JP5429707B2 - Fine particles and method for producing the same - Google Patents

Description

  The present invention relates to a novel fine particle and a method for producing the same. The present invention also relates to the growth of Plasmodium falciparum, a microparticle containing Peptide and its analogs that can elicit an immunological response against Plasmodium utilizing the immune response in humans and other animals. The present invention relates to a pharmaceutical composition for suppressing immunizing antigen and malaria parasite infection.

[Prior art and its problems]
(1) Current status of malaria as an infectious disease :
Malaria is one of the most serious protozoal infections on the planet. Each year, more than 300 million infected people and more than 2 million deaths are reported, mainly in tropical and subtropical endemic areas. In recent years, the movement of people and goods has become active due to the expansion of economic activities on a global scale. Along with this, the number of cases where Japanese travelers are infected in endemic areas and the cases of imported malaria in which people entering Japan from endemic areas develop in Japan have increased rapidly since the 1980s (Non-patent Document 1). . For this reason, malaria control has become an urgent issue not only in endemic areas but also in Japan.

  Plasmodium parasites that cause malaria in humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium ovale There are four types. Malaria parasites that enter the body due to the bite of mosquitoes that carry protozoa quickly invade hepatocytes from the blood (primary intrahepatic stage, Fig. 1a), divide and proliferate in hepatocytes, and then release into the blood Then, it enters the erythrocyte and repeats division and proliferation (intra-erythrocyte cycle, FIG. 1b), and the proliferated protozoa is further transmitted by other mosquitoes (FIG. 1c). Symptoms of fever from malaria are caused by an intraerythrocyte cycle. In particular, P. falciparum poses a risk of increasing severity and death if treatment is delayed compared to the other three.

  Malaria is not only a health problem but also contributes to the stagnation of economic activity and social unrest in African countries. The recent increase in the number of infected people in endemic areas has been pointed out to be related to tropical rainforest development and global warming. According to the International Panel on Climate Change report (1996 & 1998), in the case of global warming, the temperature rose by 2 ° C. An increase of 50-80 million is predicted. For this reason, there is a concern that malaria recurrence may occur in temperate regions including Japan, which should have been eradicated by DDT spraying and hygiene measures after World War II.

(2) Problems of drug resistance and side effects caused by therapeutic drugs:
Molecular mechanism of drug-resistant malaria Drug resistance to the specific drug chloroquine, which has spread throughout the world since the 1945s, was reported from the Colombia-Thailand-Myanmar border as early as 1957. Since then, chloroquine-resistant protozoa have spread throughout the world, making it extremely difficult to combat malaria in endemic areas.

Chloroquine is currently rarely used, but its drug resistance mechanism has long been unknown. In combination with research in endemic areas, chloroquine resistance of P. falciparum has several protein mutations (pfmdr (Non-patent document 2), cg2 (Non-patent document 3), pfcrt (Non-patent document 4)) Has been suggested and has finally begun to be understood at the molecular level (Non-patent Document 5). Chloroquine is concentrated even in small doses into the acidic phagosome in the protozoa in the erythrocytes. This phagosome digests and degrades hemoglobin and uses amino acids to detoxify the by-product heme as hemozoin. Chloroquine inhibits the formation of hemozoin, a non-nutritive heme by-product. That is, the protozoa die without being able to detoxify heme. However, in the resistant protozoa, the half-life of chloroquine is only a few minutes and develops resistance through rapid drug elimination.

  A compound developed in the Vietnam War (mefloquine) is often used to treat malaria. The latest treatment is malalone approved in the United States in 2000, which is also a diversion of an existing drug. Currently, artemisinin and its derivatives discovered from ingredients in Chinese herbal medicine are attracting attention (National Geographic 2007 July issue; Nature 2004 August 19 issue (non-patent document 6))

  However, many drugs that kill malaria parasites have very strong side effects such as headache and nausea. Therefore, preventive use is not generally recommended. In fact, existing compounds (quinine, chloroquine) are deleterious. In contrast, vaccines using antigenic peptides and biodegradable polymers as raw materials contain amino acids and hydroxycarboxylic acids as components, and thus cause only the desired preventive immune reaction and do not have the strong toxicity seen with existing therapeutic substances.

  As described above, the conventional techniques have many problems in preventing and treating malaria. Therefore, vaccination with a safe and inexpensive vaccine is desired as a preventive method. However, no vaccine has been marketed to date.

(3) Current status and problems of malaria vaccine development:
The development of malaria vaccines has started relatively recently (since 1978). Currently, vigorous research on vaccines is underway in Japan and overseas. However, there are no examples of clinical application. According to reported papers, more than a dozen groups are proceeding to clinical trials worldwide (Non-patent Document 7). In addition, approximately 3 groups in Japan are pursuing clinical trials and preparations aimed at vaccines (73rd Annual Meeting of the Parasitological Society of Japan, Symposium “Japanese Malaria Vaccine Research”, April 24, 2004, Maebashi City).

