A method of obtaining a microencapsulated form of measles vaccine for oral administration

 

The invention relates to medicine, namely to the technology of production of microcapsules, and can be used in the pharmaceutical industry. The invention consists in the following. The method includes the preparation of encapsulated material in the form of the substance of the measles virus, dispersing it in water-soluble charged polyelectrolyte, getting koatservata for the formation of shells of capsules. the introduction of the curing and drying of the finished microcapsules. The hardener is injected into the charged polyelectrolyte simultaneously with the substance of the measles virus to a final concentration of polyelectrolyte and hardener, which is not causing phase separation in the solution of the drug; microcapsules obtained from the process koatservatsii charged polyelectrolytes and hardener with the formation of the complex by freezing the solution with a speed of 0.1 to 3.0oWith a minute to a temperature below the glass transition of the amorphous phase remaining after crystallization of ice, and the process of drying the microcapsules produced by lyophilization. Before freezing liquid cake mix poured into ampoules, and after freeze drying ampoules filled with inert gas and sealed. Technical result: expanding Arsenal against measles. 5 C.p which can be used in the pharmaceutical industry for the production of vaccines and therapeutic drugs.

Known methods for producing gelatin microcapsules by emulsification capsulerebel fluid (oil) in a mixture solution of gelatin and sodium sulfate, followed by curing of the microcapsules tanning substance [1].

Known another method of producing microcapsules comprising mixing capsulerebel substances (a complex of vitamins a, D and E in the solution of oil) with a hydrophilic colloid, such as gelatinous, obtaining emulsions, getting koatservata for the formation of the shells of the microcapsules by lowering the temperature of the emulsion to +5oWith curing membranes by processing them with formaldehyde in the presence of alkali, washing, filtering and drying of the microcapsules (U.S. Patent 3539465, NC. 424-37, publ. 1977) [2].

The disadvantage of these methods [1, 2] is a significant loss (up to 90% or more) capsulerebel substances in the formation of microcapsules, if capsulerebel substance use liquid water-soluble substance, for example substance of measles virus, because the virus is in the process of forming the microcapsules can be introduced into the dispersion medium. In addition, the disadvantage of this method is the complexity of the technological process and the use of organic substances, such as solvents, which the Oia microcapsules as the delivery system for antigens in Peyer's plaques in size from 1 to 10 μm, for example, the method of obtaining the emulsion water - in - oil [3, 4] or double emulsion [5] the water - in - oil - in - water.

The disadvantages of these methods is the complexity of the technological process and the use of organic solvents, which are in the process of encapsulation of viral particles can inactivate the last.

A known method of dispersing an aqueous solution of anionic polymer containing dispersed substance (rotavirus particles) in an aqueous solution of the binding reagent, for example a dispersion of sodium alginate (negatively charged polymer) in the form of droplets of a size of 5 μm in an aqueous solution of spermine hydrochloride (amine salt) [6]. As a result of monodisperse microcapsules viral vaccines in size from 1 to 10 µm (mostly 2 μm), which cause the mice expressed the induction of humoral response.

The disadvantages of this method include: a) only 1-6% of the total number of infectious virus is included in the matrix of alginate-Perminova microcapsules, because usually microencapsulation in aqueous medium using a water-soluble polymer as the material of the shells of the microcapsules using substances not miscible or not soluble in water, then the water is here process, the increase in consumption vaccinated fluid and the need for additional equipment leads to a significant appreciation of the vaccine.

The closest technical solution (prototype) is a method of obtaining a microencapsulated form of viral vaccines, including cooking capsulerebel material in the form of a substance, such as measles virus, dispersing it in water-soluble charged polyelectrolyte polyphosphazene, getting koatservata for the formation of shells of capsules by introducing into the mixture solution of salt of monovalent metal, such as sodium chloride solution, processing the obtained microcapsules are cured by tanning them with salt multivalent ion such as calcium chloride, and drying the finished microcapsules (U.S. Patent 5807757, IPC a 61 K 39/00; a 61 K 9/50, publ. 15.09.98,) [7].

