CA2171881A1 - Single-shot vaccine formulation - Google Patents
Single-shot vaccine formulationInfo
- Publication number
- CA2171881A1 CA2171881A1 CA002171881A CA2171881A CA2171881A1 CA 2171881 A1 CA2171881 A1 CA 2171881A1 CA 002171881 A CA002171881 A CA 002171881A CA 2171881 A CA2171881 A CA 2171881A CA 2171881 A1 CA2171881 A1 CA 2171881A1
- Authority
- CA
- Canada
- Prior art keywords
- microparticle
- antigen
- mixture
- oil
- core particle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920002643 polyglutamic acid Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
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- 108090000765 processed proteins & peptides Proteins 0.000 description 2
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- 238000000935 solvent evaporation Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- KPZGRMZPZLOPBS-UHFFFAOYSA-N 1,3-dichloro-2,2-bis(chloromethyl)propane Chemical compound ClCC(CCl)(CCl)CCl KPZGRMZPZLOPBS-UHFFFAOYSA-N 0.000 description 1
- MLXDUYUQINCFFV-UHFFFAOYSA-N 2-acetyloxyacetic acid Chemical compound CC(=O)OCC(O)=O MLXDUYUQINCFFV-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 1
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
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- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 1
- NJIWVMGUAFXONX-UHFFFAOYSA-J [C+4].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [C+4].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O NJIWVMGUAFXONX-UHFFFAOYSA-J 0.000 description 1
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- 230000001580 bacterial effect Effects 0.000 description 1
- ZPHQBFRCXUIIAZ-UHFFFAOYSA-N benzene;hydrochloride Chemical compound Cl.C1=CC=CC=C1 ZPHQBFRCXUIIAZ-UHFFFAOYSA-N 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 229920006184 cellulose methylcellulose Polymers 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
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- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000002434 gonadorelin derivative Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001986 peyer's patch Anatomy 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A microparticle having a particle size ranging from 0.5 to 300 µm, which is prepared by coating an antigen or a mixture of antigens with a water-soluble substance to obtain a core particle and coating the core particle with a biodegradable polymer; and a single-shot vaccine formulation prepared by dispersing the microparticles in an injection medium.
Description
_ 2171881 SINGLE-SHOT VACCINE FORMULATION
FIELD OF THE INVENTION
The present invention relates to microparticles having a particle size ranging from 0.5 to 300 ym, which are prepared by coating an antigen or a mixture of antigens with a water-soluble substance and a bio-degradable polymer successively; and a vaccine formulation prepared by dispersing the microparticles in a medium for injection, which can accomplish the immunization effect against an infectious disease by administering a single injection only("single-shot vaccine formulation") due to the delayed release of the antigen over a period of, e.g., several months.
BACKGROUND OF THE INVENTION
Hitherto, there have been developed a large number of vaccines effective in preventing various infectious diseases.
However, most of the currently used vaccines require multiple inoculations, which requirement imposes an economical burden as well as inconveniences to the vaccinees(M. T. Aguado and P. H. Lambert, Immunobiol., 184, 113(1992); Aryward, B., et al., Vaccine, 12, 1155(1994)). In particular, it has been found that only about 30% of those who receive the first inoculation return for the second administration.
-- ~171881 Statistically, therefore, only 9 out of 100 would complete an immunization process when three inoculations are prescribed, clearly demonstrating the need for a vaccine that can accomplish immunization by a single-shot inoculation.
The prior art approach to the development of a single-shot vaccine has basically centered on the idea of using a microparticle, wherein a desired antigen is encapsulated with a biodegradable polymeric material, which releases the antigen slowly over a prescribed time period to accomplish the vaccination(R. Langer and J. Folkman, Nature, 263, 797(197~)).
Among various biodegradable polymers, polylactidetPLA), 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 in making a suture whose post-operation removal is not required; and also in formulating encapsulated leuprolide acetate, a LHRH analogue, which has been approved by FDA for human use(R. Langer and M. Mose, Science, 249, 1527 (1990); D. K. Gilding and A. M. Reed, Polymer, 20, 1459 (1979); and William Morris, et al., Vaccine, 12, 5(1994)). The degradation rates of these polymers vary with the glycolide/lactide ratio and molecular weight thereof, and, therefore, the release of the drug can be sustained over several months by adjusting the molecular weight and glycolide/lactide ratio of the polymer as well as the particle size, and coating thickness of the capsule -` ~171881 formulation(S. J. Holland, et al., J. control. Rel., 4, 155(1986)).
Since 1988, World Health Organization~WHO) has been sponsoring a number of studies to develop a single-shot vaccine for tetanus toxoid using the above-mentioned biodegradable polymers(M. T. Aguado, Vaccine, 11, 596(1993);
Proceed. Intern. Symp. Control. Rel. Bioact. Mater, 21st, Controlled Release Society, Inc., Y. Men, et al., Paper No.
126(1994); 20th, B. Gander, et al., Paper No. 135(1993);
l9th, A. M. Hazrati, et al., Paper No. 220(1992); 21st, M.
Gilley, et al., Paper No. 218(1992); 21st, Manmohan Singh, et al., Paper No. 1476(1994); 21st, C. Yan, et al., Paper No.
127(1994)).
Notwithstanding these efforts, however, no single-shot vaccine formulation has been put to practical use mainly due to the deficiencies that the amount of antibody formed by the encapsulated formulation is only about 1/10 of that produced by a conventional alum formulation and that the result i5 not reproducible(R. E. Spier, Vaccine, 11, 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 for dissolving the biodegradable polymer reduces or nullifies the antigenicity of the antigen by denaturing the antigen; secondly, when in contact with water, the antigen forms an aggregate which has a reduced antigenicity; and thirdly, the antigenicity is reduced or destroyed due to undesirable interactions between the antigen 21718~1 and the hydrophobic biodegradable polymer.
Alonso et al. prepared a single-shot vaccine by encapsulating tetanus toxoid with PLA and PLGA but only 0.5 to 20% of the original tetanus toxoid antigenicity was found to remain in the vaccine particle due to the unavoidable 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 an antibody formation capacity much lower than a control using alum as an adjuvant without boosting, showing that this vaccine formulation is not viable as a single-shot vaccine. Schwendeman et al. endeavored to solve the above problem by chemical modification of the tetanus toxoid, i.e., by S-alkylating the thiol-groups of the antigen, but the result was unsatisfactory(S. P. Schwendeman, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 21st, Paper No. 128, 1994).
Nellore et al. reported a single-shot vaccine formulation for hepatitis B which comprises microparticles prepared by encapsulating a hepatitis B surface antigen (HBsAg) with PGA by using a method involving organic solvent extraction or evaporation(R. v. Nellore, et al., J.
Parenteral Science & Technoloqy, 46, 176(1992)). Animal experiments using guinea pigs showed that a formulation comprising smaller particles in a size range of 1 to 10 ~m exhibited an antibody formation capacity which was much lower than a control using alum as an adjuvant without boosting, _ 2171881 while other formulations having particles ranging from 20 to 60 ym and from 1 to 60 ~m were essentially inactive. In these formulations, HBsAg was denatured by the organic solvent used during the encapsulation process.
Therefore, in order to prepare an efficient single-shot vaccine by encapsulating an antigen with a biodegradable polymer, one must use a biodegradable polymer which does not require the use of an organic solvent in the encapsulating step, or the antigen must be prevented from contacting an organic solvent during the process. However, there has been no known biodegradable polymer which does not require such use of an organic solvent.
For the microparticles have been typically prepared by encapsulating an organic, peptide or protein drug with a biodegradable polymer. Specifically, an oily phase is prepared by dissolving or dispersing the drug in an organic solvent wherein a biodegradable polymer is dissolved, and preparing an oil-in-water("O/W") emulsion by dispersing the oily phase into an aqueous phase in the presence of a surfactant. Then, the organic solvent is removed by a conventional method, e.g., evaporation or extraction, to solidify the biodegradable polymer to obtain 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 wherein a surfactant is dissolved. However, this conventional method has the drawbacks in that: most biodegradable polymers are apt to be - ~1718~1 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 prevent the degradation of the polymer.
In this o/w emulsion method, the drug contacts directly with the organic solvent, and, therefore, the method cannot be applied for the preparation of a vaccine formulation comprising an antigen whose antigenicity may be reduced as a result of the contact. Further, the method has a further limitation in that it cannot be employed for a water-soluble drug. Consequently, development efforts have been shifted to the discovery of a method using a W/O/W emulsion to overcome the above problems.
lS For example, European Patent Application No. 87309286.0 discloses a vaccine formulation for oral administration comprising particles of 10 ~m or less which are prepared by encapsulating various antigens with PLA, PGA, PLGA, etc.
which are known to pass the Peyer's Patches.