  In reviewing previous research reports on malaria vaccine development, malaria vaccine development is difficult because the malaria parasite has a complex immune evasion system acquired in the course of evolution, and the clinical symptoms of malaria are infections. This is considered to be because it is difficult to determine the vaccine effect because it varies greatly depending on the person's immune status. In other words, the prior art is limited in that it does not develop vaccines that take into account differences in immune status, such as different immunity status among residents in endemic areas and non-endemic travelers.

(4) Current status of polymer fine particles by emulsion method:
In recent years, polymer fine particles prepared by the double emulsion method or single emulsion method using biodegradable polymers (polylactic acid-glycolic acid copolymer, etc.) have been studied for the purpose of encapsulating bioactive substances. Yes. In the double emulsion method, fine particles encapsulating a drug are formed using three solutions of an aqueous phase, an organic phase and an aqueous phase (W / O / W). In the single emulsion method, two solutions of an organic phase and an aqueous phase (O / W) are used to form fine particles in which a drug is dissolved and encapsulated in an organic layer.

  In the conventional double emulsion method, for example, as described in Non-Patent Document 8, a small amount of an aqueous solution (250 μL) of a physiologically active substance (25 mg) is added to a biodegradable polymer (polylactic acid-glycolic acid copolymer, 250 mg) and volatile organic solvent solution (dichloromethane, 5 mL) and emulsify with stirring. Here, the inner aqueous phase containing the physiologically active substance is dispersed into the microdroplets by vigorous stirring. A large amount of polyvinyl alcohol aqueous solution (8-12%) is poured into such a W / O emulsion (25 mL) and stirring is continued. Then, the volatile organic solvent in which the biodegradable polymer is dissolved evaporates and the solubility is lowered, so that the biodegradable polymer is deposited so as to surround the inner aqueous phase to form fine particles.

  In the conventional single emulsion method, for example, a small amount of physiologically active substance (25 mg) is stirred with a volatile organic solvent solution (dichloromethane, 5 mL) of a biodegradable polymer (polylactic acid-glycolic acid copolymer, 250 mg). Dissolve. Here, the point that the physiologically active substance becomes a homogeneous dichloromethane solution is different from the double emulsion method. A large amount of polyvinyl alcohol aqueous solution (1 to 20%) is poured into such an organic phase (25 mL) and stirring is continued. Then, here, the organic phase containing the physiologically active substance and the biodegradable polymer is dispersed into the microdroplets by vigorous stirring. In such an O / W emulsion, since the volatile organic solvent in which the biodegradable polymer was dissolved evaporates and the solubility is lowered, the biodegradable polymer is precipitated while containing the bioactive substance, and the fine particles Form.

  Polyvinyl alcohol has a role of negatively charging the surface of the fine particles and preventing the formed fine particles from aggregating. A concentration of about 1 to 10% is often used. However, although polyvinyl alcohol is a pharmacologically acceptable substance, it is a vinyl polymer type polymer slightly poor in biodegradability, and therefore it is desirable that the concentration be as low as possible when considering vaccines.

(5) Current status of malaria vaccine development using microparticles:
The world's first field trial of a malaria vaccine was performed by Manuel E. Patarryo et al. In Colombia (Non-Patent Documents 9 and 10). This is called SPf66 antigen, and a chemically synthesized peptide consisting of a 45-residue amino acid sequence is used as the antigen.

  The amino acid sequence used in SPf66 is derived from a partial amino acid sequence of a protein used by protozoa at the stage of parasitizing erythrocytes and liver called merozoites and sporozoites. Target antigens generally used for vaccine development, including SPf66, are often used by malaria parasites (a) proteins that enter red blood cells and (b) proteins that are displayed on the surface of protozoa and infected red blood cells. ing. Thus, in the prior art, vaccine development often uses structural proteins that are involved in the formation of protozoan surfaces and protozoan parasite structure formation.

  SPf66 antigen was successfully protected against infection in field trials in South America and Tanzania (Non-patent Document 11). However, other field trials conducted in Gambia and Tanzania did not show a protective effect (Non-Patent Document 12). This is because, as described above, the clinical symptoms of malaria vary greatly depending on the immune status of the infected person, and thus it is difficult to determine the vaccine effect.

  On the other hand, related research groups including Patarroyo et al. Believe that this difference in vaccine efficacy is caused by the use of an aqueous suspension of aluminum hydroxide called alum used at the time of vaccination as an adjuvant to enhance the immune response. ing.

  Therefore, Patarroyo and Pedraz et al. Focused on polymer microparticles as an alternative to alum, and produced microparticles and immunologically by the double emulsion method using an aqueous solution in which SPf66 antigen was dissolved in distilled water. We are studying properties (Non-Patent Document 13). The polymer base of the fine particles reported here is a polylactic acid-glycolic acid copolymer (copolymerization ratio 50:50 or 75:25, weight average molecular weight 90,000 to 100,000), and has a diameter of about 0.1 to 2.0. Fine particles having a wide distribution of μm (average 1.3 to 1.4 μm) are obtained (Non-patent Document 14). In addition, fine particles having an average diameter reduced to 0.5 μm by making an 8% polyvinyl alcohol aqueous solution of the outer water phase into a 12% aqueous solution have been reported (Non-patent Document 15).