The disadvantage of the prototype method is that it uses this new polyelectrolyte, as polyphosphate (sheath material), which is issued by the industry, not approved for use in the pharmaceutical and food industries and has a complex synthesis that is affected by many factors (reaction temperature, the initial ratio of the reactants, the complex restorethe polymer. Polyelectrolyte with unstable physical and chemical properties cannot provide microcapsules with specified characteristics, such as size and strength of microcapsules, etc.

The technical result of the proposed method is to obtain microencapsulated forms of live measles vaccine with stable characteristics (particle size of 0.2-10 μm, the shelf life of the vaccine at least 1 year) using reagents, is permitted for use in pharmaceutical and food industries.

This result is achieved in that in a method of producing microencapsulated form of a live virus vaccine, comprising preparing capsulerebel material in the form of the substance of the measles virus, dispersing it in water-soluble charged polyelectrolyte, getting koatservata for the formation of shells of capsules, the introduction of the curing and drying of the finished microcapsules according to the invention, the hardener is injected into the charged polyelectrolyte simultaneously with the substance of the measles virus to a final concentration of polyelectrolyte and hardener, which is not causing phase separation in the solution of the drug; microcapsules receive as a result of complex koatservatsii charged polyelectrolytes and hardener with emperature glass transition of the amorphous phase, remaining after crystallization of ice, and the process of drying the microcapsules produced by lyophilization.

And before freezing liquid cake mix poured into ampoules, and after freeze drying ampoules filled with inert gas and sealed.

As a substance of measles virus substance use live virus, which is obtained by multiplication of the virus on the cell substrate, with a titer of not less than 5,2 lgT50/0.5 ml, collection vaccinated fluid, release of product from the cellular detritus and introducing stabilizing additives.

Freezing of a solution of the drug produced to a temperature of not higher than - 40oC.

The final concentration of the polyelectrolyte, the bonding agent (hardener) and a stabilizing additive in the liquid material is(0,001-2,5), (2,5-5,5) and (2,5-5,5) weight percent, respectively.

As polyelectrolyte use sodium alginate, polyacrylic acid or a copolymer of acrylic acid, or a mixture of polyacrylic acid and polyvinylpyrrolidone in the ratio (0.5 to 2.0): 1, as a binder (hardener) - gelatos or a mixture of gelatos with chitosan ratio (5,0-15,0): 1, or a mixture of gelatos with spermidine ratio (5,0-15,0):1, and as a stabilizer - Sorbi the real output koatservata with a particle size in the range of 0.2-10 μm. When joint koatservatsii water-soluble polymer or polymers that are able to dissociate into ions at pH close to the physiological, the safekeeping of the live measles virus in the process of microencapsulation. Coacervation is caused by a lowering of the solubility of the polymer (one of the polymers) with decreasing temperature and primary crystallization of ice, whereupon the polymer concentration in the mother solution (liquid phase remaining after the primary crystallization of ice) increases. The resulting koatservata together with the equilibrium liquid is subjected to a further freezing and freeze-drying. Concentration (lowering the solubility of the polymer is carried out by the primary crystallization of ice, and not due to evaporation or changes in the composition of the solution, as a rule, the addition of organic solvents or excess polymer (analogues [1-7]) that does not cause inactivation of live virus or significant dilution (no more than 1-20%). The whole process of microencapsulation is the mixing of polymer solutions with liquid semi vaccine (vaccinated liquid, stable partially hydrolyzed gelatin and sorbitol), freezing the received RA is both due to the interaction between positive and negative charges of different molecules of the reaction mixture and possibly strengthening cooperation between them, occurring during crystallization of ice. As you know, during crystallization of ice having a large pressure, which create conditions for closer convergence between oppositely charged ions and, as a consequence, the formation of a stable complex.

