MXPA96000938A - Single application vaccination formulation - Google Patents

Abstract

The present invention relates to a microparticle having a particle size ranging from 0.5 to 300 micrometers, which is prepared by dissolving an antigen or a mixture of antigens in an aqueous solution, adding a water soluble substance thereof and stirring the water to obtain a core particle, and coating the core particle with a biodegradable polymer

Description

SINGLE APPLICATION VACCINATION FORMULATION FIELD OF THE INVENTION The present invention relates to microparticles having a particle size ranging from 0.5 to 300 microns, which are prepared by successively coating an antigen or a mixture of antigens, with a water soluble substance and a biodegradable polymer; a vaccine formulation prepared by dispersing the microparticles in a medium for injection, which can achieve the effects of immunization against an infectious disease by administering a single injection only ("single application vaccine formulation") due to the delayed release of the antigen over a period, for example, of several months.

BACKGROUND OF THE INVENTION So far, a large number of effective vaccines have been developed to prevent various infectious diseases. However, most vaccines currently used require multiple inoculations, the requirement of which imposes an economic burden as well as inconveniences for people who are vaccinated (MT Aguado and PH Lambert, Im unobiol., 184, 113 (1992); ard, B., et al., Vaccine, 12, 1155 (1994)). In particular, it has been found that only about 30 percent of those receiving the first inoculation return for the second administration. Statistically, therefore, only 9 out of a hundred would complete an immunization process when three inoculations are prescribed, clearly demonstrating the need for a vaccine that can achieve immunization through a single application inoculation. The prior art approach to the development of a single application vaccine has basically focused on the idea of using a microparticle, where a desired antigen is encapsulated with a biodegradable polymeric material, which releases the antigen slowly through a designated period of time to achieve vaccination (R. Langer and J. Folkman, Nature, 263, 797 (1976)). Among the various biodegradable polymers, polylactide (PLA), polyglycolide (PGA) and poly (lactide-co-glycolide) (PLGA) are generally known to be safe because they undergo in-vivo hydrolysis to harmless lactic acid and glycolic acid. Those polymers have been used to make a suture and post-operation removal is not required; and also for formulating the encapsulated leuprolide acetate, an LHRH analogue, which has been tested by the FDA for use by humans (R. Langer and M. Mose, Science, 249, 1527 (1990); DK Gilding and AM Reed, Polymer, 20, 1459 (1979), and Willia Morris et al., Vaccine, 12_, 5 (1994)). The degradation regimes of these polymers vary with the glycolide / lactide ratio and the molecular weight thereof and, therefore, the release of the drug can be sustained over several months by adjusting the molecular weight and the glycolide ratio. lactide of the polymer as well as the size of the particle, and the coating thickness of the capsule formulation (SJ Holland, et al., J. control, Rei., 4, 155 (1986)).

Since 1988, the Worl Health Organization (WHO) has been sponsoring a number of studies to develop a one-shot vaccine for a tetanus toxoid using the aforementioned biodegradable polymers (MT Aguado, Vaccine, 11, 596 (1993); Proceed. Intern Symp. Control Reí Bioact Mater, 21st, Contolled Relay Society, Inc., Y. Men, and others, Document Number 126 (1994), 20th, B. Gander, and others, Document Number 135 (1993); 19th, AM Hazrati et al, Document Number 220 (1992); 21st, M. Gilley et al, Document Number 218 (1992); 21st, Man ohan Singh et al, Document Number 1476 (1994); 21st, C. Yan and others, Document Number 127 (1994)). Despite these efforts, however, no one-shot vaccine formulation has been put to practical use primarily due to deficiencies in that the amount of the antibody formed by the encapsulated formulation is only about 1/10 of that produced by a conventional alum formulation and that the result is not reproducible (R. E. Spier, Vaccine, U, 1450 (1993); M. T. Aguado and P.

H., Lambert, Immunobiol. , 184, 113 (1992)). The causes of these problems have been observed to be: first, an organic solvent used to dissolve the biodegradable polymers reduces or nullifies the antigenicity of the antigen by denaturing the antigen; second, when in contact with water, the antigen forms an aggregate that has a reduced antigenicity; and third, the antigenicity is reduced or destroyed due to undesirable interactions between the antigen and the biodegradable hydrophobic polymer. Alonso et al. Prepared a single application vaccine by encapsulating a tetanus toxoid with PLA and PLGA but only found that from 0.5 percent to 20 percent of the antigenicity of the original tetanus toxoid remained in the vaccine particle because to the inevitable contact of the toxoid with an organic solvent in the encapsulation process (Maria J. Alonso et al., Vaccine, 12, 299 (1994)). In an experiment using a rat, the vaccine formulation revealed a much lower antibody formation capacity than a control using alum as an adjuvant, without reinforcement, showing that this vaccine formulation is not viable as a single application vaccine. Schwendeman et al. Managed to solve the aforementioned problem by chemical modification of the tetanus toxoid, that is, S-alkylating the thiol groups of the antigen, but the result was unsatisfactory (S. P. Schwendeman et al., Proceed, Intern Symp. Control.

