to the mucous membranes and skin, chronic obstructive bronchopneumonia, asthma, etc.

Description

Pharmaceutical aerosol formulation of protease inhibitors and its preparation

Description of the Invention

Field of invention

The invention relates to medicine, and aimed at the creation of pharmaceutical aerosols with active substances of protein nature and propellant cjcction systems for thc treatment of a wide range of human diseases.

Background to the invention

The ejection system Modulite' consisting of water-insoluble ozone-saving propellant.

glycerol and ethanol, which has been used for the manufacture of anti-asthma aerosol with an active substance of synthetic non-protein nature of beelomethasone type, is known [Cabderton et al. 20021. The major medical new generation propellants from the ozone-preserving group (free from chlorinated fluorocarbons; CFC-free-chlorofluorocarbons free) are 134A (1,1,1,2-tetrafluoroethane) and 227 (1,1, ,2,3,3,3 -heptafluoropropane). l'he propellant system Modulite can partially mix with an aqueous phase. However, for the application of the Modulite system it is not known how and in what proportion the four listed components should he mixed in order to create a homogeneous mixture, in the aqueous phase of which a protein protease inhibitor, including aprotinin, has been initially dissolved, and to prevent denaturation of the protein or polypeptide molecule of the protease inhibitor on the possible phase interface wider the influence of propellant or ethanol. Protein molecules are poorly soluble in the specified fluorocarbon mixtures; they lose natural structure and functional activity, when mixed with the indicated surfactants.

A method of treatment of influenza and other respiratory infections with aerosol of protease inhibitors, preferably of aprotinin, prepared from an aqueous solution or from its dry substance, is known [patent RF 2054851]. Aprotinin is a broad spectrum protease inhibitor, which is a natural low molecL1l'\veight polypeptide consisting of 58 amino acids (MW 6 kDa) [Trautsehold Ct al. 1983j. However, it is not described in this patent, how to prepare an aerosol formulation containing a protein or polypeptide protease inhibitor, including aprotinin, as an active ingredient, and insoluble ozone-preserving propellant as an ejecting force, so that the composition would mix homogeneously and would not denature a protein and polypeptide molecule of the inhibitor when sprayed from an aerosol device with a valve. This problem is solved in the present invention, which describes a quantitative ratio of the components and their mixing procedure for the preparation of an aerosol composition allowing generating the aerosol of an active protein substance from the group of protease inhibitors.

There arc known aerosol dispensing devices consisting of a container with a special coating resistant to ozone-preserving propellant and with a dosing unit (head), which produces a certain amount of aerosol per one press of a valve. For example, devices of this type are manufactured by Bespack. Ovar 3M Pharmaceuticals, etc. The aerosol device for medical use contains pressurized propellant, active substances and additives. Pharmaceutical aerosol formulation is sprayed from the device by means of the aerosol composition sputtering through an output oracle under the action of the propellant ejecting force.

Principle of Invention The objective of the invention was the creation of a universal aerosol formulation containing an active substance of polypeptide nature from tile group of protease inhibitors and the ejecting system with water-insoluble ozone-preserving propellant, which is suitable for the application in various aerosol generating devices In order to achieve a technical result. which consists in the creation of aerosols with physiologically active proteins and polypeptides, it is necessary to select a unique ratio and mode of mixing of the indicated additional ingredients and active substance, aLlowing (I) to preserve the aerosol generating properties of the multicomponent composition and (2) not to disrupt functional properties of the active substance. In order to solve this double task, we use a unique ratio of compositional ingredients and the method of their gradual mixing, at which the aqueous solution of protease inhibitor is sequentially mixed with glycerol, ethanol, and other additives, and with propellant in the final stage.

The developed aerosol formulation based on the ozone-preserving propellants. preferentially 1 34A (1,1,1,2-preserving tetrafluoroethane), which is free of chlorine and therefore does not have an oxidative effect on polypeptide molecules, is intended for use in aerosol devices generating aerosol of an active protease inhibitor with protein and polypeptide structure, and allows to optimize an individual use of pharmaceutical protein aerosol and to reduce cross-contamination of infection among people.