Below is a description of the components used for the production of microcapsules: - polyacrylic acid (PAC) molecular mass (MM) ~ 31000 Yes, pilot production JSC "Delivery System International"; a copolymer of acrylic acid obtained by the esterification of the original PAK (synthesis differs in that it is conducted in the mode of autocatalysis, on the basis of high purity PAK, which ensures the absence of toxicity), experimental production of JSC "Delivery System International"; - sodium alginate food (viscosity of 1% solution in degrees Engler 6,5 Eoat 20oC), Arkhangelsk experienced algal plant; - chitosan MM -35000 Da ZAO Bioprogress"; - low molecular weight substance which is - N2N(CH2)4NH(CH2)3NH2- (3-aminopropyl-4-aminobutyrate), Sigma; poly - N-vinyl pyrrolidone (PVP) (mark Kollidon 90F). BASF AG.

In Fig.1 presents a graph of the dependence of the relative viscosity of the polymers from pH; Fig. 2 depicts the electronic pictures of microcapsules, storeroot (magnification 5000); in Fig.3 - structure of lyophilized measles vaccine containing complexes of sodium alginate/spermidine (increase 2500, 10000, respectively); Fig.4 shows atomic force microscopy lyophilized measles vaccine containing 0.1% of a copolymer of polyacrylic acid.

Below are examples of specific ways to obtain microencapsulated forms of live measles vaccine.

Example 1. Preparation of a liquid substance of live measles vaccine (GCV) cell Culture of fibroblasts Japanese quail produced by trypsinization embryos with a solution of trypsin - versene at 37oC. the Cell mixture is centrifuged, the cells resuspended in the growth environment and dissipate in mattresses or roller at the rate of 500 and 950 thousand cells/ml, respectively. The cultivation is carried out at 35oSince before the formation of the monolayer. During the suspension the virus added in a suspension of cells with a multiplicity of infection of 0.1 to 0.001 TCD50/class. After the formation of the monolayer of cells subjected to 6-fold the cleaning for exemption from ballast proteins, then cultured at a temperature of (351)oFrequency of rotation of the roller 2-12 rpm When situationsmanaging supportive environment. Individual viral plum with specific activity was lower at 5.2 lg50/0.5 ml are combined and freed by filtration from the cellular detritus. In the slurry, add sterile solution of 50% aqueous sorbitol to a final concentration of 5 weight percent in the liquid bulk.

Example 2. Getting microencapsulated form GCV with sheath on the basis of sodium alginate
Sterile vaccinated receive liquid as in example 1. To sterile vaccinated liquid with a specific activity of not less than 5,2 lg50/0.5 ml under continuous stirring simultaneously with a solution of sorbitol add sterile aqueous solutions of 25% of gelatos (mol. weight 3000) and 2% of sodium alginate to the final concentrations of 5.0 and 0.5 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Example 3. Getting microencapsulated form GCV with sheath on the basis of sodium alginate and chitosan
Sterile vaccinated liquid floor is/0.5 ml under continuous stirring, sterile solutions of 25% of gelatos (mol. weight 3000), 2% of sodium alginate and 2% chitosan sequentially to a final concentration of 5.0; the 0.5 and 0.35 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Example 4. Getting microencapsulated form GCV with sheath on the basis of sodium alginate and spermidine
Sterile vaccinated receive liquid as in example 1. To sterile vaccinated liquid with a specific activity of not less than 5,2 lg TCD50/0.5 ml under continuous stirring, sterile aqueous solutions of 25% of gelatos (mol. weight 3000), 2% of sodium alginate and 75% solution of spermidine sequentially to a final concentration of 5.0; the 0.5 and 0.33 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried within/p> Example 5. Getting microencapsulated form GCV shell based on polyacrylic acid
Sterile vaccinated receive liquid as in example 1. To sterile vaccinated liquid with a specific activity of not less than 5,2 lg TCD50/0.5 ml under continuous stirring, sterile aqueous solutions of 25% of gelatos (mol. weight 3000) and a 5% aqueous solution of polyacrylic acid sequentially to the final concentrations of 5.0 and 0.5 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Example 6. Getting microencapsulated form GCV with shell-based copolymer of acrylic acid
Sterile vaccinated receive liquid as in example 1. To sterile vaccinated liquid with a specific activity of not less than 5,2 lg50/0.5 ml under continuous stirring, sterile aqueous solutions of 25% of gelatos (mol. weight 3000) and 2%-aqueous solution of a copolymer of acrylic acid and ethyl Splenda. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Example 7. Getting microencapsulated form GCV with shell-based copolymer of acrylic acid
Sterile vaccinated receive liquid as in example 1. To sterile vaccinated liquid with a specific activity of not less than 5,2 lg50/0.5 ml under continuous stirring, sterile aqueous solutions of 25% of gelatos (mol. weight 3000) and 2%-aqueous solution of a copolymer of acrylic acid sequentially to the final concentrations of 5.0 and 0.1 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Example 8. Getting microencapsulated form GCV shell based on polyacrylic acid and polyvinylpyrrolidone50/0.5 ml under continuous stirring, sterile aqueous solutions of 25% of gelatos (mol. weight 3000), 5% aqueous polyacrylic acid solution and 5% aqueous solution of polyvinylpyrrolidone sequentially to a final concentration of 5.0; 0.5 and 0.75 weight percent, respectively. The obtained liquid prefabricated vaccine poured into ampoules WFC-3 at 0.5 ml into each well and frozen at a temperature of -60oC for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying ampoules filled with inert gas (argon), sealed.