However, vaccine development using polymer microparticles of vaccine antigens by the method of Patarroyo and Pedraz et al. (A) has structural proteins as antigens, (b) does not consider differences in the immune status of subjects, (c) It is necessary to dissolve the antigen in distilled water, (d) using a high-concentration polyvinyl alcohol aqueous solution, (e) the microparticle size is only a micrometer size, (f) large variation in size, There is a limit in that.

(6) Current status of polymer nano / micro particle formation by emulsion method:
As a method for producing nanometer-sized polymer fine particles using the emulsion method, there are a method using an acrylic acid polymer (Non-patent Document 16) and a method using a polylactic acid-glycolic acid copolymer (Patent Document 1). Two are known. When the drug is encapsulated in fine particles, water-containing acetone in which the drug and polymer are dissolved is used. However, this method has limitations on the drugs and polymers that can be used.

(7) Current status of malaria vaccine development using enolase:
Strangely examining the malaria epidemiological survey materials, it is strange that many of the deaths caused by malaria are “infants in endemic areas” and “travelers from non-endemic areas such as Japanese”. On the other hand, “endemic adults” can easily recover from infection with malaria. This is thought to be due to the fact that residents in endemic areas have “obtained and maintained immunity to malaria by constantly infecting malaria”. It is therefore clear that vaccine development must take into account individual differences in immune status.

  In the course of this epidemiological study, the inventors of the present invention, Suzuki and Kano, found that protozoan-derived antigens are common antigen molecules that are involved in the recovery of pathological conditions in patients with acute P. falciparum from field studies in endemic areas in South America and Southeast Asia. A glycolytic enzyme / enolase was discovered (Non-patent Document 17). That is, we found that a glycolytic enzyme, enolase, produced by Plasmodium falciparum infecting humans acts as a protective immunity molecule against P. falciparum, and started vaccine development using this.

  The vaccine antigen by enolase is greatly different from the other in that it directly inhibits the energy production system of protozoa and suppresses the growth of protozoa.

  First, the inventors studied the molecular design and chemical synthesis of artificial antigenic peptides using recombinant enolase having an amino acid sequence derived from Plasmodium falciparum and a partial amino acid sequence. Immunological studies using these enolases and artificial antigenic peptides have revealed that anti-enolase antibodies and anti-peptide antibodies inhibit the growth of P. falciparum in vitro (Patent Document 2).

  Subsequently, a vaccine test was conducted using 13 monkeys using an enolase derived from Plasmodium falciparum and an artificial antigen peptide consisting of a partial sequence (FIG. 2). The transition of the parasitism rate of the malaria parasite to the red blood cells in the peripheral blood of the monkey was plotted. As a result, it has been clarified that yolk monkeys administered with the vaccine have suppressed the rapid increase in the parasitism rate and are excellent as vaccines (Nihon Keizai Shimbun, Sankei Shimbun, Hebei Shinpo, Chugoku Shimbun, 4 (Posted on paper 2003/8/25). It was also revealed that anti-enolase antibody and anti-peptide antibody were produced in the serum.

  From the viewpoint of chemical synthesis, research on vaccine molecule synthesis was also conducted and a fragment condensation method suitable for mass synthesis was successfully developed (Patent Document 3). That is, the present inventors have found a method for synthesizing a peptide having a partial sequence of the target malaria parasite enolase or an analog thereof on a large scale by condensing five short-chain peptide segments into one protected peptide chain.

The development of the synthesis method has made it possible to obtain about 50 to 500 milligrams (about 1,000 to 10,000 people as a vaccine) at a time, even on a small scale at the laboratory level. It is possible to obtain peptides of the same scale as industrially produced in the world's largest production facility of genetic recombinants by performing laboratory-scale synthesis only 2 to 4 times. Furthermore, if production is carried out on an industrial scale, the supply of peptides for millions of people is expected, which is comparable to the annual global demand.

  In general, a chemically synthesized peptide or a recombinant peptide using a partial amino acid sequence of a protein can be used in various ways as a pharmaceutical or a test material including an artificial antigen. The problem in using these partial peptides is that there are many compounds that are hardly soluble in organic solvents and aqueous solvents. In particular, the solubility of peptides and proteins in distilled water and volatile organic solvent solutions (such as ethyl acetate, diethyl ether, and dichloromethane) is often very low.

  Similarly, the inventor's enolase partial sequence has low solubility in distilled water and volatile organic solvent solutions (such as ethyl acetate, diethyl ether, and dichloromethane). Therefore, in the known method, even if an artificial antigen having an enolase partial sequence is obtained by chemical synthesis or genetic recombination, it is difficult to encapsulate the polymer fine particles by the double emulsion method or the single emulsion method.