Further, European Patent Application No. 88302940.7 discloses an injection formulation, which is capable of maintaining its effect over 6 months, by encapsulating a peptide or protein drug such as 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 _ ~171881 surfactant are dissolved to obtain a W/O emulsion; dispersing the W/O emulsion into an aqueous phase containing a surfactant to obtain a W/O/W emulsion; and then removing 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 in comparison with the O/W
emulsion method. However, a certain degree of mixing between the inner and outer aqueous phases cannot be prevented, which may result in a reduction of the antigenicity of the vaccine.
Moreover, after a washing process to remove the surfactant, water in the inner aqueous phase is normally removed by a conventional method, e. g., lyophilization. A number of large pores may be formed in the biopolymer layer during this water removal process, and, in case of an encapsulated vaccine, these pores may act as conduits for water in human body, facilitating the formation of antigen aggregates with concomitant loss of antigenicity.
In order to overcome the above drawbacks, International Patent Publication No. WO 93/07861 discloses a process for preparing multi-phase particles, which comprises: producing a W/O/W emulsion using a highly viscous edible oil; replacing the outer aqueous phase with an acetonitrile solution of a biodegradable polymer such as PGA, PLA and PLGA; dispersing the resulting emulsion into a mineral oil; and removing acetonitrile to obtain multi-phase particles wherein a w/o microemulsion is encapsulated in a solid polymer shell.
21718~1 _ However, the process has the drawbacks in that: it is not possible to rid the finished multiphase particle of the surfactant, e.g., aluminum monostearate and Span80, which is used to increase the viscosity and dispersion stability of the edible oil thereby preventing the release of the drug from the inner aqueous phase into the outer aqueous phase;
and the drug may loss its antigenicity during the process wherein the temperature is increased up to 140C 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; adding thereto a second solvent which does not dissolve the polymer but mixes with the first solvent; and, thereby, obtaining core-shell type microparticles formed by the solidification of the polymer around the drug as the solubility of the polymer decreases. However, like the O/W emulsion method, this method cannot prevent the antigen from contacting the second organic solvent.
Another problem associated with the above-mentioned methods using emulsions is the possible loss of activity of protein drugs. Proteins are apt to be denatured by a mechanical force, as in a high energy dispersion process.
This is a SeriQuS problem considering 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 `- ~ 217~881 dissolving or dispersing a drug in an organic solvent wherein a biodegradable polymer is dissolved, and spray drying the mixture to obtain microparticles. However, this method also cannot avoid the direct contact of a drug with an organic solvent.
International Patent Publication No. WO 94/12158 and U.
S. Patent No. 5,019,400 suggest a freeze-and-extract method for preparing encapsulated protein drug(growth hormone) particles. In this method, the drug is dispersed in an organic solvent, e.g., methylene chloride, wherein a biodegradable polymer is dissolved and the solution is sprayed into a low-temperature liquid gas to form frozen particles. These particles are collected on the surface of frozen ethanol. As the frozen ethanol is melted, the frozen particles thaw and the organic solvent in the particle is extracted by ethanol, thereby forming microparticles encased in the solidified polymer. This method also allows the direct contact of the antigen with the organic solvent, and the release period of the drug is only several days.
Therefore, this method is not suitable for the preparation of a single-shot vaccine which should release the antigen over a much longer period.
International Patent Publication No. WO 92/14449 discloses a process for preparing particles containing a protein drug which comprises: dispersing a powdered protein drug in a molten fatty acid anhydride, cooling the mixture to solidify the mixture, and pulverizing the mixture to obtain _ 2171881 particles. The fatty acid anhydride melts at a temperature ranging from 45 to 75C and does not denature 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 a particle size suitable for injection.
Further, the fatty acid anhydride itself is not capable of releasing a drug over a long time, and, therefore, it is not suitable for use in the preparation of a single-shot vaccine formulation.
European Patent Application No. 88113933.1 teaches an encapsulated particle formulation designed for a zero-order or bi-phasic mode of release of a protein drug, herbicide or fertilizer, which is prepared by: coating the drug, herbicide or fertilizer with an absorptive polymer and coating again the resulting particle with a polymer, which is insoluble in water but passes the drug, herbicide or fertilizer therethrough. However, the water-insoluble polymer used in the second coating, i.e., cellulose, is not a biodegradable polymer and it releases the drug for the duration of only one day in an uncontrollable manner. Further, the particle is rapidly infiltrated by water in human body and, therefore, in case that the drug is an antigen, it would lose its antigenicity by forming aggregates. Moreover, this process is not suitable for the preparation of microparticles for injection because the size of the first-coated particle ranges from 125 to 10000 ~m and the second coating has to be _ 2171881 thick enough to prevent its rupture by the swelling of the water-absorptive polymer used in the first coating.
As described above, there have been many attempts to develop a process for preparing a single-shot vaccine which avoids undesirable interactions of a protein drug with an organic solvent, a biodegradable polymer and water in human body. Despite these efforts, a method capable of adequately protecting the antigenicity of an antigen during the process of encapsulating the antigen has not been found; and, accordingly, there has continued to exist a need to develop a single-shot vaccine formulation (William Morris, et al., Vaccine, 12, 5(1994)).
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention 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-shot vaccine formulation prepared by dispersing the microparticles in a medium for injection.
In accordance with the present invention, there is provided microparticles having an average particle size ranging from 0.5 to 300 ~m, which is prepared by coating an antigen or a mixture of antigens successively with a water-soluble substance and a biodegradable polymer; and a single-shot vaccine formulation prepared by dispersing the microparticles in a medium for injection, which can accomplish the immunization against an infectious disease by only one injection.
BRIEF DESCRIPTION OF THE DRAWING
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with Fig. 1, which shows the relationship between the particle property and antibody formation, wherein three vaccine formulations prepared in accordance with the present invention are compared with a conventional alum formulation.
DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated 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 powdered particle("a core particle") and coating the core particle with a hydrophobic biodegradable polymer to obtain the final microparticle. The microparticle has a - spherical or spheroidal shape with a size ranging from 0.5 to 300 ~m. A single-shot vaccine formulation, which can accomplish the vaccination by only one injection, may be prepared by dispersing the microparticle in an injection 2~71881 medium.
A suitable antigen which may be used in the present invention is an attenuated, killed or recombinant antigen which is used as a vaccine for a single disease("single antigen") or two or more diseases simultaneously("mixed antigen"). The mixed antigen may be a mixture of two or more antigens, or an antigen which has antigenicities for two or more diseases simultaneously, e.g., a recombinant protein.
As an antigen, there may be used an entire organism, e. g., a viral or bacterial whole cell, or a part of the organism, e. g., a certain protein having an antigenicity.
Exemplary antigens of the present invention include antigens for hepatitis, diphtheria, chickenpox, typhoid, pertussis, tetanus, tuberculosis, salmonellosis, cholera, HIV, herpes, yellow fever, measles, poliomyelitis, 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, ricin 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 a suitable aqueous solvent, e.
g., water or a ~uffer, and drying the mixture by a spray drying or a freeze drying method. A suitable adjuvant may be added to the solution, if necessary, and examples thereof include alum; muramyl dipeptide, muramyl tripeptide and --` 21718~1 derivatives thereof; tymosin alpha; monophosphoryl lipid A;
saponin; an immunostimulating complex; a polyelectrolyte such as a copolymer of polyoxyethylene and polyoxypropylene; and a mixture thereof.
The water-soluble substance used for the preparation of the core particle does not bring about an undesirable interaction with the 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, saccharose, alginate, dextran, hyaluronic acid, chondroitin sulfate and water-soluble cellulose derivatives, e.g., hydroxypropylmethyl cellulose, hydroxypropyl cellulose(HPC), carboxymethyl cellulose(CMC) and sodium carboxymethyl cellulose(CMC-Na); proteins such as albumin and gelatin; amino acids such as glycine, alanine, glutamic acid, arginine, lysine and a salt thereof; and a mixture thereof; while HPC, CMC, CMC-Na, gelatin, and a mixture thereof are preferred.
The water-soluble substance may be used in an amount ranging from 1 to 50, preferably, from 5 to 15 times the weight of total antigen.