Method viscosity dependence of conformation on pH (Fig.1) polyacrylic acid (PAC), its copolymer and chitosan (pH-dependent polymers). Defined ranges of pH at which the polymer most dissociative and is in the "expanded" state, the transition region, the region "collapsed" state, and the pH value at which it precipitates.

Example 9. Determination of specific activity of the drug
The specific activity of the vaccinated material was determined by cytopathic effect on the cell culture Vero in accordance with FS 42-3092-00 "Vaccine measles culture alive dry". To determine specific is titrated in 96 - hole cultural tablets on the culture of VERO cells by cytopathic effect in accordance with [8]. For microencapsulated forms GCV of examples 3 and 4 cannot determine the specific activity in cell culture (titer2 lgT50/0.5 ml) because of the difficulty of disclosure of microcapsules in the model system in vitro. The specific activity of these samples determined in the system in vivo.

For samples of the vaccine, manufactured in examples 1-8, the results of determination of specific activity are shown in table.1. The data show that for samples 5 and 8 the decline of specific activity in the test for accelerated aging does not exceed 1,5 lg50/0.5 ml, and for sample 7-1 lg50/0.5 ml (meets the who requirement for thermostable vaccines).

Example 10. The study of the immunogenicity of drugs on animals
The immunogenicity of the samples of the vaccine in the system in vivo was determined by oral immunization of Guinea pigs. For this lyophilized samples were dissolved in 0.5-1 ml of distilled water, and then use the eyedropper is administered orally Guinea pigs (weighing 200-250 g), 6 animals in the group, one or twice. In parallel control the nutrient dissolved in 0.5 ml of solvent for measles vaccine. At 28 days after immunization was performed blood sampling; blood serum was investigated in the reaction of inhibition of haemagglutination with 4 AE twin-over-antigen measles virus in accordance with FS 42-3092-00 "Vaccine measles culture alive dry."

The results are shown in table. 2. As seen in the results table, microencapsulated form of the vaccine is causing the pigs pronounced induction of a humoral response that exceeds the response to the introduction of traditional forms GCV.

Example 11. The results of morphological studies of microcapsules
To determine the structure of lyophilised preparations used the method of cromatografia using cromatografia install BAF-400D (Balzers, Liechtenstein). To fill the voids of the study drug were made in a specially selected matrix; drop obtained substance was frozen to a temperature of minus 100oWith and break the vacuum with a knife, cooled to liquid nitrogen temperature. Then on the surface of the replica without breaking vacuum formed platinum-carbon replica, which was further purified from the residue of the drug and the matrix in the appropriate stain. The resulting preparation was studied in the electron microscope N (Hitachi, Japan).

D is eparate was applied to the surface of the glass. After drying of water-soluble components were removed by washing and examined the specimen using the atomic force microscope Solver P47BIO (NT-MDT, Russia) in tapping mode.