I laughed Bioact. Mater., 21st, Document Number 128, 1994).

Nellore et al., Reported a one-shot hepatitis B vaccine formulation comprising microparticles prepared by encapsulating a hepatitis B surface antigen (HBsAg) with PGA, using a method involving extraction or evaporation of the organic solvent (RV Nellore and others, J. Parenteral Science &Technology, 46, 176 (1992)). Animal experiments using guinea pigs showed that a formulation comprising smaller particles within the size range of 1 to 10 micrometers exhibited an antibody formation capacity that was much lower than a control using alum as an adjuvant without reinforcement, while other formulations having particles ranging from 20 to 60 micrometers and 1 to 60 micrometers were essentially inactive. In these formulations, HBsAg was denatured by the organic solvent, used during the encapsulation process. Therefore, in order to prepare a single efficient application vaccine by encapsulating an antigen with a biodegradable polymer, a biodegradable polymer must be used that does not require the use of an organic solvent in the encapsulation step, or it must be prevented from the antigen is contacted with an organic solvent during the process. However, no biodegradable polymer is known that does not require the use of an organic solvent. Microparticles have typically been prepared by encapsulating an organic drug, peptide or protein with a biodegradable polymer. Specifically, an oily phase is prepared by dissolving or dispersing the drug in an organic solvent in which a biodegradable polymer is dissolved, and preparing an oil-in-water emulsion ("0 / W") dispersing the oily phase to an aqueous phase in presence of a surfactant. Then, the organic solvent is removed by a conventional method e.g., evaporation or extraction, to solidify the biodegradable polymer in order to obtain the microparticles. At the end of the process, the drug exists to be dispersed in the polymer matrix and the resulting microparticles exist as a dispersion in the aqueous phase, where a surfactant is dissolved. However, this conventional method has the disadvantages that: most biodegradable polymers are likely to be hydrolyzed by water; surfactants are generally unsuitable for injection and therefore, must be removed by a washing process; and the particles must be subjected to a drying process after the washing process to avoid degradation of the polymer.