Medical Applicability

Influenza and other respiratory infections of viral and bacterial nature cause an enormous harm to human health. Inhalation of zanamivir powder aerosol, which is a neuraminidase inhibitor, is proposed for the treatment of influenza [Moscona, 2005]. Aerosol zanamivir inhibits virus replication in the respiratory tract through inhibition of viral neuraminidase. It is well known that respiratory infections, including influenza infection, are accompanied by disturbances of proteolytie balance in the respiratory tract [Kido et at. 20071. To normalize this dysbalance we suggest the application of protease inhibitors of protein nature or with polypeptide structure consisting of amino acids and their derivatives. The most rational therapeutic method of application is a direct treatment of the infection focus in the respiratory tract with an aerosol lorni olactive protease inhibitors, which typical representative is aprotinin.

Such effect of the protease inhibitor, firstly, blocks virus replication by inhibition of viral protcase activation, and secondly, suppresses the disease pathogenesis by lowering the level of harmful proteases directly in the focus and in the infected organism. As a result, unlike zananlivir. the aerosol of protease inhibitors has a binary antiviral and pathogenetic therapeutic effect.

The developed pharmaceutical aerosol formulation of protease inhibitors, preferentially aprotinin -a broad spectrum protease inhibitor, will be widely used in medicine, since the disturbance of proteolytic balance, requiring correction with protease inhibitors, develops in many human and animal diseases. In particular. an antiprotease aerosol formulation may be used for such diseases as respiratory infections, including influenza, keratoconjunctivitis of viral and bacterial etiology, herpetie lesions of skin and mucous membranes, chronic obstructive bronchial pneumonia, asthma and others.

Examples of the invention embodiment Example 1. Preparation of aerosol formulation from an aqueous solution of aprotinin and ozone-saving propellant.

Composition NI is presented for the aerosol formulation preparation. For its preparation individual components are mixed together sequentially in the following order and proportions: 1.2 ml of 96% aqueous solution of glycerol arc added to 0.12 ml of an aqueous solution containing 3.64 mg (or 25,000 kallikrein-inhihiting units (KTIJ)) ofaprotinin protein; then 2.0 ml of 96% ethanol are added to the obtained mixture. followed by the addition of 0.013 mL of peppermint oil, and at the final stage 13.5 ml of 1 34A propellant under pressure in a sealed container are added with continuous stirring. As an additional example we present an aerosol formulation N2, which was obtaincd by mixing a grcatcr volume of the componcnts in the ordcr.

specified for the Ni composition. The resulting aerosol formulations have the following ratio of ingredients (Table 1).

lähle 1. Composition olcomponents in aerosol formulation containing water-soluble aprotinin and water-insoluble propellant A 134 % composition Components Formulation NI Formulation N2 Of components Water solution of aprotinin: Aprotinin, Water phase Stock concentration 0,12 ml (contains 0,18 ml (contains 0,5-5 vol.% 250001ClUor3,64mg 3S000IClIJor solution of aprotinin protein) 5,45 mg of aprotinin protein) Ejecting system of water-insoluble propellant: Propellant 134A Propellant system (glycerol, ethanol.

(1,1,1,2-tetra-ftor-13,5 ml 20,0 il propellant) ethan) CI-T1FCF3 98,9-94,1 vol.% Glycerol (96 % water solution) 1,2m1 l,Sml Ethanol (96 % water solution) 2,0 nil 3.3 ml Misccllancous substances: Additives Peppermint oil 0,013 ml 0,02 in! 0,9-0,05 vol. % The obtained aerosol formulation is forcibly dispensed under pressure into sealed containers made of aluminum aHoy mid equipped with a metered discharge valve with a h&e of 0.3 mm in diameter. The filled containers are stored at 18-20°C for 0.5 and 4 years.

Example 2. Polypeptide protease inhibitors for medical purposes.

The example in Table 2 illustrates a list of various protease inhibitors of polypeptide nature, which are dissolved in an aqueous-glycerin-akoholic phase of the proposed aerosol formulation prepared according to the dependent claim 1 for the application as an active component in pharmaceutical aerosol formulation.

Fable 2. Polypcptidc protcasc inhibitors for medical usage.