The study of the structure of the microcapsules was performed using transmission electron microscopy according to the method of negative contrast. For this study samples of the vaccine was dissolved in water, were applied to the mesh-covered formarum. A contrast study was performed in solution phosphorus-tungsten acid at pH=7,0.

Using transmission electron microscopy, it was found that the microcapsules are rounded and clear boundaries (Fig.2). The study drugs by the method of cromatografia showed the presence of polymorphic particles of irregular shape with a size of 1-3 μm (Fig.3). In the atomic force microscope was determined both the single and groups oval structure with a height of 0.6-1 µm with a horizontal size of 1-2 μm (Fig.4).

According to electron microscopy viewed in the fields of free viral particles (not prisoners inside the microcapsules) is not detected.

Microcapsules live measles vaccine, obtained by the proposed method is stable in aqueous medium at pH from 2 to 9 within 24 hours and in life. microcapsulation. - Moscow, Chemistry, 1980, S. 54-57.

2. U.S. patent 3539465, NC. 424-37, 1977

3. J. H. Eldridge, J. K. Staas, J. A. Meulbroek, J. R. McGhee, T. R. Tice and R. M. Gilley. Biodegradable microspheres as a vaccine delivery system. Molecular Immunology, Vol.28, No.3, pp.287-294, 1991.

4. U.S. patent 5075109, IPC a 61 K 9/50, 24.12. 1991.

5. Conwey, C. R., Alpar, H. 0., Lewis, D. A. Studies on the optimization of loading and release kinetics of interferon-gamma from polylactide microspheres. Proc. Int. Symp. Contr. Rel. Bioact. Mater. 21:284-285, 1994.

6. P. A. Offit, P. A. Khoury, S. A. Moser, H. F. Clark, J. E. Kirm and T. J. Speaker. Enhancement of Rotavirus Immunogenicity by Microencapsulation. Virology, 203(1), 134-143, 1994/
7. U.S. patent 5807757, IPC a 61 K 39/00; a 61 K 9/50, 15.09.98, (prototype).

8. F. S. 42-3092-00 "Vaccine measles culture alive dry".


Claims

1. A method of obtaining a microencapsulated form of measles vaccine for oral administration, comprising preparing capsulerebel material in the form of the substance of the measles virus, dispersing it in water-soluble charged polyelectrolyte, getting koatservata for the formation of shells of capsules, the introduction of the curing and drying of the finished microcapsules, characterized in that the hardener is injected into the charged polyelectrolyte simultaneously with the substance of the measles virus to a final concentration of polyelectrolyte and hardener, which is not causing phase separation in the solution pre-formation of the complex by freezing the solution with a speed of 0.1 to 3.0oWith a minute to a temperature below the glass transition temperature of the amorphous phase remaining after crystallization of ice, and the process of drying the microcapsules produced by lyophilization.

2. The method according to p. 1, characterized in that as the substance of measles virus substance use live virus, which is obtained by multiplication of the virus on the cell substrate, with a titer of not less than 5,2 lg50/0.5 ml collection vaccinated fluid, release of product from the cellular detritus and introducing stabilizing additives.

3. The method according to p. 1, characterized in that the freezing of a solution of the drug produced to a temperature of not higher than -40oC.

4. The method according to p. 1, wherein the final concentration of the polyelectrolyte, the bonding agent (hardener) and stabilizing additives to the liquid material is(0,001-2,5), (2,5-5,5) and (2,5-5,5) weight. % respectively.

5. The method according to PP. 1 and 4, characterized in that the polyelectrolyte used sodium alginate, polyacrylic acid, or a copolymer of acrylic acid, or a mixture of polyacrylic acid and polyvinylpyrrolidone in the ratio (0.5 to 2.0): 1, as a binder (hardener) - gelatos, or a mixture of gelatos with chitosan ratio (5,0-15,0): 1, or a mixture of gelatos with whom, before freezing liquid cake mix poured into ampoules, and after freeze drying ampoules filled with inert gas and sealed.

 

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