In this 0 / W emulsion method, the drug is contacted directly with the organic solvent and, therefore, the method can not be applied for the preparation of a vaccine formulation comprising an antigen whose antigenicity can be reduced as a result of the contact. In addition, the method has an additional limitation that it can not be used for a water-soluble drug. Consequently, efforts for development have shifted towards the discovery of a method using a W / O / W emulsion to overcome the aforementioned problems. For example, European Patent Application Number 87309286.0 discloses a vaccine formulation for oral administration comprising particles of 10 microns or less that are prepared by encapsulating the various antigens with PLA, PGA, PLGA, etc. that Peyer's Patches are known to pass. In addition, European Patent Application Number 88302940.7 discloses an injection formulation that is capable of maintaining its effect over 6 months by encapsulating a peptide or protein drug such as an LHRH analog with a biodegradable polymer, e.g. , PLGA. More specifically, the microparticles are prepared by: dissolving or dispersing the drug in an aqueous phase; mixing the aqueous phase with an organic solvent wherein a biodegradable polymer and a surfactant are dissolved to obtain a W / 0 emulsion; dispersing the W / 0 emulsion into an aqueous phase containing a surfactant to obtain a W / O / W emulsion; and then remove the organic solvent to prepare microparticles comprising the drug. According to this method, the contact of the drug with the organic solvent can be reduced compared to the 0 / W emulsion method. However, a certain degree of mixing between the internal and external aqueous phases can not be avoided, which can result in a reduction of the antigenicity of the vaccine. In addition, after a washing process to remove the surfactant, the water in the internal aqueous phase is usually removed by a conventional method e.g., lyophilization. A large number of large pores can form in the bipolymer layer during this water removal process and in the case of an encapsulated vaccine, these pores can act as conduits for water in the human body facilitating the formation of antigen aggregates with the loss of concomitant antigenicity. In order to overcome the aforementioned inconveniences, International Patent Publication Number WO 93/07861 discloses a process for preparing multi-phase particles comprising: producing a W / O / W emulsion, using a highly viscous edible oil; replacing the external aqueous phase with an acetonitrile solution of a biodegradable polymer such as PGA, PLA and PLGA; dispersing the resulting emulsion in a mineral oil; and removing acetonitrile to obtain multi-phase particles wherein a W / O microemulsion is encapsulated in a solid polymer shell. However, the process has the disadvantages that: it is not possible to separate the finished multi-phase particle from the surfactant, e.g., aluminum monostearate and Span80 which is used to increase the viscosity and dispersion stability of the edible oil, thus avoiding the release of the drug from the internal aqueous phase to the external aqueous phase; and the drug may lose its antigenicity during the process, where the temperature is increased to 140 ° C to facilitate the dispersion of the emulsion. The phase separation method (J.C. Wu et al., J. Microencapsulation, 11 (3), 297-308 (1994)) comprises dissolving a polymer in a first solvent; add to it a second solvent that does not dissolve the polymer but mixes it with the first solvent; and from this mnaera obtain core-shell type microparticles formed by the solidification of the polymer around the drug, as the solubility of the polymer decreases. However, like the 0 / W emulsion method, this method can not prevent the antigen from coming into contact with the second organic solvent. Another problem associated with the aforementioned methods using emulsions is the possible loss of activity of the protein drugs. Proteins have a tendency to be denatured by mechanical force such as in a high energy dispersion process. This is a serious problem, taking into account that most antigens are proteins. On the other hand, a spray-drying method (B. Gander et al., J. Microencapsulation, 12_ (1), 83-97 (1995)) comprises dissolving or dispersing a drug in an organic solvent in which a biodegradable polymer is dissolved. , and spray drying the mixture to obtain microparticles. However, this method also can not avoid direct contact of a drug with an organic solvent. International Patent Publication Number WO 94/12158 and US Patent Number 5,019,400 suggest a freezing-and-extraction method for preparing particles of the encapsulated protein drug. (growth hormone) . In this method, the drug is dispersed in an organic solvent, eg, methylene chloride, where a biodegradable polymer is dissolved and the solution is sprayed into the liquid gas at a low temperature to form frozen particles. These particles are collected on the surface of the frozen ethanol. As the frozen ethanol is melted, the frozen particles are defrosted and the organic solvent in the particle is extracted by ethanol, thereby forming microparticles enclosed in the solidified polymer. This method also allows direct contact of the antigen with the organic solvent, and the period of release of the drug is only several days. Therefore, this method is not appropriate for the preparation of a single application vaccine that would release the antigen through a much longer period. International Patent Publication Number WO 92/14449 discloses a process for preparing particles containing a protein drug comprising: dispersing a powdered protein drug in a molten fatty acid anhydride, cooling the mixture to solidify the mixture and pulverizing the mixture to obtain the particles. The fatty acid anhydride is melted at a temperature ranging from 45 ° to 75 ° C and is not denatured in a protein unlike a conventional organic solvent. However, in this process, the use of a fatty acid anhydride as the encapsulating material makes it difficult to obtain microparticles having an appropriate particle size for injection. In addition, the fatty acid anhydride itself is not capable of releasing a drug over a prolonged period and, therefore, is not suitable for use in the preparation of a single application vaccine formulation. European Patent Application Number 88113933.1 discloses an encapsulated particle formulation designed for a zero or two-phase release mode of a protein drug, a herbicide or a fertilizer, which is prepared by: coating the drug, the herbicide or the fertilizer with an absorptive polymer and again coating the resulting particle with a polymer, which is insoluble in water but passes through it the drug, the herbicide or fertilizer. However, the water-insoluble polymer used in the second coating, i.e., cellulose, is not a biodegradable polymer and releases the drug through the duration of only one day in an uncontrollable manner. In addition, the particle is rapidly infiltrated by water in the human body and therefore in case the drug is an antigen, it would lose its antigenicity forming aggregates. Furthermore, this process is not suitable for preparation of microparticles for injection because the size of the coated particle first varies from 125 to 10000 microns and the second coating has to be thick enough to prevent its rupture by swelling the absorptive polymer of water used in the first coating. As described above, many attempts have been made to develop a process for preparing a single application vaccine that avoids the undesirable interactions of a protein drug with an organic solvent, a biodegradable polymer and water in the human body. Despite their efforts, a method capable of adequately protecting the antigenicity of an antigen during the process of encapsulating the antigen has not been found; and, consequently, there continues to be a need to develop a single application vaccine formulation (William Morris, et al., Vaccine, 12, 5 (1994)).

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide microparticles comprising an intact antigen or a mixture of antigens encapsulated in a polymer. Another object of the present invention is to provide a single application vaccine formulation, prepared by dispersing the microparticles in a medium for injection. In accordance with the present invention, microparticles are provided having an average particle size ranging from 0.5 to 300 micrometers, which is prepared by coating an antigen or a mixture of antigen successively with a water soluble substance and a biodegradable polymer, and a single vaccine formulation. application prepared by dispersing the microparticles in a medium for injection which can achieve immunization against an infectious disease by means of only one injection.BRIEF DESCRIPTION OF THE DRAWING The above objects and features and others of the present invention will become apparent from the following description of the invention, when taken together with Figure 1, which shows the relationship between the property of the particle and the formation of the antibody, wherein three vaccine formulations prepared in accordance with the present invention compare with a conventional alum formulation.