Japanese Unexamined Patent Publication No. 5-58882 Japanese Patent Laid-Open No. 2002-371098 WO2006 / 035815

Kano, S. and Kimura, M., Acta Tropica, 2004, 89, 271-278. Foote, S. J .; Thompson, J. K .; Cowman, A. F .; Kemp, D. J .; Cell, 1989, 57, 921-930. Su, X .; Kirkman, L. A .; Fujioka, H .; Wellems, T. E., Cell, 1997, 91, 593-603. Johnson, D. J .; Fidock, D. A .; Mungthin, M .; Lakshmanan, V .; Sidhu, A. B. S .; Bray, P. G .; Ward, S. A., Molecular Cell, 2004, 15, 867-877. Oku Hiroyuki, Chemistry, 2002, 57, 64-65. J. L. Vennerstrom et al., Nature, vol. 430, pp. 900-904. Moorthy, V. S .; Good, M.F .; Hill, A. V., Lancet, 2004, vol. 363, pp. 150-156. Rosas et al., Vaccine 2001, 19, 4445-4451 Patarroyo et al., Nature 1987, 328, 629-632 Valero et al., Lancet 1993, 341, 705-710 Noya et al., Journal of Infectious Disease 1994, 170, 396-402 D'Alessandro et al., Lancet 1995, 346, 462-467 Rosas et al., Vaccine 2002, 20, 1707-1710 Carcaboso et al., International Journal of Pharmaceutics 2003, 260, 273-282 Carcaboso et al., Vaccine 2004, Vol. 22, pages 1423-1432 J. Pharm. Sci. 1989, 78, 68-72 Jpn. J. Trop. Med. Hyg. 18, pp. 317-24, pp. 475-481 (1990)

The subject of this invention is providing the novel microparticles | fine-particles containing physiologically active substances, such as protein and a peptide, and its manufacturing method.
The present invention also provides for the growth of Plasmodium falciparum, a microparticle comprising a peptide or analog thereof capable of inducing an immunological response against Plasmodium utilizing the immune response in humans and other animals. It is an object of the present invention to provide a pharmaceutical composition for preventing immunizing antigen and malaria parasite infection.

The inventors of the present invention are the first to use the double emulsion method or the single emulsion method for the solubility by using a volatile organic acid aqueous solution or a volatile organic acid solution that can dissolve artificial antigen peptides, which are often poorly soluble. We succeeded in producing microparticles encapsulating a low antigenic peptide. Selection of the production of fine particles in the double emulsion method the volatile organic acid used in the internal aqueous phase W _1, organic acid concentration and volume of the organic acid aqueous solution, the production of fine particles in the single emulsion method was investigated the capacity of the organic acid solution . In addition, the temperature control of the emulsion being produced at low temperature, the method of removing the organic solvent in the emulsion, etc. were studied. Thus, the present invention has been completed by finding a method for obtaining fine particles containing a hardly soluble artificial antigenic peptide.

The present invention is as follows.
(1) Double emulsion method or single emulsion method using a volatile organic acid aqueous solution containing a physiologically active substance or a volatile organic acid solution of the physiologically active substance, which is a fine particle containing a physiologically active substance and a biodegradable polymer Fine particles made by
(2) The fine particles according to (1), wherein the volatile organic acid is acetic acid or a mixture containing acetic acid. (3) The fine particles according to (1), wherein the volatile organic acid is formic acid or a mixture containing formic acid. (4) The fine particles according to any one of (1) to (3), wherein the biodegradable polymer is a polylactic acid-glycolic acid copolymer.
(5) The fine particles according to any one of (1) to (3), wherein the biodegradable polymer is polylactic acid.
(6) The fine particles according to any one of (1) to (3), wherein the biodegradable polymer is a polydepsipeptide.
(7) The fine particles according to any one of (1) to (6), wherein the physiologically active substance is a protein or a peptide.
(8) The microparticle according to (7), wherein the protein or peptide is a protein or peptide derived from Plasmodium falciparum.
(9) The microparticle according to (8), wherein the P. falciparum protein or peptide is P. falciparum-derived enolase or a partial peptide thereof.
(10) The peptide according to (9), wherein the partial peptide contains a structural difference between SEQ ID NO: 1 and SEQ ID NO: 2 and can induce a cellular or humoral immunological response against Plasmodium falciparum. The fine particles described.
(11) The fine particles according to (10), wherein the partial peptide is a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 6.
(12) A pharmaceutical composition comprising the microparticles according to any one of (1) to (11) and a pharmaceutically acceptable carrier.
(13) A pharmaceutical composition for preventing malaria parasite infection, comprising the fine particles according to any one of (8) to (11) and a pharmaceutically acceptable carrier.
(14) An antigen for immunization containing the fine particles according to any one of (8) to (11) as an active ingredient for suppressing the growth of malaria parasites.
(15) A step of preparing an emulsion by mixing an organic acid aqueous solution containing a bioactive substance or an organic acid solution of a bioactive substance with a volatile organic solvent containing a biodegradable polymer, and the resulting emulsion is negatively charged. A method for producing bioactive substance-containing microparticles, which comprises a step of mixing with an aqueous solution of a polymer having a pH

The “volatile organic acid” described in the present invention refers to an organic compound having a carboxylic acid group, or an ammonium salt or ester derivative thereof, which can be removed under reduced pressure.
The volatile organic acid can be selected from organic compounds having a carboxylic acid group that is liquid at room temperature. Examples include acetic acid and formic acid, and other examples include lactic acid having a hydroxyl group as a functional group other than carboxylic acid, fumaric acid having a plurality of carboxylic acid groups, malonic acid, malic acid, and the like. Absent.
Here, as a method of using the volatile organic acid, it is considered that the peptide or protein serving as an antigen can be dissolved, and that the prepared fine particles can be easily removed when freeze-dried, but is not limited thereto.