inhibitors Accession Number SW1SS-UniPROT Aprotinin P00974 Alpha-protease inhibitor P01009 Alpha 1 -antichirntrypsin P01011 Cystatin A P01040 Cystatin C P01034 Tiostatin P01042 Calpastatin P20810 Alpha 2 -macroglobulin P01023 Alpha I Ovicroglohulin P02760 Tissue factor pathway inhibitor P10646 Tissue factor pathway inhibitor 2 P48307 Mucus protease inhibitor P00995 Elafin P19957 Leupeptin Acetyl-Leu-Leu-Arg-aldehyde Inhibitor of apoptosis BIRC 5 015392 Inhibitor of apoptosis XIAP P98 170 Caspase lithibitort c-FLIP 015519 Tissue Inhibitor of metaloproease (1-4) X01683 Serpin Al (alpha 1 antiprypsin) P0 1009 Scrpin A3 (anti-chymotrypsin) P01011 Serpin F2 (alpha 2-antiplasmin) P08697 Serpin Gl (Cl inhibitor) P05 155 I eucocyte secretory inhibitor P03973 Proteasc inhibitor Bowman-I3IRK X68704 Angiotcnsin-3 P01019 Protease Inhibitor SPINK-1 NMOO3 122 Example 3. Lack of physical denaturation of aerosol formulation.

In order to evaluate the mixability of the constituents in the obtained samples of aerosol formulation, optical properties, i.e. transparency of the solution, were examined. For this purpose the aerosol formulation was discharged from the container by valve opening and release of the aerosol into 15 ml test tubes (Falcon; Germany). Immediately after the release a portion of the collected solution was traimferred from the 15 ml test tube into a cuvette for the measurement (0.5 nil), and the optical density of the solution in a stream of visible light on the spectrophotorneter Ultraspec-2 (Pharmacia, Sweden) was determined. The lack of optical density (transparency) of the aerosol solution is compared with the optical density of distilled water..

which is taken as a zero value. Three portions of the aerosol formulation are tested: from a full containcr (emissions number 10-40), emissions 120-150 (half-filled container); emissions 190-

D

240 (last fraction of the container). The container contained a total of 25 ml of aerosol Ibrmulation, which allowed to make about 300 emissions of 85 iii, each from one container. The results of the optical density of the aerosol formulations of three fractions are demonstrated in

Table 3.

Table 3. Optical density of early and late fractions of Aerosol formulation Fractions of Period of keeping Aerosol formulation 0 0.5 years 4 years Early 0,0+0,03 0.0+0,03 0.0+0,06 Middle 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 Table 3 data show that the aerosol solution generated from the early and later fractions of the container has a full transparency similar to distilled water. This result indicates good compatibility of the components and lack of precipitation of suspended particles components in the aerosol formulation both in the container and in the generated aerosol.

Example 4. Biophysical stability of aprotinin in aerosol formulation.

Method of protein fractionation in polyacrylarnide gel in electric field, so-called polypeptide electrophoresis in polyacrylarnide gel (PAGE), is used for the aprotinin biophysical properties testing. Early and late portions of the aerosol formulation from one container are tested initially just after preparation. and portions from the containers stored for 0.5 and 4 years at a temperature of 18-22°C are tested secondly. Early, middle and late portions of the aerosol formulation are obtained for testing purposes by the collection of the indicated fractions as described in section n 3.

Equal aliquots (15 R1) are selected from the collected portions of the aerosol formulation.

Then these aliquots arc mixed with 5 jil of the dissociating solution containing 5% sodium dodecyl sulfate (SDS) and 200 RM of dithiothreitol (DTT). heated for 10 mm at 70°C and applied on 3% focusing polyacrylamide gel prepared on 0.12 M tris-HCI (pH 6.8) and 0.1% SDS. Focusing gel has a thickness of 1.2mm and a height of 1 cm. Separating gel had a height of 7 cm and contained 17.5% acrylamide. 0.3% methylene hisacrylarnide, 0.4 M Tris-HCI (p1-I 8.3), 0.1% SDS. The focusing and separating gels are polymerized using a catalyst system -ammonium persulfate (0.05%) and tetramethylethylene diamine (TEMED, 0.2%). Buffer for the elecliodes contained tris-hydroxyaminomethane (0.03 M), glycine (0.2 M), 0.1% SDS. and had a pH of 8.3; it is placed by 75 ml in anode and cathode chambers, electrophoresis buffer system according to Laemmli method (1970). Electrophoretic fractionating is performed at 70V on PAGE plate with a width of 8 cm for 2 hours. After the completion of electrophoresis.

polypeptides in gel are stained with Coomassie Blue R-350 (0.1%), which was dissolved in a mixture of water: ethanol: acetic acid in the ratio 5:5:1 by volume for 2 hours at room temperature. Non-bound dye is washed out from the gel in a mixturc of water: ethanol: acetic acid in the ratio 88:5:7, respectively. For comparison, a commercial formulation of natural purified aprotinin isolated from bovine lungs (Sigma, USA) is used as a standard sample of aprotirnn.