DETAILED DESCRIPTION OF THE INVENTION All references mentioned herein are incorporated herein in their entirety by reference. The microparticle of the present invention is prepared by coating an antigen with a water soluble substance to obtain a pulverized particle ("a core particle") and coating the core particle with a biodegradable hydrophobic polymer in order to obtain the final particle. The microparticle has a spherical or spheroidal configuration with a size ranging from 0.5 to 300 micrometers. A single application vaccine formulation that can be achieved by vaccination by only one injection can be prepared by dispersing the microparticle in an injection medium. An appropriate antigen that can be used in the present invention is an attenuated, deleted or recombinant antigen that is used as a vaccine for a single disease ("a single antigen") or two or more diseases simultaneously ("mixed antigen"). The mixed antigen can be a mixture of two or more antigens, or an antigen that has antigenicities for two or more diseases simulataneously, e.g., a recombinant protein. As an antigen, a whole organism can be used, eg, a whole viral or bacterial cell or a part of the organism, eg, a certain protein having an antigenicity. Exemplary antigens of the present invention include antigens for hepatitis, diphtheria, varicella, typhoid, whooping cough, tetanus, tuberculosis, salmonellosis, cholera, HIV, herpes, yellow fever, measles, polio, rubella, mumps, rabies, plaque, schistosomiasis, influenza , tumor, trypanosomiasis, leishmaniasis, leprosy, meningitis and malaria. More specifically, they include hepatitis B surface antigen, tetanus toxoid, staphylococcal enterotoxin B toxoid, castor toxoid and attenuated influenza virus. The core particle is prepared by dissolving or dispersing the antigen in a solution obtained by dissolving a water soluble substance in an appropriate aqueous solvent, e.g., water or a stabilizer, and drying the mixture by spray drying or a method of freeze drying An appropriate adjuvant may be added to the solution if necessary, and examples thereof include alum; muramyl dipeptide, muramyl tripeptide and derivatives thereof; alpha-thymosin; lipid A of mofosphoryl; saponin; an immunostimulatory complex, a polyelectrolyte such as an II polyoxyethylene and polyoxypropylene copolymer; and a mixture of them. The water-soluble substance used for the preparation of the core particle does not effect an undesirable interaction with a protein antigen and is practically insoluble in the organic solvent used in the second coating step. Exemplary water soluble substances include water-soluble saccharides such as glucose, xylose, galactose, fructose, lactose, maltose, sucrose, alginate, dextran, hyaluronic acid, chondroitin sulfate, and water soluble cellulose derivatives, e.g. hydroxypropylmethylcellulose, hydroxypropylcellulose (HPC), carboxymethylcellulose (CMC) and sodium carboxymethylcellulose (CMC-Na); proteins such as albumin and gelatin; amino acids such as glycine, alanine, glutamic acid, arginine, usin and a salt thereof; and a mixture thereof; while HPC, CMC, CMC-Na, gelatin and a mixture thereof are preferred. The water soluble substance can be used in an amount ranging from 1 to 50, preferably 5 to 15 times the weight of the total antigen. The core particle prepared in this manner has a particle size ranging from 0.1 to 200 micrometers, preferably from 0.5 to 20 micrometers. In order to prepare the final microparticle, the core particle is dispersed in an organic solvent in which a biodegradable hydrophobic polymer is dissolved using an appropriate apparatus, eg, a magnetic stirrer, homogenizer, microliterizer and sonicator. The biodegradable polymer is used in an amount ranging from 1 to 100, preferably, 5 to 30 times the weight of the core particle. The coating of the core particle is made of a water-soluble substance that is insoluble in the organic solvent and therefore prevents the reduction or loss of the antigenicity of the antigen, blocking the contact of the antigen with the organic solvent. Exemplary hydrophobic biodegradable polymers that can be used in the present invention include poly (lactide-co-glycolide) (PLGA), polyglycolide (PGA), Polylactide (PLA), copolyoxalates, polycaprolactone, poly (lactide-co-caprolactone), polyesteramides, polyorthoesters, poly (beta-hydroxybutyric acid) and polyanhydride; while PLGA and PLA are preferred. Any of the organic solvents well known in the art can be used to dissolve the biodegradable polymer and these include carbon tetrachloride, methylene chloride, acetone, chloroform, ethyl acetate and acetonitrile.