“Derivatives of proteins and peptides” described in the present invention are proteins and peptides produced by substitution, deletion and / or insertion of one to several amino acids constituting them. A peptide having the same activity as the original protein or peptide with respect to the response.
For example, in the peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 6, 1 to 5, preferably 1 to 3, more preferably 1 to 2 amino acids are substituted, deleted, and / or inserted. It may be a thing.
Here, as a method of using the derivative, it is conceivable to make the solubility and crystallinity in fine particle preparation, the solubility in use in an immune reaction, and the immunological response more effective, but it is not limited to these. It is not something.

  In the present invention, “immunological response” is a concept including both a cellular immunological response and a humoral immunological response. Among these, cellular immunological response refers to immunity caused by macrophages, natural killer cells (NK cells), eosinophils, T cells, etc., and is a cellular immunological response to Plasmodium falciparum. As such, those involving killer T cells are known. Further, humoral immunological responses are known that are caused by host-derived antibodies that can specifically bind to P. falciparum-derived proteins, sugar chains, and the like. It is desirable to induce antibodies as a humoral immunological response from microparticles containing an antigenic peptide produced according to the present invention. It is also desirable to induce a cellular immunological response.

According to the present invention, fine particles can be efficiently produced even when a hardly soluble physiologically active substance is used.
Plasmodium falciparum (P. falciparum) that can induce an immunological response against Plasmodium using the immune response in humans and other animals by making the physiologically active substance a partial sequence and an analogous sequence of Plasmodium enolase It can be used as an antigen for immunity that suppresses the growth of Plasmodium falciparum), a pharmaceutical composition for the prevention of Plasmodium falciparum infection, and a diagnostic material for an immune state against malaria infection.

The figure which shows the life cycle of the malaria parasite in a human body. (a) Infection by mosquito blood sucking and transfer of parasitic protozoa into the liver. It is released into the blood after dividing and proliferating in hepatocytes. (b) Intra-erythrocyte cycle in which protozoan parasites enter erythrocytes and repeat division and growth. (c) Proliferated protozoa are transmitted by blood sucking by other mosquitoes. The figure which showed typically about the discovery of the Enolase derived from a Plasmodium falciparum, the molecular design of the artificial antigen peptide which has a partial amino acid sequence (AD22, sequence number 6), and the animal experiment of the vaccine using a monkey. The figure which shows the molecular structure of the artificial antigen peptide used in the Example. AD22 is the amino acid sequence set forth in SEQ ID NO: 2. The figure which shows the preparation methods of the artificial antigen fine particle by the double emulsion method. In the figure, MAPs represents an artificial antigen peptide, PLGA represents a polylactic acid glycolic acid copolymer, DCM represents dichloromethane, and PVA represents polyvinyl alcohol. In the single emulsion method, since an organic acid solution is used instead of the aqueous phase W ₁ , the artificial antigen peptide is uniformly dissolved in the biodegradable polymer solution O. SEM photograph of microparticles containing artificial antigen peptide by single emulsion method using acetic acid solution (magnification 3000 times, average particle size = 0.7 mm). SEM photograph of fine particles containing artificial antigenic peptide by formic acid solution using single emulsion method (magnification 3000 times, average particle size = 0.8 mm). SEM photograph of fine particles containing artificial antigenic peptide by double emulsion method using formic acid aqueous solution (magnification 3000 times, average particle size = 5 mm). Since the inner aqueous phase droplets remain during the formation of the emulsion, it has double-structured fine particles (W / O / W double emulsion). Since the average particle size is large, long-term survival is expected under the skin. Release behavior of fluorescently labeled artificial antigenic peptide from 1.0 mg of fine particles placed on the bottom of a container containing 40 mL of 0.2% SDS-phosphate buffered saline at 37 ° C. The figure which shows the process (x) which antigen peptide 4.0microgram disperse-dissolves. The figure which shows the comparison of the amino acid sequences of P. falciparum enolase (P. falciparum enolase, GenBank accession number AB026051) and human alpha-enolase (Human alpha enolase, GenBank accession number M14328). The four partial amino acid sequences shown in the specification (GG14, AA13, GL16, AD22) are underlined. The fluorescence image from the artificial antigen peptide in the microparticles | fine-particles administered to the mouse | mouth subcutaneously. The distribution of the artificial antigen peptide encapsulated in the microparticles produced by the same method as in Example 1 is observed by in vivo imaging. Immunization experiment with microparticles administered subcutaneously in mice and transition of antibody titer1. Experimental group 1 has an antigen amount of 1.6 μg / animal (0.4 mg microparticles / animal), experimental group 2 has an antigen amount of 16 μg / animal (4.0 mg microparticles / animal), and experimental group 3 has an antigen amount of 16 μg / animal (antigen alone and no microparticles) ), Experimental group 4 has an antigen amount of 0.0 μg / animal (without antigen, 4.0 mg microparticles only / animal). 1. Immunization experiment with microparticles administered subcutaneously in mice and transition of antibody titer Here, 4 mice in experimental group 2 described in FIG. 11 were re-administered with fine particles (antigen amount 16 μg / animal, 4.0 mg fine particle / animal) at 2 weeks (mouse 3 and 5), and the remaining 2 mice. (Mouse 1, 4) was observed for up to 27 weeks without administering anything.