The core particle so prepared has a particle size ranging from 0.1 to 200 ~m, preferably, from 0.5 to 20 ~m.
In order to prepare the final microparticle, the core particle is dispersed in an organic solvent, wherein a hydrophobic biodegradable polymer is dissolved, by using a -- ~171881 suitable apparatus, e.g., a magnetic stirrer, homogenizer, microfluidizer and sonicator.
The biodegradable polymer is used in an amount ranging from 1 to 100, preferably, from 5 to 30 times the weight of the core particle. The coating of the core particle is made of a water-soluble substance which is insoluble in the organic solvent, and therefore, it prevents the reduction or loss of the antigenicity of the antigen by blocking the contact of the antigen with the organic solvent.
Exemplary hydrophobic biodegradable polymers which may 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(~-hydroxybutyric acid), and polyanhydride; while PLGA and PLA
are preferred.
Any of the organic solvents well-known in the art may 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 evenly dispersed in an organic solution of a biodegradable polymer("the core particle dispersed system").
The core particle dispersed system is advantageous in that a microparticle can be prepared therefrom in accordance with a -- ` 2171881 conventional method, while avoiding the contact of the antigen with the organic solvent or with the biodegradable polymer. Further, the surface area of the core particle in contact with the organic solvent is sufficiently low such that a physical contact of the antigen with the organic solvent may not be occurred.
Specifically, the microparticle of the present invention may be prepared from the core particle dispersed system in accordance with any one 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 lS prior arts in that the core particle dispersed system, wherein the contact of the antigen with the organic solvent is prevented, is employed in place of an aqueous solution wherein the antigen is dissolved or dispersed.
Specifically, the microparticle may be prepared by dispersing the core particle dispersed system in an aqueous solution comprising a surfactant to obtain an O/W emulsion and then removing the organic solvent from the core particle dispersed system, or by dispersing the core particle dispersed system in a solvent, which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer, to prepare an O/O emulsion and removing the organic solvent from the core particle dispersed - 21718~1 system. When acetonitrile is used as the organic solvent of the core particle dispersed system, a mineral oil can be used as the solvent which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer.
2) Solvent extraction method This method is also well-known in the art for the preparation of a microparticle, but the present invention differs from the prior arts in that the core particle dispersed system is employed. Specifically, the microparticle may be prepared by extracting the organic solvent of the core particle dispersed system by using a solvent, which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer, such as a mineral oil and paraffin oil.
FIELD OF THE INVENTION
The present invention relates to microparticles having a particle size ranging from 0.5 to 300 ym, which are prepared by coating an antigen or a mixture of antigens with a water-soluble substance and a bio-degradable polymer successively; and a vaccine formulation prepared by dispersing the microparticles in a medium for injection, which can accomplish the immunization effect against an infectious disease by administering a single injection only("single-shot vaccine formulation") due to the delayed release of the antigen over a period of, e.g., several months.
BACKGROUND OF THE INVENTION
Hitherto, there have been developed a large number of vaccines effective in preventing various infectious diseases.
However, most of the currently used vaccines require multiple inoculations, which requirement imposes an economical burden as well as inconveniences to the vaccinees(M. T. Aguado and P. H. Lambert, Immunobiol., 184, 113(1992); Aryward, B., et al., Vaccine, 12, 1155(1994)). In particular, it has been found that only about 30% of those who receive the first inoculation return for the second administration.
-- ~171881 Statistically, therefore, only 9 out of 100 would complete an immunization process when three inoculations are prescribed, clearly demonstrating the need for a vaccine that can accomplish immunization by a single-shot inoculation.
The prior art approach to the development of a single-shot vaccine has basically centered on the idea of using a microparticle, wherein a desired antigen is encapsulated with a biodegradable polymeric material, which releases the antigen slowly over a prescribed time period to accomplish the vaccination(R. Langer and J. Folkman, Nature, 263, 797(197~)).
Among various biodegradable polymers, polylactidetPLA), 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 in making a suture whose post-operation removal is not required; and also in formulating encapsulated leuprolide acetate, a LHRH analogue, which has been approved by FDA for human use(R. Langer and M. Mose, Science, 249, 1527 (1990); D. K. Gilding and A. M. Reed, Polymer, 20, 1459 (1979); and William Morris, et al., Vaccine, 12, 5(1994)). The degradation rates of these polymers vary with the glycolide/lactide ratio and molecular weight thereof, and, therefore, the release of the drug can be sustained over several months by adjusting the molecular weight and glycolide/lactide ratio of the polymer as well as the particle size, and coating thickness of the capsule -` ~171881 formulation(S. J. Holland, et al., J. control. Rel., 4, 155(1986)).
Since 1988, World Health Organization~WHO) has been sponsoring a number of studies to develop a single-shot vaccine for tetanus toxoid using the above-mentioned biodegradable polymers(M. T. Aguado, Vaccine, 11, 596(1993);
Proceed. Intern. Symp. Control. Rel. Bioact. Mater, 21st, Controlled Release Society, Inc., Y. Men, et al., Paper No.
126(1994); 20th, B. Gander, et al., Paper No. 135(1993);
l9th, A. M. Hazrati, et al., Paper No. 220(1992); 21st, M.
Gilley, et al., Paper No. 218(1992); 21st, Manmohan Singh, et al., Paper No. 1476(1994); 21st, C. Yan, et al., Paper No.
127(1994)).
Notwithstanding these efforts, however, no single-shot vaccine formulation has been put to practical use mainly due to the deficiencies that the amount of antibody formed by the encapsulated formulation is only about 1/10 of that produced by a conventional alum formulation and that the result i5 not reproducible(R. E. Spier, Vaccine, 11, 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 for dissolving the biodegradable polymer reduces or nullifies the antigenicity of the antigen by denaturing the antigen; secondly, when in contact with water, the antigen forms an aggregate which has a reduced antigenicity; and thirdly, the antigenicity is reduced or destroyed due to undesirable interactions between the antigen 21718~1 and the hydrophobic biodegradable polymer.
Alonso et al. prepared a single-shot vaccine by encapsulating tetanus toxoid with PLA and PLGA but only 0.5 to 20% of the original tetanus toxoid antigenicity was found to remain in the vaccine particle due to the unavoidable 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 an antibody formation capacity much lower than a control using alum as an adjuvant without boosting, showing that this vaccine formulation is not viable as a single-shot vaccine. Schwendeman et al. endeavored to solve the above problem by chemical modification of the tetanus toxoid, i.e., by S-alkylating the thiol-groups of the antigen, but the result was unsatisfactory(S. P. Schwendeman, et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 21st, Paper No. 128, 1994).
Nellore et al. reported a single-shot vaccine formulation for hepatitis B which comprises microparticles prepared by encapsulating a hepatitis B surface antigen (HBsAg) with PGA by using a method involving organic solvent extraction or evaporation(R. v. Nellore, et al., J.
Parenteral Science & Technoloqy, 46, 176(1992)). Animal experiments using guinea pigs showed that a formulation comprising smaller particles in a size range of 1 to 10 ~m exhibited an antibody formation capacity which was much lower than a control using alum as an adjuvant without boosting, _ 2171881 while other formulations having particles ranging from 20 to 60 ym and from 1 to 60 ~m were essentially inactive. In these formulations, HBsAg was denatured by the organic solvent used during the encapsulation process.
Therefore, in order to prepare an efficient single-shot vaccine by encapsulating an antigen with a biodegradable polymer, one must use a biodegradable polymer which does not require the use of an organic solvent in the encapsulating step, or the antigen must be prevented from contacting an organic solvent during the process. However, there has been no known biodegradable polymer which does not require such use of an organic solvent.
For the microparticles have been typically prepared by encapsulating an organic, peptide or protein drug with a biodegradable polymer. Specifically, an oily phase is prepared by dissolving or dispersing the drug in an organic solvent wherein a biodegradable polymer is dissolved, and preparing an oil-in-water("O/W") emulsion by dispersing the oily phase into an aqueous phase in the presence of a surfactant. Then, the organic solvent is removed by a conventional method, e.g., evaporation or extraction, to solidify the biodegradable polymer to obtain 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 wherein a surfactant is dissolved. However, this conventional method has the drawbacks in that: most biodegradable polymers are apt to be - ~1718~1 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 prevent the degradation of the polymer.