The microparticle of the present invention is composed of the core particle coated with a biodegradable polymer, and is obtained from an organic suspension wherein the core particle is uniformly dispersed in an organic solution of a biodegradable polymer ("the dispersed particle system"). of core "). The dispersed core particle system is advantageous since a microparticle thereof can be prepared in accordance with a conventional method, while avoiding contact of the antigen with the organic solvent or the biodegradable polymer. In addition, the surface area of the core particle in contact with the organic solvent is sufficiently low that physical contact of the antigen with the organic solvent can not occur. Specifically, the microparticle of the present invention can be prepared from the dispersed core particle system in accordance with any of the following conventional methods. 1) Solvent evaporation method This method is well known for the preparation of a microparticle, but the present invention differs from the prior art since the dispersed core particle system wherein contact of the antigen with the organic solvent is avoided, it is used instead of an aqueous solution in which the antigen dissolves or disperses. Specifically, the microparticle can be prepared by dispersing the dispersed core particle system in an aqueous solution comprising a surfactant to obtain a 0 / W emulsion and then removing the organic solvent from the dispersed core particle system or dispersing the dispersed system of core particle in a solvent, which is immiscible with the dispersed core particle system and is a non-solvent material for the biodegradable polymer, to prepare an O / O emulsion and remove the organic solvent from the dispersed core particle system. When acetonitrile is used as the organic solvent of the dispersed core particle system, a mineral oil can be used as the solvent which is immiscible with the dispersed core particle system and is not a solvent for the biodegradable polymer. 2) Solvent extraction method This method is well known in the art for the preparation of a microparticle, but the present invention differs from the prior art in that the dispersed core particle system is employed. Specifically, the microparticle can be prepared by extracting the organic solvent from the dispersed core particle system using a solvent, which is immiscible with the dispersed core particle system and is a non-solvent for the biodegradable polymer, such as a mineral oil or a paraffin oil. 3) Rapid Freezing Method and Solvent Removal The present invention is different from the prior art in that the dispersed core particle system is employed. Specifically, the dispersed core particle system is sprayed into a liquid-gas phase at a low temperature using an ultrasonic apparatus to form a frozen particle. This particle is collected on the surface of the frozen ethanol. As the frozen ethanol is melted, the molten particles are thawed and the organic solvent in the particle is extracted into the ethanol phase with the concomitant formation of a microparticle coated with the biodegradable polymer. 4) Spray drying method This method is especially preferred for use in the present invention and specifically, the microparticle is prepared by spraying the dispersed core particle system using a spray dryer. This method is advantageous due to its high productivity and speed. In addition, it is also advantageous since the removal of water is unnecessary because the water is not used in the process; no surfactant is required; and the washing and drying processes can be omitted. The particle size of the microparticle prepared in this manner varies from 0.5 to 300 micrometers, preferably from 1 to 180 micrometers. Those microparticles having a particle size smaller than 180 microns can be dispersed in an injection medium to prepare an injection formulation for subcutaneous, intramuscular and intraperitoneal injections. Those particles having a particle size greater than 180 microns can be used to prepare a formulation for oral administration. Therefore, the present invention also provides a single application vaccine formulation that is prepared by dispersing the microparticles in an appropriate injection medium. The vaccine formulation may comprise a single antigen alone or two or more classes of antigens put together. The vaccine formulation comprising two or more antigens can be prepared by using core particles comprising a mixture of two or more kinds of antigens, or by employing a mixture of two or more kinds of core particles each comprising one antigen different from the other . The single application vaccine formulation of the present invention can achieve a vaccination against the antigen comprised therein by only one injection and the amount of antigen in the vaccine formulation is equal to or less than that of a conventional alum formulation which You need several injections to achieve vaccination. Exemplary injection means that can be used in the present invention include a stabilizer with or without dispersing agents and / or preservatives, an edible oil, a mineral oil, bacalus liver oil, shark, mono-, di- or tri-glyceide and a mixture of the same; the edible oil being corn oil, sesame oil, olive oil, soy bean oil, safflower oil, cottonseed oil, peanut oil or a mixture thereof. The following Examples are intended to further illustrate the present invention without limiting its scope. In addition, the percentages given below for the solid in the mixture of liquid solids in the liquid and the solid in the liquid are on a weight / weight, volume / volume and weight / volume basis respectively, unless specifically stated otherwise. The materials and methods used in the Examples are the following: • Antigen A recombinant hepatitis B surface antigen (HBsAg) dissolved in phosphate stabilized saline (PBS), which is prepared according to the method of Korean Patent Publication Number 93-2735 (Publication date: 9 April 1994). • PLGA biodegradable polymer (50/50) produced by BPI, E.U.A. -Antigen quantity: determined by the Lowry method. • Antigenicity: determined using the test kit AUZYME (Abbott, E.U.A.). • Antibody titration Geometric average of titers obtained from the sera of 11 guinea pigs using the AUSAB test kit (Abbott, E.U.A.). • Guinea pig Those who weigh 300 to 400 grams and who were confirmed to have no antibodies in their blood two days before the administration of the sample. The blood is collected from the heart of the guinea pig using a cardiac function technique.

Example 1: Preparation of the core particle using the freeze-drying method.

The recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 micrograms per milliliter and hydroxypropylcellulose was added thereto to a concentration of 0.3 milligram per milliliter (Sample 1), 1.5 milligrams per milliliter (Sample 2) and 3.0 milligrams per milliliter. (Sample 3), respectively. Each solution was frozen at -70 ° C for 30 minutes using dry ice and acetone, and then dried by freezing for 24 hours using an EYELA FD-81 freeze dryer (Tokyo Rikakikai, Japan), at 0.05 torr and a temperature of capacitor of -80 ° C in order to obtain core particles coated with hydroxypropylcellulose. The average particle sizes of the core particles prepared in this manner were 1.0, 1.2 and 1.5 micrometers, respectively.