Hereinafter, the present invention will be described in more detail.
The fine particles of the present invention are fine particles containing a physiologically active substance and a biodegradable polymer, and are produced by a double emulsion method or a single emulsion method using a volatile organic acid aqueous solution or a volatile organic acid solution. .

  The physiologically active substance is not particularly limited, and examples thereof include proteins or peptides, nucleic acids such as DNA and RNA, pharmaceutical compounds, or combinations thereof. Of these, proteins or peptides are preferred. The type of protein or peptide is not particularly limited, and examples thereof include enzymes, cytokines, antibodies, and heterologous protein immunization fragments.

When the microparticles of the present invention are used as a diagnostic material for an immune state against malaria infection, an immunizing antigen that suppresses the growth of Plasmodium falciparum and a medicament for the prevention of Plasmodium falciparum infection, the physiologically active substance is a Plasmodium falciparum It can be a derived protein or a partial peptide thereof.
Examples of the protein derived from Plasmodium falciparum or a partial peptide thereof include enolase derived from Plasmodium falciparum or a partial peptide thereof. The amino acid sequence of enolase derived from Plasmodium falciparum is exemplified in SEQ ID NO: 1.
When the microparticle of the present invention is used as a vaccine against Plasmodium falciparum infection, it contains a structural difference between SEQ ID NO: 1 and SEQ ID NO: 2 (amino acid sequence of human enolase) (FIG. 9), Peptides that can induce cellular or humoral immunological responses are preferably contained in the microparticles as physiologically active substances.
There are no particular restrictions on the structural differences between SEQ ID NO: 1 and SEQ ID NO: 2, but the following four types of peptides are exemplified.
GG14: Gly Ser Lys Asn Glu Trp Gly Trp Ser Lys Ser Lys Leu Gly (SEQ ID NO: 3)
AA13: Ala Ala Pro Asn Lys Val Ser Leu Tyr Lys Tyr Leu Ala (SEQ ID NO: 4)
GL16: Gly Phe Ala Pro Asn Ile Leu Asn Ala Asn Glu Ala Leu Asp Leu Leu (SEQ ID NO: 5)
AD22: Ala Ser Glu Phe Tyr Asn Ser Glu Asn Lys Thr Tyr Asp Leu Asp PheLys Thr Pro
Asn Asn Asp (SEQ ID NO: 6)

  On the other hand, when a fine particle contains a pharmaceutical compound as a physiologically active substance, an antibiotic that inhibits cell wall synthesis (penicillin, cephem / oxacephem, penem / carbapenem, monobactam, peptide, fosfomycin), cell membrane inhibitory action Type antibiotics (polypeptides, polyenes), nucleic acid synthesis inhibitory antibiotics (quinolone, rifampicin, interferon), protein synthesis inhibitory antibiotics (aminoglycoside, macrolide ketolide, tetracycline, Chloramphenicol, lincomycin, folate metabolic pathway inhibitor antibiotics (trimethoprim), β-lactamase inhibitors, sulfa drugs, anti-infectives (antibacterial, antitubercular, antifungal, antiviral) Medicine, parasite medicine, protozoan medicine), adjuvant (aluminum hydroxide) Um and aluminum salts such as aluminum phosphate, mineral salts, Freund's incomplete adjuvant, ISCOMs such, PRR / TLR ligands, pharmaceutical compounds such as saponins) and preservatives are exemplified.

Next, the biodegradable polymer contained in the fine particles will be described.
Examples of the biodegradable polymer contained in the fine particles include polylactic acid-glycolic acid copolymer, polylactic acid, and polydepsipeptide.

Here, examples of the polydepsipeptide include the following (I) to (V).
(F ₁ −F ₂ ) _n (I)
(F ₁ −F ₂ −F ₃ ) _n (II)
(F ₁ −F ₂ −F ₃ −F ₄ ) _n (III)
(F ₁ −F ₂ −F ₃ −F ₄ −F ₅ ) _n (IV)
(F ₁ −F ₂ −F ₃ −F ₄ −F ₅ −F ₆ ) _n (V)

In formulas (I) to (V), F ₁ , F ₂ , F ₃ , F ₄ , F ₅ and F ₆ constituting the depsipeptide structure part represent amino acid or hydroxycarboxylic acid residues, and in formula (I) At least one of F ₁ and F _2, at least one of the formulas (II) in F _1, F ₂ and F _3, at least one of formula (II) in F _1, F ₂ and F _3, the formula (III) Is at least one of F ₁ , F ₂ , F ₃ and F ₄ , Formula (IV) is at least one of F ₁ , F ₂ , F ₃ , F ₄ and F ₅ , Formula (V) is F ₁ , F _At least one of ₂ , F ₃ , F ₄ , F ₅ and F ₆ is a hydroxycarboxylic acid residue. The amino acid constituting the depsipeptide structure portion is appropriately selected according to the use of the fine particles and the type of physiologically active substance, but is preferably an L-form amino acid.
The hydroxycarboxylic acid is preferably valic acid ((S) -2-hydroxy-3-methylbutanoic acid), lactic acid or glycolic acid.