In this o/w emulsion method, the drug contacts directly with the organic solvent, and, therefore, the method cannot be applied for the preparation of a vaccine formulation comprising an antigen whose antigenicity may be reduced as a result of the contact. Further, the method has a further limitation in that it cannot be employed for a water-soluble drug. Consequently, development efforts have been shifted to the discovery of a method using a W/O/W emulsion to overcome the above problems.
lS For example, European Patent Application No. 87309286.0 discloses a vaccine formulation for oral administration comprising particles of 10 ~m or less which are prepared by encapsulating various antigens with PLA, PGA, PLGA, etc.
which are known to pass the Peyer's Patches.
Further, European Patent Application No. 88302940.7 discloses an injection formulation, which is capable of maintaining its effect over 6 months, by encapsulating a peptide or protein drug such as 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 _ ~171881 surfactant are dissolved to obtain a W/O emulsion; dispersing the W/O emulsion into an aqueous phase containing a surfactant to obtain a W/O/W emulsion; and then removing 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 in comparison with the O/W
emulsion method. However, a certain degree of mixing between the inner and outer aqueous phases cannot be prevented, which may result in a reduction of the antigenicity of the vaccine.
Moreover, after a washing process to remove the surfactant, water in the inner aqueous phase is normally removed by a conventional method, e. g., lyophilization. A number of large pores may be formed in the biopolymer layer during this water removal process, and, in case of an encapsulated vaccine, these pores may act as conduits for water in human body, facilitating the formation of antigen aggregates with concomitant loss of antigenicity.
In order to overcome the above drawbacks, International Patent Publication No. WO 93/07861 discloses a process for preparing multi-phase particles, which comprises: producing a W/O/W emulsion using a highly viscous edible oil; replacing the outer aqueous phase with an acetonitrile solution of a biodegradable polymer such as PGA, PLA and PLGA; dispersing the resulting emulsion into a mineral oil; and removing acetonitrile to obtain multi-phase particles wherein a w/o microemulsion is encapsulated in a solid polymer shell.
21718~1 _ However, the process has the drawbacks in that: it is not possible to rid the finished multiphase particle of the surfactant, e.g., aluminum monostearate and Span80, which is used to increase the viscosity and dispersion stability of the edible oil thereby preventing the release of the drug from the inner aqueous phase into the outer aqueous phase;
and the drug may loss its antigenicity during the process wherein the temperature is increased up to 140C 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; adding thereto a second solvent which does not dissolve the polymer but mixes with the first solvent; and, thereby, obtaining core-shell type microparticles formed by the solidification of the polymer around the drug as the solubility of the polymer decreases. However, like the O/W emulsion method, this method cannot prevent the antigen from contacting the second organic solvent.
Another problem associated with the above-mentioned methods using emulsions is the possible loss of activity of protein drugs. Proteins are apt to be denatured by a mechanical force, as in a high energy dispersion process.
This is a SeriQuS problem considering 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 `- ~ 217~881 dissolving or dispersing a drug in an organic solvent wherein a biodegradable polymer is dissolved, and spray drying the mixture to obtain microparticles. However, this method also cannot avoid the direct contact of a drug with an organic solvent.
International Patent Publication No. WO 94/12158 and U.
S. Patent No. 5,019,400 suggest a freeze-and-extract method for preparing encapsulated protein drug(growth hormone) particles. In this method, the drug is dispersed in an organic solvent, e.g., methylene chloride, wherein a biodegradable polymer is dissolved and the solution is sprayed into a low-temperature liquid gas to form frozen particles. These particles are collected on the surface of frozen ethanol. As the frozen ethanol is melted, the frozen particles thaw and the organic solvent in the particle is extracted by ethanol, thereby forming microparticles encased in the solidified polymer. This method also allows the direct contact of the antigen with the organic solvent, and the release period of the drug is only several days.
Therefore, this method is not suitable for the preparation of a single-shot vaccine which should release the antigen over a much longer period.
International Patent Publication No. WO 92/14449 discloses a process for preparing particles containing a protein drug which comprises: dispersing a powdered protein drug in a molten fatty acid anhydride, cooling the mixture to solidify the mixture, and pulverizing the mixture to obtain _ 2171881 particles. The fatty acid anhydride melts at a temperature ranging from 45 to 75C and does not denature 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 a particle size suitable for injection.
Further, the fatty acid anhydride itself is not capable of releasing a drug over a long time, and, therefore, it is not suitable for use in the preparation of a single-shot vaccine formulation.
European Patent Application No. 88113933.1 teaches an encapsulated particle formulation designed for a zero-order or bi-phasic mode of release of a protein drug, herbicide or fertilizer, which is prepared by: coating the drug, herbicide or fertilizer with an absorptive polymer and coating again the resulting particle with a polymer, which is insoluble in water but passes the drug, herbicide or fertilizer therethrough. However, the water-insoluble polymer used in the second coating, i.e., cellulose, is not a biodegradable polymer and it releases the drug for the duration of only one day in an uncontrollable manner. Further, the particle is rapidly infiltrated by water in human body and, therefore, in case that the drug is an antigen, it would lose its antigenicity by forming aggregates. Moreover, this process is not suitable for the preparation of microparticles for injection because the size of the first-coated particle ranges from 125 to 10000 ~m and the second coating has to be _ 2171881 thick enough to prevent its rupture by the swelling of the water-absorptive polymer used in the first coating.
As described above, there have been many attempts to develop a process for preparing a single-shot vaccine which avoids undesirable interactions of a protein drug with an organic solvent, a biodegradable polymer and water in human body. Despite these efforts, a method capable of adequately protecting the antigenicity of an antigen during the process of encapsulating the antigen has not been found; and, accordingly, there has continued to exist a need to develop a single-shot vaccine formulation (William Morris, et al., Vaccine, 12, 5(1994)).
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention 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-shot vaccine formulation prepared by dispersing the microparticles in a medium for injection.
In accordance with the present invention, there is provided microparticles having an average particle size ranging from 0.5 to 300 ~m, which is prepared by coating an antigen or a mixture of antigens successively with a water-soluble substance and a biodegradable polymer; and a single-shot vaccine formulation prepared by dispersing the microparticles in a medium for injection, which can accomplish the immunization against an infectious disease by only one injection.
BRIEF DESCRIPTION OF THE DRAWING
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with Fig. 1, which shows the relationship between the particle property and antibody formation, wherein three vaccine formulations prepared in accordance with the present invention are compared with a conventional alum formulation.
DETAILED DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated 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 powdered particle("a core particle") and coating the core particle with a hydrophobic biodegradable polymer to obtain the final microparticle. The microparticle has a - spherical or spheroidal shape with a size ranging from 0.5 to 300 ~m. A single-shot vaccine formulation, which can accomplish the vaccination by only one injection, may be prepared by dispersing the microparticle in an injection 2~71881 medium.
A suitable antigen which may be used in the present invention is an attenuated, killed or recombinant antigen which is used as a vaccine for a single disease("single antigen") or two or more diseases simultaneously("mixed antigen"). The mixed antigen may be a mixture of two or more antigens, or an antigen which has antigenicities for two or more diseases simultaneously, e.g., a recombinant protein.
As an antigen, there may be used an entire organism, e. g., a viral or bacterial whole cell, or a part of the organism, e. g., a certain protein having an antigenicity.
Exemplary antigens of the present invention include antigens for hepatitis, diphtheria, chickenpox, typhoid, pertussis, tetanus, tuberculosis, salmonellosis, cholera, HIV, herpes, yellow fever, measles, poliomyelitis, 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, ricin 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 a suitable aqueous solvent, e.
g., water or a ~uffer, and drying the mixture by a spray drying or a freeze drying method. A suitable adjuvant may be added to the solution, if necessary, and examples thereof include alum; muramyl dipeptide, muramyl tripeptide and --` 21718~1 derivatives thereof; tymosin alpha; monophosphoryl lipid A;
saponin; an immunostimulating complex; a polyelectrolyte such as a copolymer of polyoxyethylene and polyoxypropylene; and a mixture thereof.
The water-soluble substance used for the preparation of the core particle does not bring about an undesirable interaction with the 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, saccharose, alginate, dextran, hyaluronic acid, chondroitin sulfate and water-soluble cellulose derivatives, e.g., hydroxypropylmethyl cellulose, hydroxypropyl cellulose(HPC), carboxymethyl cellulose(CMC) and sodium carboxymethyl cellulose(CMC-Na); proteins such as albumin and gelatin; amino acids such as glycine, alanine, glutamic acid, arginine, lysine and a salt thereof; and a mixture thereof; while HPC, CMC, CMC-Na, gelatin, and a mixture thereof are preferred.