Example 2: Preparation of the core particle using the spray-drying method.

The recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 micrograms per milliliter, and each of hydroxylpropylcellulose (Sample 4), sodium carboxymethylcellulose (Sample 5) and gelatin (Sample 6) was added thereto at a concentration of 3.0. milligrams per milliliter. Each solution was provided to a spray dryer (Buchi 190) at a flow rate of 3 milliliters per minute in order to obtain core particles coated with hydroxypropylcellulose, sodium carboxymethylcellulose and gelatin, respectively. In this step, the flow rate of the impellent nitrogen was 600 liters per minute and the inlet air temperature was 70 ° C. The average particle sizes of the core particles prepared in this manner were 4.6, 6.3 and 4.9 microns, respectively.

Test example 1: Protection of the antigen in the core particle.

To confirm whether the core particle coated with the water soluble substance protects the antigenicity of the antigen in contact with the organic solvent, the core particles prepared in Examples 1 and 2 were dispersed in dissolving ethyl acetate or acetonitrile. the PLGA but not the hydroxypropyl cellulose, and the resulting solutions were mixed using a magnetic stirrer to allow the core particle to come into contact with the organic solvent. The core particles were separated and dried to remove the organic solvent. The dried core particles were dissolved in a stabilizer (10mM phosphate, pH 7.5) and the antigenicity was determined by an AUZYME kit and compared to that of the recombinant HBsAg solution of Korean Patent Publication Number 93-2735 stored in a cryogenic state. In addition, the HBsAg was not coated with the water soluble substance that was used as a control in the following manner. The recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 micrograms per milliliter and the same volume of each of the various organic solvents listed in Table 1 was added to the solution. The resulting solutions were mixed using a magnetic stirrer during 10 minutes. The aqueous phases were separated from the solutions and the antigenicities thereof were determined according to the same procedure as above. The precipitated material separated, dried and added to PBS for the determination of antigenicity. However, the precipitated material did not dissolve in PBS due to the denaturation thereof which resulted in an insoluble form. As a result, the antigen in the core particle that is coated with hydroxypropylcellulose maintained its antigenicity after its contact with the organic solvent, while the antigen that was not coated with the polymer lost its antigenicity completely after its contact with the organic solvent. .

Table 1 Mues Solvent Antigeppt Polymer. Organic soluble obsertra nicidad insoluvación No. in water (%) ble Solu- - - 100 original HBsAg hydroxypropyl acetonitrile cellulose | 0.3 mg / ml) ethyl acetate 75 hydroxypropyl acetonitrile 80 cellulose (1.5 mg / ml) ethyl acetate 80 hydroxypropyl acetonitrile 91 cellulose (3.0 mg / ml) ethyl acetate 88 hydroxypropyl 90 cellulose (3.0 mg / ml) carboxymethyl 85 ethyl acetate cellulose sodium (3.0 mg / ml) gelatin 82 (3.0 mg / ml) acetonitrile formed ethyl acetate 0 formed - carbon tetrachloride 0 formed - chloroform «1. .0 formed * D chloride of «1,, 0 formed * D methylene benzene «1. .0 formed * D Hexane «1.0 formed * D the amount of trace was redissolved Example 3: Preparation of Microparticles PLGA (50/50) was dissolved in ethyl acetate to a concentration of 1 percent and the core particle of Sample 3 prepared as in Example 1 was dispersed therein to a concentration of 1 milligram per milliliter. The resulting solution was provided to a spray dryer (Buchi 190) at a flow rate of 3 milliliters per minute to obtain the final microparticles which were further coated with PLGA. In this step, the nitrogen flow rate was 600 liters per minute and the inlet air temperature was 60 ° C. The average particle size of the microparticles prepared in this way was 7 microns and the weight ratio of the PLGA / core particle was 10. The amount of the antigen determined by the Lowry method was 6 micrograms protein / milligram of particle.

Example 4: Preparation of Microparticles PLGA-coated microparticles were prepared using Sample 4 of Example 2 as the core particle, in accordance with the same procedures described in Example 3. The weight ratio of PLGA / core particle was 10. The size of The average particle of the microparticles prepared in this way was 9 microns and the amount of antigen was 3 micrograms of protein per milligram of particle.

Example 5: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 2 milligrams per milliliter, and microparticles coated with PLGA were prepared according to the same procedures described in Example 3. The weight ratio of PLGA / particle of The core was 5. The average particle size of the microparticles prepared in this manner was 7 microns and the amount of the antigen was 6 micrograms of protein per milligram of particle.

Example 6: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 0.5 milligram per milliliter, and microparticles coated with PLGA were prepared according to the same procedures described in Example 3. The weight ratio of PLGA / particle of core was 20. The average particle size of the microparticles prepared in this manner was 10 microns and the amount of the antigen was 1.5 micrograms protein / milligram particle.