Figure 1 shows the results of the analysis of the aerosol formulation samples, obtained from a containerjust after its filling and after storage for 0,5 and 4 years at room temperature (18- 22°C). First, as can he seen, aprotinin from the aerosol formulation of early and late fractions had a profile of clcctrophorctic mobility typical for a polypcptidc with a molecular wcight of about 7kD. and was thlly consistent with electrophoretic characteristics of standard aprotinin. Second! no high molecular weight protein aggregates have been detected in the aprotinin aerosol samples.

liiis high-molecular zone (HMZ), corresponding to a molecular weight of 150-250 kl)a is shown in Figure 1 in a frame on the top of the separating gel. As shown in the Figure. no protein material were detected in the area of aggregates in the samples obtained immediately after filling of the containers, and in the samples from the containers stored for 4 years. These results indicate that aprotiriin in aerosol formulation prcscrvcs its natural structural propcrtics, and docs not form aggregates during the process of aerosol formulation storage and its subsequent spraying.

The electrophoresis is followed by the evaluation of proteins, which is performed in gel by gel scanning method. For this purpose, the gel is stained with Coomassie Blue and scanned in visible light using ScanJet 6300 scanner. Quantitative assessment of the intensity of the protein bands on the scans is determined using a program that allows to scan optical density of the regions. Using the obtained intensity values of the bands on the eleetrophoregram, the area ratios of the regions are calculated per unit area of thc scanned zone for aprotinin and protein impurities in the high molecular weight zone corresponding to molecular weights from 150 to 250 kDa. The intensity of the main peak of aprotinin (molecular weight of about 7000 Daltons) is taken as 100% NA/Bx 100.

where: A -optical intensity of high molecular weight zone (BM3); B -optical intensity of aprotinin band; N -percentage of high molecular weight impurities.

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

Example 5. Immunolozical stability of aprotinin in aerosol formulation.

Immunological properties of aprotinin in aerosol formulation are tested by its interaction with specific antibodies by the western blot" method. Examine the Three samples are examined: standard aprotinin (Sigma, USA), and aerosol formulation from a container that had been stored for 0.5 and 4 years at a temperature of 20-22°C. After the eleetrophorcsis on PAGE, proteins arc transferred from gel to nitrocellulose protran membrane with a pore diameter of 0.45 microns (Shleiher & Sehull. Germany), and then the protein adsorbed on the membralle is tested by the interaction with antibodies specific to aprotinin. Interaction of aprotinin with antibodies on the membrane is identified by enhanced chemiluminescence method using peroxidase conjugate with a secondary anti-species antibody. A commercial solution West Dura produccd by Pierce (USA) is used as a substrate for the peroxidase, and its luminescence is registered on Kodak (USA) X-ray film. First, Figure 2 shows that aprotinin of the standard and of both test samples of the aerosol formulation interacts well with the antibodies against aprotinin, conlirming its immi.rnological stability in the aerosol formulation. Second, after 4 years of storage of the aerosol formulation aprotinin maintains the ability to react with specific anti-aprotinin antibodies. This resuli indicates that aprotinin preserves native immun&ogical structure during the aerosol fonnulation storage for 4 years.

Example 6. Preservation of anti-protease activity of aprotinin in the aerosol formulation.

Anti-protease activity of the aerosol formulation is tested by its ability to inhibit the hydrolytic function of trypsin. For this purpose, a quantity standard preparation of trypsin (Sigma; USA), which hydrolyzes a chromogenic substrate L-ZAPA-Arg-pNA; BACHEM, Switzerland) with the formation of nitroanilide (NA) with the intensity of yellow color of about 0.8 units at a wavelength of 405 nm (0D405) was prepared. This amount is approximately 100 ng of trypsin with a total volume of the reaction mixture of 150R1* Then this amount of trypsin in the volume of 50 jil is mixed with serial dilutions of the aerosol formulation samples (volume 50 pi). and incubated for 30 mm at 20°C for aprotinin binding with trypsin and its inhibition.