The water-soluble substance may be used in an amount ranging from 1 to 50, preferably, from 5 to 15 times the weight of total antigen.
The core particle so prepared has a particle size ranging from 0.1 to 200 ~m, preferably, from 0.5 to 20 ~m.
In order to prepare the final microparticle, the core particle is dispersed in an organic solvent, wherein a hydrophobic biodegradable polymer is dissolved, by using a -- ~171881 suitable apparatus, e.g., a magnetic stirrer, homogenizer, microfluidizer and sonicator.
The biodegradable polymer is used in an amount ranging from 1 to 100, preferably, from 5 to 30 times the weight of the core particle. The coating of the core particle is made of a water-soluble substance which is insoluble in the organic solvent, and therefore, it prevents the reduction or loss of the antigenicity of the antigen by blocking the contact of the antigen with the organic solvent.
Exemplary hydrophobic biodegradable polymers which may 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(~-hydroxybutyric acid), and polyanhydride; while PLGA and PLA
are preferred.
Any of the organic solvents well-known in the art may 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 evenly dispersed in an organic solution of a biodegradable polymer("the core particle dispersed system").
The core particle dispersed system is advantageous in that a microparticle can be prepared therefrom in accordance with a -- ` 2171881 conventional method, while avoiding the contact of the antigen with the organic solvent or with the biodegradable polymer. Further, the surface area of the core particle in contact with the organic solvent is sufficiently low such that a physical contact of the antigen with the organic solvent may not be occurred.
Specifically, the microparticle of the present invention may be prepared from the core particle dispersed system in accordance with any one 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 lS prior arts in that the core particle dispersed system, wherein the contact of the antigen with the organic solvent is prevented, is employed in place of an aqueous solution wherein the antigen is dissolved or dispersed.
Specifically, the microparticle may be prepared by dispersing the core particle dispersed system in an aqueous solution comprising a surfactant to obtain an O/W emulsion and then removing the organic solvent from the core particle dispersed system, or by dispersing the core particle dispersed system in a solvent, which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer, to prepare an O/O emulsion and removing the organic solvent from the core particle dispersed - 21718~1 system. When acetonitrile is used as the organic solvent of the core particle dispersed system, a mineral oil can be used as the solvent which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer.
2) Solvent extraction method This method is also well-known in the art for the preparation of a microparticle, but the present invention differs from the prior arts in that the core particle dispersed system is employed. Specifically, the microparticle may be prepared by extracting the organic solvent of the core particle dispersed system by using a solvent, which is immiscible with the core particle dispersed system and is a nonsolvent for the biodegradable polymer, such as a mineral oil and paraffin oil.
3) Rapid freezing and solvent extraction method The present invention is different from the prior arts in that the core particle dispersed system is employed.
Specifically, the core particle dispersed system is sprayed into a low-temperature liquid gas phase using an ultrasonic apparatus to form a frozen particle. This particle is collected on the surface of frozen ethanol. As the frozen ethanol is melted, the frozen particle thaws and the organic solvent in the particle is extracted into the ethanol phase with concomitant formation of a microparticle coated with the ~-~ 2171881 biodegradable polymer.
Specifically, the core particle dispersed system is sprayed into a low-temperature liquid gas phase using an ultrasonic apparatus to form a frozen particle. This particle is collected on the surface of frozen ethanol. As the frozen ethanol is melted, the frozen particle thaws and the organic solvent in the particle is extracted into the ethanol phase with concomitant formation of a microparticle coated with the ~-~ 2171881 biodegradable polymer.
4) Spray drying method This method is most preferable for use in the present invention and, specifically, the microparticle is prepared by spraying the core particle dispersed system by employing a spray-dryer. This method is advantageous due to its high productivity and rapidity. Further, it is also advantageous in that removal of water is unnecessary because 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 thus prepared ranges from 0.5 to 300 ~m, preferably, from 1 to 180 ~m.
Those microparticles having a particle size smaller than 180 ~m may be dispersed in an injection medium to prepare an injection formulation for subcutaneous, intramuscular, and intraperitoneal injections. Those particles having a particle size larger than 180 ~m may be used for preparing a formulation for oral administration.
Therefore, the present invention further provides a single-shot vaccine formulation which is prepared by dispersing the microparticles in a suitable injection medium.
The vaccine formulation may comprise single antigen alone, or two or more kinds of antigens together. The vaccine formulation comprising two or more antigens may be prepared by employing core particles comprising a mixture of two or -- ` ~17188 1 more kinds of antigens, or by employing a mixture of two or more kinds of core particles each comprising an antigen different from each other.
The single-shot vaccine formulation of the present invention can accomplish a vaccination against the antigen comprised therein by only one injection and the amount of antigen in the vaccine formulation is the same or less than that of the conventional alum formulation which needs several injections to accomplish the vaccination.
Exemplary injection media which can be used in the present invention include a buffer with or without dispersing agents and/or preservatives, an edible oil, mineral oil, cod liver oil, squalene, squalane, mono-, di- or triglyceride and a mixture thereof; said edible oil being corn oil, sesame oil, olive oil, soybean oil, safflower oil, cotton seed oil, peanut oil or a mixture thereof.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.
The materials and methods used in the Examples are as follows:
Antigen A recombinant hepatitis B surface antigen(HBsAg) dissolved in a phosphate buffered saline(PBS), which i5 prepared in accordance with the method of Korean Patent Publication No. 93-2735(Publication date: April 9, 1994).
Biodegradable polymer PLGA(50/50) produced by BPI, U.S.A.
Amount of antigen: Determined by Lowry method.
Antigenicity: Determined by using AUZYME test kit(Abbott, U.S.A).
Antibody titer Geometric average of titers obtained from the sera of 11 guinea pigs by using AUSAB test kit(Abbott, U.S.A).
Guinea pig Those weighing 300-400g and confirmed not to have antibodies in their blood 2 days before the administration of sample. Blood is collected from the heart of the guinea pig by using the cardiac puncture technique.
Example 1: Preparation of Core particle by Using Freeze Drying Method Recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 ~g/mQ and hydroxypropyl cellulose was added thereto to a concentration of 0.3 mg/mQ(Sample 1~, 1.5 --` 2171~81 mg/mQ(Sample 2), and 3.0 mg/mQ(Sample 3), respectively. Each solution was frozen at -70C for 30 minutes by using dry ice and acetone, and then freeze dried for 24 hours by using EYELA FD-81 freeze dryer(Tokyo Rikakikai, Japan) at 0.05 torr and a condenser temperature of -80C to obtain core particles coated with hydroxypropyl cellulose. The average particle sizes of the core particles thus prepared were 1.0, 1.2 and 1.5 ~m, respectively.
Example 2: Preparation of Core particle by Using Spray Drying Method Recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 ~g/mQ, and each of hydroxypropyl cellulose(Sample 4), sodium carboxymethyl cellulose(Sample 5) and gelatin(Sample 6) was added thereto to a concentration of 3.0 mg/mQ. Each solution was provided to a spray dryer(Buchi 190) at a flow rate of 3 mQ/minute to obtain core particles coated with hydroxypropyl cellulose, sodium carboxymethyl cellulose and gelatin, respectively. In this step, the flow rate of propellant nitrogen was 600 Q/minute and the inflow air temperature was 70 C. The average particle sizes of the core particles thus prepared were 4.6, 6.3 and 4.9 ~m, respectively.
Test Example 1: Protection of Antigen in Core particle To confirm whether the core particle coated with the water-soluble substance protects the antigenicity of the antigen therein from the contact with the organic solvent, the core particles prepared in Example 1 and 2 were dispersed in ethyl acetate or acetonitrile, which dissolves PLGA but not hydroxypropyl cellulose, and the resulting solutions were mixed by using a magnetic stirrer to allow the core particle to contact the organic solvent. The core particles were separated and dried to remove the organic solvent. The dried core particles were dissolved in a buffer(10 mM phosphate, pH
7.5) and the antigenicity was determined by AUZYME kit and compared to that of recombinant HBsAg solution of Korean Patent Publication No. 93-2735 stored in a cryogenic state.
Further, the HBsAg which was not coated with the water-soluble substance was used as a control, as follows.