In the formulas (I) to (V), n representing the repeat of depsipeptide is preferably an integer of 2 to 100, more preferably an integer of 5 to 50.
The repeating unit F ₁ -F ₂ or F ₁ -F ₂ -F ₃ or F ₁ -F ₂ -F ₃ -F ₄ or F _1-
F ₂ -F ₃ -F ₄ -F ₅ or F ₁ -F ₂ -F ₃ -F ₄ -F ₅ -F ₆ may be two or more types of sequences. In addition, repeating sequences having different numbers of residues may be arranged in an arbitrary order. For example, the following can be considered in which a sequence of 5 residues and a sequence of 6 residues are arranged in an arbitrary order.
(F ₁ −F ₂ −F ₃ −F ₄ −F ₅ ) _o − (F ₁ −F ₂ −F ₃ −F ₄ −F ₅ −F ₆ ) _p − (F ₁ −F ₂ −F ₃ −F ₄ −F ₅ ) _q − (F ₁ −F ₂ −F ₃ −F ₄ −F ₅ −F ₆ ) _r
o represents an integer of 1 to 19, p represents 19 to 1, q represents 19 to 0, and r represents an integer of 19 to 0.

Examples of the sequence of the depsipeptide which is a repeating unit are as follows.
As F ₁ -F ₂ -F ₃ -F ₄ -F ₅ in the formula (IV), -Xaa ¹ -Xaa ² -Gly-Lac-Pro- or -Xaa ¹ -Xaa ² -Gly-Hmb-Pro- Examples of F ₁ -F ₂ -F ₃ -F ₄ -F ₅ -F ₆ in the formula (V) include -Xaa ¹ -Xaa ² -Gly-Hmb-Ala-Pro- (WO2006 / 43644) .
Here, Xaa ¹ and Xaa ² represent arbitrary amino acid residues, Hmb represents a valic acid residue represented by the formula (VI), and Lac represents a lactic acid residue represented by the formula (VII).

More preferably, F ₁ -F ₂ -F ₃ -F ₄ -F ₅ in formula (IV) is -Ala-Ile-Gly-Lac-Pro-, -Gly-Ile-Gly-Hmb-Pro- or -Gly -Val-Gly-Hmb-Pro- where F ₁ -F ₂ -F ₃ -F ₄ -F ₅ -F ₆ in formula (V) is -Gly-Val-Gly-Hmb-Ala-Pro- .

  Depsipeptides can be obtained using two methods: synthesis of a polymer or oligomer by a polymerization reaction, or synthesis of a compound having an appropriate chain length by a unit-by-unit extension reaction called segment condensation (WO2006 / 43644). ).

  The particle size of the fine particles of the present invention is not particularly limited and is appropriately adjusted according to the application, but is preferably 100 nm to 50 μm.

The fine particles of the present invention can be obtained by a double emulsion method or a single emulsion method.
Specifically, a step of preparing an emulsion by mixing a volatile organic acid solution containing a physiologically active substance or a volatile organic acid solution of a physiologically active substance with a volatile organic solvent containing a biodegradable polymer, and The obtained emulsion can be obtained by mixing with an aqueous solution of a negatively charged polymer.

As the volatile organic solvent, an organic solvent that can dissolve the biodegradable polymer and is volatile and hardly mixed with water is used, and dichloromethane, chloroform, ethyl acetate, diethyl ether, and the like are preferably used.
Examples of the negatively charged polymer include a polymer containing an oxygen atom, and polyvinyl alcohol, carboxymethyl cellulose, and polyethylene glycol are preferably used.

The double emulsion method can be performed, for example, as described in Rosas et al., Vaccine 2001, Vol. 19, pages 4445-4451. An example of a specific production method is shown below.
Stir emulsify bioactive substance (25 mg) in acetic acid solution (250 μL) with biodegradable polymer (polylactic acid-glycolic acid copolymer, 250 mg) volatile organic solvent solution (dichloromethane, 5 mL). . Here, the inner aqueous phase containing the physiologically active substance is dispersed into the microdroplets by vigorous stirring. A large amount of an aqueous solution (8-12%) of a negatively charged polymer (such as polyvinyl alcohol) is poured into such a W / O emulsion (25 mL) and stirring is continued. As a result, the volatile organic solvent in which the biodegradable polymer was dissolved evaporates and the solubility is lowered, so that the biodegradable polymer is deposited so as to surround the inner aqueous phase, thereby forming fine particles. .