Example 7: Preparation of Microparticles milligrams of Sample 4 (core particle) prepared in Example 2 were dispersed in 1 milliliter of ethyl acetate, where 200 milligrams of PLGA was dissolved, using an ultrasonic. The dispersed core particle system was mixed with 1 milliliter of a 1 percent aqueous solution of polyvinyl alcohol (PVA) using a magnetic stirrer and the resulting mixture was dispersed in 100 milliliters in a 0.3 percent aqueous solution of PVA to obtain a 0 / W emulsion. The emulsion was mixed continuously for 5 hours to evaporate the ethyl acetate and the microparticles formed in this way were separated using a 0.5 micron capacity filter, washed and then dried. The average particle size was 110 microns.

Test Example 2: In Vivo Effect of Water Soluble Substance The microparticle prepared in Example 3 was dispersed in PBS (Sample 7), and Sample 7 was injected once intraperitoneally into guinea pigs in an amount where the amount of antigen is converted to 40 micrograms of protein / head. Blood samples were taken from the guinea pigs for 2 months after the injection at 15-day intervals and the concentration (mlU / milliliter) of the antibody formed in each sample was determined. As a control, particles coated with PLGA only without a core layer of a water-soluble substance were prepared in the following manner. The original HBsAg solution was frozen in accordance with the method of Example 1 without using a water soluble substance as the core layer and the HBsAg particles directly coated with PLGA only (Comparison sample) were prepared therefrom in accordance with The method of Example 3. The Comparison Sample was administered to guinea pigs according to the same method as the previous one, and then the concentration of the antibody formed was determined. As a result, Sample 7 showed an antibody concentration higher than that of the Comparison Sample, as can be seen from Table 2. This result demonstrates that, without the core coating of a water soluble substance, the antigen lost its antigenicity due to the undesirable interactions of the antigen with the organic solvent and the biodegradable polymer.

Table 2 Ab Conc. Time After Injection (Months) (mlU / ml) Sample 0.5 1.5 Sample 7 0.0 2.4 10.6 24.6 34.0 Comparison sample 0.0 1.1 8.1 10.2 14.3 Test Example 3: In Vivo Effect of Single Application Vaccine Formulation The following experiment was carried out to confirm whether the vaccine formulation of the present invention has a superior effect as a single application vaccine formulation. The microparticles prepared in Example 5 were dispersed in 1.0 milliliter of the injection medium (PBS containing 0.02 weight percent Tween 80) to form a vaccine formulation (Sample 8), which was injected once subcutaneously into guinea pigs from India. using a 26G syringe in an amount where the amount of the antigen is converted to 20 micrograms of protein / head. Blood samples were taken from the guinea pigs for 5 months after the injection at intervals of fifteen days or one month and the concentration (mlU / milliliter) of the antibody formed in each sample was determined. A comparison formulation using alum as an adjuvant was prepared in the following manner. PBS containing HBsAg and an alum dispersion solution (Superfos Biasector, Vedbaek, Denmark) were mixed to prepare a vaccine formulation containing HBsAg in an amount of 10 micrograms of protein / 1.5 milligrams of alum. The vaccine formulation was injected once into two groups of guinea pigs using a 26G syringe in an amount where the amount of the antigen is converted to 10 micrograms of protein / milliliter / injection (primary injection). Then, a group underwent a first booster treatment using the same amount of antigen at fifteen days after the primary injection (Comparison Example 1). The other group also underwent a second booster treatment at 1.5 months after they received the first booster treatment in accordance with the same procedures as those previously cited (Comparison Example 2). In addition, a further group of guinea pigs received a primary injection only using the alum formulation and the same method as the previous one in an amount wherein the amount of the antigen is converted to 20 micrograms of protein / milliliter / injection (Example 3 of Comparison, Sample 9) The concentration (mlU / milliliter) of the antibody formed of Examples 1, 2 and 3 of Comparison were determined as above and the results were compared with those of Sample 8. The result is shown in Table 3, where it can be seen that the concentration of the antibody formed by an injection of Sample 8 is more higher than those formed by up to three times injections of the alum formulation. This result demonstrates that the vaccine formulation of the present invention has a superior effect as a single application vaccine formulation, i.e., a single injection of the vaccine formulation of the invention using 20 micrograms of protein, shows an effect of higher vaccination than three injections of the alum formulation using 30 micrograms of protein.