Recombinant HBsAg was dissolved in lOmM PBS to a concentration of 300 ~g/mQ and to the solution was added a same volume of each of various organic solvents listed in Table 1. The resulting solutions were mixed by using a magnetic stirrer for 10 minutes. The aqueous phases were separated from the solutions-and antigenicities thereof were determined according to the same procedure as above. The precipitate was separated, dried and added to PBS for the determination of the antigenicity. However, the precipitate was not dissolved in PBS due to denaturation thereof which resulted in an insoluble form.
As a result, the antigen in the core particle which is coated with hydroxypropyl cellulose maintained its antigenicity after its contact with the organic solvent, while the antigen which was not coated with the polymer lost its antigenicity completely after its contact with the organic solvent.
~17188~
Table l Sample Water-soluble Organic Antigen- Insoluble No. - polymer solvent icity ppt. Note (%) Original HBsAg - - l00 Solution aceto- 78 hydroxypropyl nitrile l cellulose (0.3 mg/mQ) ethyl 75 acetate aceto- 80 - -hydroxypropyl nitrile 2 cellulose (l.5 mg/mQ) ethyl 80 acetate aceto- 9l - -hydroxypropyl nitrile 3 cellulose (3.0 mg/mQ) ethyl 88 acetate hydroxypropyl 90 4 cellulose (3.0 mg/mQ) sodium ethyl 85 carboxymethyl acetate cellulose (3.0 mg/mQ) 6 gelatin 82 3.0 mg/mQ) aceto- 0 formed nitrile ethyl 0 formed acetate carbon 0 formed Control not used tetra-chloride chloroform <<l.0 formed *
methylene <<l.0 formed *
chloride benzene <<l.0 formed *
hexane <<l.0 formed *
1)trace amount was redissolved.
' - ~
Example 3: Preparation of Microparticles PLGA(50/50) was dissolved in ethyl acetate to a concentration of 1 % and the core particle of Sample 3 prepared in Example 1 was dispersed therein to a concentration of 1 mg/mQ. The resulting solution was provided to a spray dryer(Buchi 190) at a flow rate of 3 mQ/minute to obtain the final microparticles which were coated further with PLGA. In this step, the flow rate of nitrogen was 600 Q/minute and the inflow air temperature was 60 C.
The average particle size of the microparticles thus prepared was 7 ~m and the weight ratio of PLGA/the core particle was 10. The amount of antigen determined by Lowry method was 6 ~g protein/mg of particle.
Example 4: Preparation of Microparticles Microparticles coated with PLGA were prepared by using Sample 4 of Example 2 as the core particle, in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 10.
The average particle size of the microparticles thus prepared was 9~m and the amount of antigen was 3 ~g protein/mg of particle.
21718~1 Example 5: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 2 mg/mQ, and microparticles coated with PLGA were prepared in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 5.
The average particle size of the microparticles thus prepared was 7 ~m and the amount of antigen was 6 ~g protein/mg of particle.
Example 6: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 0.5 mg/mQ, and microparticles coated with PLGA were prepared in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 20.
The average particle size of the microparticles thus prepared was 10 ~m and the amount of antigen was 1.5 ~g protein/mg of particle.
Example 7: Preparation of Microparticles 5 mg of Sample 4(the core particle) prepared in Example 2 was dispersed in 1 mQ of ethyl acetate, wherein 200 mg of PLGA was dissolved, by using an ultrasonicator. The core particle dispersed system was mixed with 1 mQ of 1 % aqueous polyvinylalchol(PVA) solution by using a magnetic stirrer and the resulting mixture was dispersed in 100 mQ of 0.3 %
aqueous PVA solution to obtain an O/W emulsion. The emulsion was mixed continuously for 5 hours to evaporate ethyl acetate and microparticles thus formed were separated by using a 0.5 ym filter, washed and then dried.
The average particle size was 110 ~m.
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 to guinea pigs in a quantity that the amount of antigen becomes 40 ~g protein/head. Blood samples were taken from the guinea pigs for 2 months after the injection at fifteen-day intervals and the concentration (mIU/mQ) of formed antibody in each sample was determined.
As a control, particles coated with PLGA alone without a core coat of a water-soluble substance was prepared as follows. Original HBsAg solution was frozen in accordance with the method of Example 1 without using a water-soluble substance as the core coat and the HBsAg particles coated directly with PLGA alone(Comparative sample) were prepared therefrom in accordance with the method of Example 3. The Comparative sample was administered to guinea pigs in accordance with the same method as above, and then the 217188 l concentration of formed antibody was determined.
As a result, Sample 7 showed an antibody concentration higher than that of Comparative sample, as can be seen from Table 2. This result shows 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 the Injection(Months) tmIU/mQ ) Sample 0 0.5 1 1.5 2 Sample 7 0.0 2.410.6 24.6 34.0 Comparative sample 0.0 1.1 8.1 10.2 14.3 Test Example 3: In vivo Effect of Single-Shot Vaccine Formulation The following experiment was carried out to confirm if the vaccine formulation of the present invention has a superior effect as a single-shot vaccine formulation.
The microparticles prepared in Example 5 were dispersed in 1.0 m~ of an injection medium(PBS containing 0.02 wt% of Tween 80) to form a vaccine formulation(Sample 8), which was injected once subcutaneously to guinea pigs by using a 26G
syringe in a quantity that the amount of antigen becomes 20 yg protein/head. Blood samples were taken from the guinea pigs for 5 months after the injection at fifteen-day or a month intervals and the concentration(mIU/mQ) of formed antibody in each sample was determined.
A comparative formulation which employs alum as an adjuvant was prepared as follows. 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 ~g protein/1.5 mg alum.
The vaccine formulation was injected once to two groups of guinea pigs by using a 26G syringe in a quantity that the amount of the antigen becomes lOyg protein/mQ/injection (primary injection). Thereafter, one group was subjected to the first boosting using the same antigen amount fifteen days after the primary injection(Comparative Example 1). The other group was further subjected to the second boosting 1.5 months after they received the first boosting in accordance with the same procedures as above(Comparative Example 2).
Further, one more group of guinea pigs were received a primary injection only by using the alum formulation and the same method as above, in a quantity that the amount of the antigen becomes 20 yg protein/mQ/injection (Comparative Example 3, Sample 9).
The concentrations(mIU/mQ) of formed antibody of Comparative Examples 1, 2 and 3 were determined as above and the results were compared with that of Sample 8.
2171~81 The result is shown in Table 3, wherein it can be seen that the concentration of antibody formed by an injection of Sample 8 is higher than those formed by injections up to three times of the alum formulation. This result shows that the vaccine formulation of the present invention has a superior effect as a single-shot vaccine formulation, i.e., a single injection of the inventive vaccine formulation using 20 yg protein shows a superior vaccination effect than three injections of the alum formulation using 30 yg of protein.
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Test Example 4: Relationship between the Particle Property of the Vaccine Formulation and Antibody Formation In order to investigate whether the time and amount of antibody formation are controllable in accordance with the weight ratio of the biodegradable polymer and the core particle, the following test was carried out.
Each of the microparticles prepared in Examples 4, 5 and 6 was suspended in PBS to prepare a single-shot vaccine formulation(Samples 10, 8 and 11), which was injected once subcutaneously to guinea pigs in a quantity that the amount of antigen becomes 20 ~g protein/ head. Blood samples were taken from the guinea pigs for 5 months after the injection at fifteen-day intervals and the concentration~mIU/mQ) of formed antibody in each sample was determined.
Further, the alum formulation prepared in Test Example 3 was injected once to guinea pigs in a quantity that the amount of antigen becomes 20 ~g protein/head(Sample 9) and the concentration(mIU/mQ) of formed antibody was determined in accordance with the same method as above.
The antibody concentration changes with time after the injection are shown in Fig. 1, wherein Sample 8, which has a PLGA/core particle weight ratio of 5, shows the highest rate of increase in the antibody concentration. Therefore, it was confirmed that the rate of antibody formation is controllable by regulating the thickness of the biodegradable polymer coating. Therefore, a single-shot vaccine containing mixed antigens or a single-shot vaccine which can release an antigen in different patterns can be prepared by using various particles having different properties.
As described in the above Examples, the single-shot vaccine formulation of the present invention can accomplish vaccination by administering only one injection and further has a higher antibody forming capacity than conventional vaccine formulations.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.
The particle size of the microparticle thus prepared ranges from 0.5 to 300 ~m, preferably, from 1 to 180 ~m.
Those microparticles having a particle size smaller than 180 ~m may be dispersed in an injection medium to prepare an injection formulation for subcutaneous, intramuscular, and intraperitoneal injections. Those particles having a particle size larger than 180 ~m may be used for preparing a formulation for oral administration.