Thereafter, the L-ZAPA substrate (25 pA of solution with concentration of I mg/mi) is introduced into the mixture, and incubated for 15 mm at 20°C. Then a hydrolytic reaction is stopped by the addition of 25 pA of 1M solution of hydrochloric acid, and optical density is measured at 405 rnn for the determination of residual trypsin activity (shown in the Figure 3). Protein concentrations oltrypsin and aprotinin in the initial solutions are determined ftr calculation olthe molar ratio of trypsin and aprotinin. in which 50% trypsin inhibition is retained. Standard Uradford procedure, in which standard bovine serum albumin (Sigma, USA) and Cooinassie Blue G-250 (Sigma, USA) are taken as a standard, is used to determine the aprotinin protein concentration. The molar ratio trypsin/aprotinin is calculated considering the dilution factor of the tested aerosol formulation, at which a 50% reduction of the optical density 0D405 of the released nitroanilide substrate was obtained.

The titration curves of aprotinin in the aerosol formulation immediately after preparation of the containers and after 4 years of storage are shown in Figure 3. Evaluations show that 50% trypsin inhibition is observed with the original substance of aprotinin at a molar ratio of trypsin/aprotinin = 1/1. Testing of the aerosol formulation shows a similar anti-trypsin activity of aprotinin, and 50% inhibition (shown in Figure 4 by an arrow) is also registered at a molar ratio of trypsin/aprotinin = 1/1. Thus, this testing indicates that aprotinin remains stable in the aerosol formulation, and stably maintains anti-protease activity at the level of initial aprotinin during the 4 year storage.

Example 7. Preservation of protease inhibitors' activity in a three-phase water-.Uvcerol alcohol suspension. mixed with the propellant.

The aqueous solution of protease inhibitors containing polypeptide aprotillin, leupeptin (oligopeptide: acetyl-Leu-Leu-Arg-aldehyde (Sigma, USA)), or protein alphal-antitrypsin (Boehringer, Germany) with concentration of 1 mg of dry substance per I ml of solution is mixcd succcssivcly with an aqucous solution of 99% glycerol solution (Fishcr Biotcch, USA) and 96% ethanol solution of medicai qualification. After that the obtained three-component water-containing mixtures were mixed with the 134A propellant at a temperature of 27°C, in which the propellant is in a liquid state in the volume ratio of 13% of mixture and 87% of propellant. After mixing, the mixture was exposed at 20°C and left until complete evaporation of the propellant. The mixture residue containing a protease inhibitor was tested for the presence of anti-protease activity Two ranges of the ingredients' ratios are tested -A and B. After the incubation for 1 hour at 20°C. anti-protease activity is determined by the trypsin hydrolytic activity inhibition.

Hydrolytic activity of trypsin is determined by the ability to cleave a chromogenic substrate [-zapa (UACHEM, Switzerland) according to the procedure described above in Example 6.

Positive inhibition reaction is considered to be a result, when a reduction of the optical signal of free nitroaniuide by 15% or more in comparison with the control trypsin sample without inhibitor is determined in the tested inhibitor mixture samples. It has been established that at mixing of protein and polypeptide protease inhibitors with the aerosol four-phase aqueous-glycerol-alcohol propellant mixture their activities are preserved (Table 4).

Table 4. Preservation of protease inhibitor activity in a three-phase water-glycerol-alcohol suspension mixed with the propellant.

A B

Iligretheilts _________________ _________________ _________________ __________________ Volume (ml) Composition Volume (ml) Composition ___________ _________ (%) _________ (%) Water solution Of protease inhibitor 0.05 1.5 0.05 1,5 (1 mg/ml) Glycerol (99%) 1,0 30,8 2,5 77,0 Ethanol (95%) 2,2 67,7 0,7 21,5 Anti-trypsm activity Aprotinin + + Leupeptin + + Alpha 1-antitrypsin + + Example 8. Preservation of antiviral activity of aprotinin in the aerosol fonnulation.

For testing of antiviral properties of the aerosol formulation the marker of the viral protein HAD cleavage and of its inhibition by aprotinin in reproduction of human influenza virus A/Puerto Rico/34 (Hi Ni) and A/Aichi/2/68 (H3 N2) in chicken embryos was used. 9-day-old enibryonated chicken eggs were infected by injection olvirus into the allanloic cavity olan embryo in an amount of about 1000 of viral particles. Immediately after the infection the tested aerosol formulation condcnsate was additionally introduccd into the allantoic cavity of thc embryo. After 24 hours of incubation of the embryos at 37°C, the allantoic fluid was obtained from chick embryos, and the amount of accumulated synthesized infectious virus and viral protein composition were examined.

In order to prove the antiviral effect of the aerosol formulation, protein composition of the virus synthesized in chicken embryos in the presence of the aerosol formulation was studied.