Table 3 Ab Conc. Time After Injection (Months) (mlU / ml) Sample 0 0.5 1 1.5 2 2.5 3 4 5 Sample 8 0.0 2.5 18.9 40.0 90.0 97.0 113.0 132.5 149.4 Example 1 of Comparison 0.0 3.3 9.6 30.6 72.0 86.0 86.0 108.6 131.5 Comparison Example 2 0.0 - 18.0 - 92.2 - 99.8 Comparison Example 3 (Sample 9) 0.0 6.8 10.7 9.2 14.4 18.0 22.0 22.0 30.2 Test Example 4: Relationship between the Property of the Vaccine Formulation Form and the Antibody Formation In order to investigate whether the time and the amount of antibody formation according to the weight ratio of the biodegradable polymer and the core particle are controllable, the following test was carried out. Each of the microparticles prepared in Examples 4, 5 and 6 were suspended in PBS to prepare a single application vaccine formulation (Samples 10, 8 and 11), which was injected once subcutaneously into guinea pigs in an amount where the amount of the antigen becomes 20 micrograms of protein / head. The blood samples were taken from the guinea pigs for 5 months after the injection at 15-day intervals and the concentration (mlU / milliliter) of the antibody formed in each sample was determined. In addition, the alum formulation prepared in Test Example 3 was injected once into guinea pigs in an amount where the amount of the antigen was converted to 20 micrograms of protein / head (Sample 9) and the concentration was determined (mlU / milliliter) of the antibody formed in accordance with the same method as the previous one. Changes in concentration of the antibody over time after injection are shown in Figure 1, where Sample 8, which has a weight ratio of PLGA / core particle of 5, shows the highest rate of increase. elevated in the concentration of the antibody. Therefore it was confirmed that the antibody formation rate is controllable by regulating the thickness of the biodegradable polymer coating. Therefore, a single application vaccine containing mixed antigens or a single application vaccine that can release an antigen in different standards can be prepared using the different particles having different properties. As described in the above-cited Examples, the single application vaccine formulation of the present invention can achieve vaccination by administering only one injection and also has a higher antibody-forming capacity than conventional vaccine formulations. Although the invention has been described with respect to specific embodiments mentioned above, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which are also within the scope of the invention as defined by the appended claims. .

Claims (19)

N O V E D A D I N V E N C L I N Having described the invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property:

1. A microparticle having a particle size ranging from 0.5 to 300 micrometers, which is prepared by coating an antigen or a mixture of antigens with a water-soluble substance in order to obtain a core particle, and coating the core particle with a biodegradable polymer.

2. The microparticle according to claim 1, wherein the antigen is an attenuated, deleted or recombinant antigen.

3. The microparticle according to claim 1, wherein the core particle further includes an adjuvant or an inorganic salt.

4. The microparticle according to claim 1, wherein the antigen is one for one or more diseases that are selected from the group consisting of: hepatitis, diphtheria, varicella, typhoid, whooping cough, tetanus, tuberculosis, salmonellosis, cholera, HIV , herpes, yellow fever, measles, poliomyelitis, rubella, mumps, rabies, plaque, schistosomiasis, influenza, tumor, trypanosomiasis, leishmaniasis, leprosy, meningitis and malaria.

5. The microparticle according to claim 3, wherein the adjuvant is alum; muramyl dipeptide, muramyl tripeptide and derivatives thereof; alfatimosine; lipid A of monophosphoryl; saponin; an immunostimulatory complex; a polyelectrolyte; or a mixture of them.

6. The microparticle according to claim 1, wherein the water soluble substance is used in an amount ranging from 1 to 50 times the weight of the antigen. The microparticle according to claim 1, wherein the water soluble substance is a saccharide, a protein, an amino acid or a mixture thereof. The microparticle according to claim 7, wherein the saccharide is a cellulosic polymer, glucose, xylose, galactose, fructose, lactose, maltose, sucrose, alginate, dextran, hyaluronic acid, chondroitin sulfate or a mixture thereof . 9. The microparticle according to claim 8, wherein the cellulosic polymer is hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose or a mixture thereof. 10. The microparticle according to claim 7, wherein the protein is gelatin, albumin or a mixture thereof. 11. The microparticle according to claim 7, wherein the amino acid is glycine, alanine, glutamic acid, arginine, or a salt or mixture thereof. 12. The microparticle according to claim 1, wherein the particle size of the core particle varies from 0.1 to 200 microns. The microparticle according to claim 1, wherein the biodegradable polymer is used in an amount ranging from 1 to 100 times the weight of the core particle. The microparticle according to claim 1, wherein the biodegradable polymer is a polyglycolide, polylactide, poly (lactide-co-glycolide) or a mixture thereof. The microparticle according to claim 1, wherein the biodegradable polymer is copolyoxalate, polycaprolactone, poly (lactide-co-caprolactone), polyesteramide, polyorthoester, poly (beta-hydroxybutyric acid), polyanhydride or a mixture thereof. 16. A single application vaccine formulation prepared by dispersing particles in accordance with any of the 1 to 15, in an injection medium. The single application vaccine formulation according to claim 16, wherein the injection medium is a stabilizer optionally containing a dispersing agent and / or a preservation agent. 18. The one-shot vaccine formulation according to claim 17, wherein the injection medium is an edible oil, mineral oil, cod liver oil, shark oil, mono-, di- or tri-glyceride or a mixture of them. 19. The one-shot vaccine formulation according to claim 18, wherein the edible oil is corn oil, sesame oil, olive oil, soybean oil, safflower oil, cottonseed oil. , peanut oil or a mixture of them.