Therefore, the present invention further provides a single-shot vaccine formulation which is prepared by dispersing the microparticles in a suitable injection medium.
The vaccine formulation may comprise single antigen alone, or two or more kinds of antigens together. The vaccine formulation comprising two or more antigens may be prepared by employing core particles comprising a mixture of two or -- ` ~17188 1 more kinds of antigens, or by employing a mixture of two or more kinds of core particles each comprising an antigen different from each other.
The single-shot vaccine formulation of the present invention can accomplish a vaccination against the antigen comprised therein by only one injection and the amount of antigen in the vaccine formulation is the same or less than that of the conventional alum formulation which needs several injections to accomplish the vaccination.
Exemplary injection media which can be used in the present invention include a buffer with or without dispersing agents and/or preservatives, an edible oil, mineral oil, cod liver oil, squalene, squalane, mono-, di- or triglyceride and a mixture thereof; said edible oil being corn oil, sesame oil, olive oil, soybean oil, safflower oil, cotton seed oil, peanut oil or a mixture thereof.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, unless specifically indicated otherwise.
The materials and methods used in the Examples are as follows:
Antigen A recombinant hepatitis B surface antigen(HBsAg) dissolved in a phosphate buffered saline(PBS), which i5 prepared in accordance with the method of Korean Patent Publication No. 93-2735(Publication date: April 9, 1994).
Biodegradable polymer PLGA(50/50) produced by BPI, U.S.A.
Amount of antigen: Determined by Lowry method.
Antigenicity: Determined by using AUZYME test kit(Abbott, U.S.A).
Antibody titer Geometric average of titers obtained from the sera of 11 guinea pigs by using AUSAB test kit(Abbott, U.S.A).
Guinea pig Those weighing 300-400g and confirmed not to have antibodies in their blood 2 days before the administration of sample. Blood is collected from the heart of the guinea pig by using the cardiac puncture technique.
Example 1: Preparation of Core particle by Using Freeze Drying Method Recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 ~g/mQ and hydroxypropyl cellulose was added thereto to a concentration of 0.3 mg/mQ(Sample 1~, 1.5 --` 2171~81 mg/mQ(Sample 2), and 3.0 mg/mQ(Sample 3), respectively. Each solution was frozen at -70C for 30 minutes by using dry ice and acetone, and then freeze dried for 24 hours by using EYELA FD-81 freeze dryer(Tokyo Rikakikai, Japan) at 0.05 torr and a condenser temperature of -80C to obtain core particles coated with hydroxypropyl cellulose. The average particle sizes of the core particles thus prepared were 1.0, 1.2 and 1.5 ~m, respectively.
Example 2: Preparation of Core particle by Using Spray Drying Method Recombinant HBsAg was dissolved in 10 mM PBS to a concentration of 300 ~g/mQ, and each of hydroxypropyl cellulose(Sample 4), sodium carboxymethyl cellulose(Sample 5) and gelatin(Sample 6) was added thereto to a concentration of 3.0 mg/mQ. Each solution was provided to a spray dryer(Buchi 190) at a flow rate of 3 mQ/minute to obtain core particles coated with hydroxypropyl cellulose, sodium carboxymethyl cellulose and gelatin, respectively. In this step, the flow rate of propellant nitrogen was 600 Q/minute and the inflow air temperature was 70 C. The average particle sizes of the core particles thus prepared were 4.6, 6.3 and 4.9 ~m, respectively.
Test Example 1: Protection of Antigen in Core particle To confirm whether the core particle coated with the water-soluble substance protects the antigenicity of the antigen therein from the contact with the organic solvent, the core particles prepared in Example 1 and 2 were dispersed in ethyl acetate or acetonitrile, which dissolves PLGA but not hydroxypropyl cellulose, and the resulting solutions were mixed by using a magnetic stirrer to allow the core particle to contact the organic solvent. The core particles were separated and dried to remove the organic solvent. The dried core particles were dissolved in a buffer(10 mM phosphate, pH
7.5) and the antigenicity was determined by AUZYME kit and compared to that of recombinant HBsAg solution of Korean Patent Publication No. 93-2735 stored in a cryogenic state.
Further, the HBsAg which was not coated with the water-soluble substance was used as a control, as follows.
Recombinant HBsAg was dissolved in lOmM PBS to a concentration of 300 ~g/mQ and to the solution was added a same volume of each of various organic solvents listed in Table 1. The resulting solutions were mixed by using a magnetic stirrer for 10 minutes. The aqueous phases were separated from the solutions-and antigenicities thereof were determined according to the same procedure as above. The precipitate was separated, dried and added to PBS for the determination of the antigenicity. However, the precipitate was not dissolved in PBS due to denaturation thereof which resulted in an insoluble form.
As a result, the antigen in the core particle which is coated with hydroxypropyl cellulose maintained its antigenicity after its contact with the organic solvent, while the antigen which was not coated with the polymer lost its antigenicity completely after its contact with the organic solvent.
~17188~
Table l Sample Water-soluble Organic Antigen- Insoluble No. - polymer solvent icity ppt. Note (%) Original HBsAg - - l00 Solution aceto- 78 hydroxypropyl nitrile l cellulose (0.3 mg/mQ) ethyl 75 acetate aceto- 80 - -hydroxypropyl nitrile 2 cellulose (l.5 mg/mQ) ethyl 80 acetate aceto- 9l - -hydroxypropyl nitrile 3 cellulose (3.0 mg/mQ) ethyl 88 acetate hydroxypropyl 90 4 cellulose (3.0 mg/mQ) sodium ethyl 85 carboxymethyl acetate cellulose (3.0 mg/mQ) 6 gelatin 82 3.0 mg/mQ) aceto- 0 formed nitrile ethyl 0 formed acetate carbon 0 formed Control not used tetra-chloride chloroform <<l.0 formed *
methylene <<l.0 formed *
chloride benzene <<l.0 formed *
hexane <<l.0 formed *
1)trace amount was redissolved.
' - ~
Example 3: Preparation of Microparticles PLGA(50/50) was dissolved in ethyl acetate to a concentration of 1 % and the core particle of Sample 3 prepared in Example 1 was dispersed therein to a concentration of 1 mg/mQ. The resulting solution was provided to a spray dryer(Buchi 190) at a flow rate of 3 mQ/minute to obtain the final microparticles which were coated further with PLGA. In this step, the flow rate of nitrogen was 600 Q/minute and the inflow air temperature was 60 C.
The average particle size of the microparticles thus prepared was 7 ~m and the weight ratio of PLGA/the core particle was 10. The amount of antigen determined by Lowry method was 6 ~g protein/mg of particle.
Example 4: Preparation of Microparticles Microparticles coated with PLGA were prepared by using Sample 4 of Example 2 as the core particle, in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 10.
The average particle size of the microparticles thus prepared was 9~m and the amount of antigen was 3 ~g protein/mg of particle.
21718~1 Example 5: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 2 mg/mQ, and microparticles coated with PLGA were prepared in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 5.
The average particle size of the microparticles thus prepared was 7 ~m and the amount of antigen was 6 ~g protein/mg of particle.
Example 6: Preparation of Microparticles Sample 4 of Example 2 was dispersed in ethyl acetate to a concentration of 0.5 mg/mQ, and microparticles coated with PLGA were prepared in accordance with the same procedures as described in Example 3. The weight ratio of PLGA/the core particle was 20.
The average particle size of the microparticles thus prepared was 10 ~m and the amount of antigen was 1.5 ~g protein/mg of particle.
Example 7: Preparation of Microparticles 5 mg of Sample 4(the core particle) prepared in Example 2 was dispersed in 1 mQ of ethyl acetate, wherein 200 mg of PLGA was dissolved, by using an ultrasonicator. The core particle dispersed system was mixed with 1 mQ of 1 % aqueous polyvinylalchol(PVA) solution by using a magnetic stirrer and the resulting mixture was dispersed in 100 mQ of 0.3 %
aqueous PVA solution to obtain an O/W emulsion. The emulsion was mixed continuously for 5 hours to evaporate ethyl acetate and microparticles thus formed were separated by using a 0.5 ym filter, washed and then dried.
The average particle size was 110 ~m.
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 to guinea pigs in a quantity that the amount of antigen becomes 40 ~g protein/head. Blood samples were taken from the guinea pigs for 2 months after the injection at fifteen-day intervals and the concentration (mIU/mQ) of formed antibody in each sample was determined.