Samples of allantoic fluid (AF) were subjected to 2-phase centrifugation for the purification of viral particles. For the precipitation of cellular debris AF samples were centrifuged at 4000 rev/mm for 30 mm, and then 3 ml of clarified AF were centrifuged in the ultracentrifuge Spinko L7-50 (rotor SW 55.1) at 25,000 rev/ mm for 2.5 hours in 5.5 ml tube, on the bottom of which 2 ml of 18% sucrose solution prepared on phosphate buffer saline (PBS: 10mM Na21-1P04/NaH2PO4 p1-I 7.2, 2.7mM KC1; 137mM NaCl) were underlaid. During centrilugation the virus passed though the sucrose layer and pelleted on the bottom of the tube (virus preparation), while contaminating host cell proteins remained in the supernatant liquid.

Polypeptidcs of viral precipitations wcrc elcctrophorescd in PAGE by thc Lacminli (1970) method, described in section 3 above, and analyzed by western blot technique (WB) with antibodies to the HA protein. For this purpose, the proteins were transferred from the gel to a niocellulose membrane (Protran; Shleiher & Schull, Germany) in a buffer for semidry transfer of proteins (0.05 M Tris; 0.01 M glycinc, pH 9.7; 0.01% SDS; 17.5% ethanol). Transfer was performed at 0.8 mA per cm2 of the membrane for 1 hour. After the transfer the n1embrane was saturated overnight in 3% solution of skimmed milk of cows. and then incubated for 1 hour at 20°C in PB containing 0.5% BSA and guinea pig antibodies against viral HA protein, immune complexes formed on the membrane were identified using peroxidase conjugate against pig immunoglohulin (Pierce; USA) using enhanced cherniluminescence (ECL) method with ECL-substrate (Pierce; LISA).

Profile of polypeptides IIAO (molecular weight 75 lcD) and hAl (molecular weight 55 kD) in viral preparations is shown in Figure 4. It should be considered that the polypeptide HAO is a part of noninfectious virions, while HAl makes virions infectious. Transition HAO -*HAI is performed by trypsin-like proteases of chick emhiyo, at which the injected aprotinin action developed. As seen in Figure 4, active hAl (about 95%) was detected in the virus of embryos treated with the aerosol formulation, which indicated the formation of infectious virus in such embryos. In contrast. uncleaved HAO (about 50%) prevailed in the virus from the embryos treated with the aerosol formulation, which showed preferential accumulation of non-infectious virus in these embryos. These results indicate that aprotinin in aerosol formulation retains its anti-protease activity even after 4 years of storage, and blocks the activation of Influenza virus by inhibiting the cleavage I-lAO HAl.

In order to assess the effect of aerosol composition on virus reproduction, the infectious virus yicld in chickcn cmbryos trcatcd and not trcatcd with thc acrosol formulation was compared. Aerosol formulation stored for 5 months and 4 years at room teniperature (18-22°C) was tested. Influenza virus yield was determined by the standard technique of titration of infectious virus by the method of infectious foci in MDCK cell culture. Each sample of the aerosol formulation was introduced into 3 embryos. and viral yield was independently examined in each embryo. The results are shown in Table 5. As can he seen, both samples of the aerosol formulation from the containers had a pronounced virus-inhibiting effect, and reduced the accumulation of infcctious virus by morc than 100 timcs. It is significant that virus-inhibiting activity of the aerosol formulation stored for 4 years was equal to that of the initial sample of the aerosol formulation before storage. These results demonstrate that the aerosol formulation has antiviral activity for infectious virus reproduction inhibition, and this activity of the formulation remains at a high initial level for at least 4 years of storage.

Table 5. Inhibition virus replication by the aerosol formulation.

Number Control * . . 0,5 years 4 years (of egg sample) (no aprotinin) 1. 2,7 x lO 1,3 x 10' 2,0 x l0 2. 1,9x io 1,5x10 1,7 xl0 3. 1,2 x l0 0,7 x l0 3,3 x 4. 0,9 x l0 4,3 x l0 5,1 x 5. 2,9 x107 1,8 x104 2,8 xl0 Mean value 1,92+ 2,0 x107 1,14+ 1,46 xl0 1,47+ 1,1 x i05 (*) 9-day old chickcn cggs wcrc infcctcd with 1000 virus particlcs of influcnza virus A/Aichi/2/68 (113N2) and 75 ul of aerosol formulation kept for 0,5 and 4 years (early and late outputs from aerosol-formulation containing container) were injected into allantoic cavity of

II

infected eggs. After incubation at 37 grad C for 24 hrs, allantoic Iluid was withdrawn and virus infectious activities in these samples were measured by plaque assay test under agar overlayer in MDCK cells.