As a control, particles coated with PLGA alone without a core coat of a water-soluble substance was prepared as follows. Original HBsAg solution was frozen in accordance with the method of Example 1 without using a water-soluble substance as the core coat and the HBsAg particles coated directly with PLGA alone(Comparative sample) were prepared therefrom in accordance with the method of Example 3. The Comparative sample was administered to guinea pigs in accordance with the same method as above, and then the 217188 l concentration of formed antibody was determined.
As a result, Sample 7 showed an antibody concentration higher than that of Comparative sample, as can be seen from Table 2. This result shows 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 the Injection(Months) tmIU/mQ ) Sample 0 0.5 1 1.5 2 Sample 7 0.0 2.410.6 24.6 34.0 Comparative sample 0.0 1.1 8.1 10.2 14.3 Test Example 3: In vivo Effect of Single-Shot Vaccine Formulation The following experiment was carried out to confirm if the vaccine formulation of the present invention has a superior effect as a single-shot vaccine formulation.
The microparticles prepared in Example 5 were dispersed in 1.0 m~ of an injection medium(PBS containing 0.02 wt% of Tween 80) to form a vaccine formulation(Sample 8), which was injected once subcutaneously to guinea pigs by using a 26G
syringe in a quantity that the amount of antigen becomes 20 yg protein/head. Blood samples were taken from the guinea pigs for 5 months after the injection at fifteen-day or a month intervals and the concentration(mIU/mQ) of formed antibody in each sample was determined.
A comparative formulation which employs alum as an adjuvant was prepared as follows. 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 ~g protein/1.5 mg alum.
The vaccine formulation was injected once to two groups of guinea pigs by using a 26G syringe in a quantity that the amount of the antigen becomes lOyg protein/mQ/injection (primary injection). Thereafter, one group was subjected to the first boosting using the same antigen amount fifteen days after the primary injection(Comparative Example 1). The other group was further subjected to the second boosting 1.5 months after they received the first boosting in accordance with the same procedures as above(Comparative Example 2).
Further, one more group of guinea pigs were received a primary injection only by using the alum formulation and the same method as above, in a quantity that the amount of the antigen becomes 20 yg protein/mQ/injection (Comparative Example 3, Sample 9).
The concentrations(mIU/mQ) of formed antibody of Comparative Examples 1, 2 and 3 were determined as above and the results were compared with that of Sample 8.
2171~81 The result is shown in Table 3, wherein it can be seen that the concentration of antibody formed by an injection of Sample 8 is higher than those formed by injections up to three times of the alum formulation. This result shows that the vaccine formulation of the present invention has a superior effect as a single-shot vaccine formulation, i.e., a single injection of the inventive vaccine formulation using 20 yg protein shows a superior vaccination effect than three injections of the alum formulation using 30 yg of protein.
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Test Example 4: Relationship between the Particle Property of the Vaccine Formulation and Antibody Formation In order to investigate whether the time and amount of antibody formation are controllable in accordance with the weight ratio of the biodegradable polymer and the core particle, the following test was carried out.
Each of the microparticles prepared in Examples 4, 5 and 6 was suspended in PBS to prepare a single-shot vaccine formulation(Samples 10, 8 and 11), which was injected once subcutaneously to guinea pigs in a quantity that the amount of antigen becomes 20 ~g protein/ head. Blood samples were taken from the guinea pigs for 5 months after the injection at fifteen-day intervals and the concentration~mIU/mQ) of formed antibody in each sample was determined.
Further, the alum formulation prepared in Test Example 3 was injected once to guinea pigs in a quantity that the amount of antigen becomes 20 ~g protein/head(Sample 9) and the concentration(mIU/mQ) of formed antibody was determined in accordance with the same method as above.
The antibody concentration changes with time after the injection are shown in Fig. 1, wherein Sample 8, which has a PLGA/core particle weight ratio of 5, shows the highest rate of increase in the antibody concentration. Therefore, it was confirmed that the rate of antibody formation is controllable by regulating the thickness of the biodegradable polymer coating. Therefore, a single-shot vaccine containing mixed antigens or a single-shot vaccine which can release an antigen in different patterns can be prepared by using various particles having different properties.
As described in the above Examples, the single-shot vaccine formulation of the present invention can accomplish vaccination by administering only one injection and further has a higher antibody forming capacity than conventional vaccine formulations.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.
Claims (19)
1. A microparticle having a particle size ranging from 0.5 to 300 µm, which is prepared by coating an antigen or a mixture of antigens with a water-soluble substance to obtain a core particle and coating the core particle with a biodegradable polymer.
2. The microparticle of claim 1, wherein the antigen is an attenuated, killed or recombinant antigen.
3. The microparticle of claim 1, wherein the core particle further includes an adjuvant or an inorganic salt.
4. The microparticle of claim 1, wherein the antigen is for one or more of diseases selected from the group consisting of: hepatitis, diphtheria, chickenpox, typhoid, pertussis, 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 of claim 3, wherein the adjuvant is alum; muramyl dipeptide, muramyl tripeptide and derivatives thereof; tymosin alpha; monophosphoryl lipid A;
saponin; an immunostimulating complex; a polyelectrolyte; or a mixture thereof.
saponin; an immunostimulating complex; a polyelectrolyte; or a mixture thereof.
6. The microparticle of claim 1, wherein the water-soluble substance is used in an amount ranging from 1 to 50 times the weight of the antigen.
7. The microparticle of claim 1, wherein the water-soluble substance is a saccharide, a protein, an amino acid or a mixture thereof.
8. The microparticle of claim 7, wherein the saccharide is a cellulosic polymer, glucose, xylose, galactose, fructose, lactose, maltose, saccharose, alginate, dextran, hyaluronic acid, chondroitin sulfate or a mixture thereof.
9. The microparticle of claim 8, wherein the cellulosic polymer is hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose or a mixture thereof.
10. The microparticle of claim 7, wherein the protein is gelatin, albumin or a mixture thereof.
11. The microparticle of claim 7, wherein the amino acid is glycine, alanine, glutamic acid, arginine, lysine, or a salt or a mixture thereof.
12. The microparticle of claim 1, wherein the particle size of the core particle ranges from 0.1 to 200 µm.
13. The microparticle of claim 1, wherein the biodegradable polymer is used in an amount ranging from 1 to 100 times the weight of the core particle.
14. The microparticle of claim 1, wherein the biodegradable polymer is polyglycolide, polylactide, poly(lactide-co-glycolide) or a mixture thereof.
15. The microparticle of 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-shot vaccine formulation prepared by dispersing microparticles recited in any one of claims 1 to 15 in an injection medium.
17. The single-shot vaccine formulation of claim 16, wherein the injection medium is a buffer which optionally contains a dispersing agent and/or a preservative.
18. The single-shot vaccine formulation of claim 17, wherein the injection medium is an edible oil, mineral oil, cod liver oil, squalene, squalane, mono-, di- or triglyceride, or a mixture thereof.
19. The single-shot vaccine formulation of claim 18, wherein the edible oil is corn oil, sesame oil, olive oil, soybean oil, safflower oil, cotton seed oil, peanut oil or a mixture thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019950005424A KR100201352B1 (en) | 1995-03-16 | 1995-03-16 | Single shot vaccine formulation |
| KR95-5424 | 1995-03-16 |
Publications (1)
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|---|---|
| CA2171881A1 true CA2171881A1 (en) | 1996-09-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002171881A Abandoned CA2171881A1 (en) | 1995-03-15 | 1996-03-15 | Single-shot vaccine formulation |
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|---|---|
| US (1) | US5753234A (en) |
| EP (1) | EP0737472A1 (en) |
| JP (1) | JP2739570B2 (en) |
| KR (1) | KR100201352B1 (en) |
| CN (1) | CN1213736C (en) |
| AR (1) | AR002969A1 (en) |
| BR (1) | BR9601032A (en) |
| CA (1) | CA2171881A1 (en) |
| PE (1) | PE57797A1 (en) |
| TW (1) | TW495361B (en) |
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| KR960033469A (en) | 1996-10-22 |
| MX9600938A (en) | 1997-07-31 |
| EP0737472A1 (en) | 1996-10-16 |
| CN1136918A (en) | 1996-12-04 |
| JP2739570B2 (en) | 1998-04-15 |
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| US5753234A (en) | 1998-05-19 |
| AR002969A1 (en) | 1998-05-27 |
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| CN1213736C (en) | 2005-08-10 |
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| BR9601032A (en) | 1998-01-06 |
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