Example 9. Particle-size distribution profile at the aerosol formulation spraying.

The determination is carried out microscopically after aerosol release on a glass. Aerosol container is shaken, and 1 dose is dispersed on a pure dry skimmed slide glass, located perpendicular to the spraying direction at a distance of 6 cm from the spray nozzle outlet.

Determination of the moist particles size is carried out with a microscope at 450-fold magnification right after the aerosol dispersion on the glass. The particle diameter is determined using a transparent matrix with a marked grid. Calculation is carried out on 10 fields of view (Figure 5). The measurement is performed 3 timcs from one container at different terms of production of the aerosol. 3 containers are used in the trial. The distribution of particle sizes by fractions with a specific range is calculated by the particle diameter as a percentage of the total amount olparticles on the glass in the field olview.

References 1. Patent RF 2054180 "Method of treatment of viral respiratory infections, aerosol for its preparation" Priority date August 21, 1991.

2. (ianderton 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: 53-8.

3. Moseona A. 2005. Oseltarnivir resistance -disabling our influenza defenses. N Engl J Mcd.

353(25):2633-6.

4. Trautschold 1., Werle E.. Zickgraf-Rudel G. 1967. Trasylol. Biochein. Pharmacol. 16: 59-72.

5. Kido II, Okumura Y, Yarnada II. Le TQ. Yano M. Proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents. Curr Pharm Des. 2007; 1 3(4):405-l 4. Review PubMed PM ID: 17311557.

6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage 14. Nature. 1970 Aug 15;227(5259):680-5. PubMedPMID: 5432063.

Brief explanations of Fi2ures Fig.1. Structural stability of aprotinin in aerosol formulation.

Equivalent amounts of aerosol formulation of standard aprotinin and aerosol thrmulation stored for 0,5 and 4 years (early and late container outputs) were analyzed in 15% PAGE. Gels were stained with Coomassic R350 and photographed. Molecular weight markers (Invitrogen, USA) are shown in a separate lane.

Fig.2. Immunological stability of aprotinin in aerosol formulation.

Samples of aerosol formulation stored for 0.5 and 4 years were electrophoresed and tested by Western using anti-aprotinin antibody developed in guinea pig and anti-species HRP conjugate. Protein amounts loaded in the gel wells are shown at the bottom. Molecular weight markers (Invitrogen, USA) are shown in a separate lane. Positive band were developed with the ECL West Dura substrate (Pierce, USA).

Fig.3. Antiprotease activity of aerosol formulation.

ug aliquots containing different an1ounts of aerosol forn1ulation solution obtained just after preparation (A) and kept for 0,5 (*) and 4 (.) years were mixed with 50 ul of PBS containing 100 ng of trypsin (Sigma. IJSA) and incubated for 30 mm at 20 grad C. After incubation, 25 ug of substrate L-zapa (1 mg/ml) were added and samples were additionally incubated for 30 mm at 20 grad C. Reactions were stopped with 25 ul of IM hydrochloric acid.

Activity of the residual non-inhibited trypsin in samples was evaluated by the intensity of the released ehroniogenie nitroanilide at 405 nni. Ordinate shows mean values of 3 independent measurements of 0D405. Abscissa shows protein amounts in tested samples. Arrow indicates a zone of 50% inhibition of trypsin hydrolytic activity Fig.4. Inhibition of influenza virus protein cleavage HAO->I-IAI by aerosol formulation.

9-day old chicken eggs were infected with 1000 virus particles of influenza virus A/Aiehi/2/68 (H3N2) and 75 ul of aerosol formulation kept for 0,5 and 4 years (early and late outputs from aerosol-formulation containing container) were injected into allantoic cavity of infected eggs. After incubation at 37 grad C for 24 hrs, virus was withdrawn from allantoie cavity and polypeptides HAO and HAl were tested by PAGE-WB using anti-HA antibody and anti-species-HRP conjugate with ECL West Dura substrate (Pierce, USA). Samples from 5 independent eggs are shown. IIAO/IIA1 ratios measured by WB membrane scanning are outlined at the bottom. Percentage values were normalized regarding the sum oIHAO+HAI (100%) in each lane.

Fig.5. Particle-size distribution profile in the aerosol cloud generated from the aerosol formulation.

Aerosol formulation prepared according to Table 1 was sprayed from the aerosol container onto defatted glass plate located at 4,5 em from the aerosol generator oracle. Dimeters of droplets on the glass surface were measured with the help of light microscope (magnification x400). Ordinate shows % of droplet content in the fractions; abscissa shows diameter fractions ofdroplets: 1(0,1-10 microns), 2(10-50), 3(50-100), 4(100-1000).

1. A pharmaceutical aerosol formulation of a protease inhibitor with am ozone-saving propellant comprising a homogenous mixture of an aqueous solution of the active substance from the group of protease inhibitors containing 0.05-250 mg of protease inhibitor per ml of solution, and ingredients of a three-component mixture of the ejecting system. namely, glycerol, ethanol and a water-insoluble ozone-saving propellant, wherein the ratio of the aqueous phase and three-component ejecting system is 0.02-5 and 95- 99.98 vol.%, and allowing generating the aerosol of active protease inhibitor due to the ejecting force of the propellant spraying the obtained mixture from an aerosol vaJve device.

2. A mixture according to claim 1. characterized in that the aqueous solution of the protease inhibitor, consequently admixed with the components of the ejecting system -glycerol, ethanol, and fluorine-containing propellant, preFerentially 134A, taken in a volume ratio of 0.1-10: 1-13: 77-98.9%. respectively 3. A mixture according to claim 1, characterized in that the active substance is selected from the group of protease inhibitors, and protein substances represent polypeptide nature, such as aprotinin, alpha 2-antiplasmin, alpha-i antitrypsin, antitrypsin, cystatin, preferentially such as aprotinin, or their modified molecules and combination.

4. A mixture according to claim 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 derivatives.

5. A mixture according to claim 1, comprising one or more organoleptic additives from the group of vegetable oils consisting of peppermint oil, mint fragrant. menthol oil in an amount oIO.Ol-0.9% olthe total volume.

6. A mixture according to claim 1, comprising one or more additives from the group of surfactants, including Tween 20, Tween-80, Sran-20, Sran-80, oleic acid, citric acid, g1ycery monooleate, dirnethylsulfoxide. polyethylene glycol, at amount 0.001-3.0% of the total volume.

7. Aerosol according to claim I has a particle diameter in the range of 0.5-1000 microns.

Claims (8)

1. CLAIMSI. A pharmaceutical aerosol fommlation of a protease inhibitor with an ozone-saving propellant comprising a homogenous mixture of an aqueous solution of the active substance from the group of protease inhibitors containing 0.05-250 mg of protease inhibitor per ml of solution, and ingrcdicnts of a thrcc-componcnt mixturc of the ejecting system, namely, glycerol, ethanol and a water-insoluble ozone-saving propellant, and allowing generating the aerosol of active protease inhibitor due to the ejecting force of the propellant spraying the obtained mixture from an aerosol valve device.
2. 2. A mixture according to claim 1 characterized in that the ratio of the aqueous phase and three-component ejecting system is 0.02-S and 95-99.98 vol.%.
3. 3. A mixture according to claim 1, characterized in that the aqueous solution of the protease inhibitor, consequently admixed with the components of the ejecting system -glycerol, ethanol, and fluorine-containing propellant, preferentially 134A. taken in a volume ratio of 0.1-10: 1-13: 77-98.9%, respectively.
4. 4. A mixture according to claim 1. characterized in that the active substance is selected from the group of protease inhibitors, and protein substances represent polypeptide nature, such as aprotinin, alpha 2-antiplasmin, alpha-1 antitrypsin. antitrypsin, cystatin, preferentially such as aprotinin, or their modified molecules and combination.
5. 5. A mixture according to claim 1, characterized in that the active substance is selected from the group of antiprotcasc oligopeptides consisting of two or more amino acid residues and their modified derivatives.
6. 6. A mixture according to claim 1, comprising one or more organoleptic additives from the group of vcgctablc oils consisting of peppermint oil, mint fragrant, menthol oil in an amount of 0.01-0.9% of the total volume.
7. 7. A mixture according to claim 1, comprising one or more additives from the group of surfactants, including Tween 20, Twccn-80. Sran-20. Sran-80. olcic acid, citric acid, glyceiyl monooleate. dimethylsulfoxide, polyethylene glycol, at amount 0.001-3.0% of the to1a volume.
8. 8. Aerosol according to claim I has a particle diameter in the range of 0.5-1000 microns.