An example of a specific production method by the single emulsion method is shown below.
First, a small amount of physiologically active substance (25 mg) is dissolved in acetic acid, and this acetic acid solution is dissolved in a volatile organic solvent solution (dichloromethane, 5 mg of a biodegradable polymer (such as polylactic acid-glycolic acid copolymer). and dissolve with stirring. It differs from the double emulsion method in that the physiologically active substance becomes a uniform volatile organic solvent solution. A large amount of an aqueous solution (1 to 12%) of a negatively charged polymer (such as polyvinyl alcohol) is injected into the organic phase thus obtained (25 mL) and stirring is continued. Thereby, the organic phase containing the physiologically active substance and the biodegradable polymer is dispersed in the microdroplets by vigorous stirring. In such an O / W emulsion, since the volatile organic solvent in which the biodegradable polymer was dissolved evaporates and the solubility is lowered, the biodegradable polymer is precipitated while containing the bioactive substance, and the fine particles Is formed.

The fine particles of the present invention may contain aluminum ions. Since salts containing aluminum ions are easily soluble in volatile organic acids such as acetic acid and formic acid, double emulsion method or single emulsion method using salts containing aluminum ions together with physiologically active substances as volatile organic acid aqueous solution or volatile organic acid solution By performing the above, fine particles containing a physiologically active substance and aluminum can be obtained.
By containing aluminum ions, the antigenicity of the antigenic peptide is further promoted. For example, by referring to Lindblad, Vaccine 2004, Vol. 22, pages 3658-3668 and cited references, it is possible to select an optimal aluminum salt and design fine particles.

The fine particles of the present invention may contain an adjuvant other than aluminum ions. Adjuvants are easily soluble in volatile organic acids such as acetic acid and formic acid or volatile organic acid aqueous solutions or organic solvents. Therefore, volatile organic acid solutions or volatile organic acid aqueous solutions or organic solutions containing these adjuvants together with physiologically active substances are used. By performing the double emulsion method or the single emulsion method, fine particles containing a physiologically active substance and an adjuvant can be obtained.
By including an adjuvant, the antigenicity of the antigen peptide is further promoted. For example, referring to Guy, Nature Reviews Microbiology 2007, Vol. 5, pp. 505-517 and cited references, it is possible to select an optimal adjuvant and design microparticles.

The microparticles of the present invention can be made into a pharmaceutical composition by blending a pharmaceutically acceptable carrier. By including the fine particles of the present invention, a sustained-release pharmaceutical composition capable of controlling the release of a physiologically active substance can be obtained.
Here, examples of the pharmaceutically acceptable carrier include components that are acceptable in the preparation process such as a buffer, a lyophilization aid, a stabilization aid, a solubilization aid, and an antibacterial agent.
Examples of the buffer include phosphate, citrate, sulfosalicylate, acetate, and the like.
Lyophilization aids include mannitol, lactose, sorbitol, dextran,
Examples include Ficoll and polyvinyl pyrrolidine (PVP).
Examples of stabilizing aids include ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, inositol and the like.
Examples of solubilizers include ethanol, glycerin, polyoxyethylene sorbitan monooleate, sorbitan monooleate, polysorbates, poly (oxyethylene) poly (oxypropylene) poly (oxyethylene) block copolymers (Pluronics), and lecithin. Is mentioned.
Antibacterial agents include benzyl alcohol, benzalkonium chloride, chlorobutanol, methyl paraben, propyl paraben, butyl paraben and the like.
The dosage form of the pharmaceutical composition is not particularly limited and includes injections, oral preparations, coating agents and the like, and injections are preferred. An injection can be produced by dissolving fine particles in a diluent or the like and adding a stabilizer, a preservative, a buffering agent or the like as necessary. Examples of the administration form include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection and the like.
By using a physiologically active substance as a protein or peptide derived from Plasmodium falciparum, a pharmaceutical composition for preventing malaria parasite infection can be obtained.
The dosage of the pharmaceutical composition of the present invention to humans varies depending on the age, sex, body weight, immune status, administration method, ratio of physiologically active substance contained in the pharmaceutical composition, etc. Is preferably 1 μg to 100 mg, more preferably 100 μg to 10 mg.
The frequency of administration is not particularly limited, and multiple administrations over a certain period can be expected to increase and maintain the antibody titer due to the boost effect. The fixed period is preferably 2 weeks to 6 months, more preferably 3 weeks to 3 months. The number of times is preferably 2 to 20 times, more preferably 2 to 4 times.

  The fine particles of the present invention containing a protein or peptide derived from Plasmodium falciparum as a physiologically active substance can also be used as an active ingredient of an antigen for immunization (vaccine) that suppresses the growth of malaria parasites. It can be used in combination with a pharmaceutically acceptable carrier or adjuvant as described above.

The fine particles of the present invention can also be bound to a solid surface such as a polymer carrier, film, latex particle, ultrafine metal particle, or plastic plate.
For example, by linking the microparticles of the present invention containing a protein or peptide derived from Plasmodium falciparum as a physiologically active substance to the solid phase surface, it can be used as a diagnostic material or research reagent for an immune state against malaria infection. it can.

  Hereinafter, the fine particles produced by the double emulsion method or the single emulsion method, which is an embodiment of the present invention, are described in detail in Examples 1 to 3, and an alkaline glycine buffer (150 mM, pH 8.5) is used for the inner aqueous phase. Details are shown in Comparative Example 1, and details using distilled water for the inner aqueous phase are shown in Comparative Example 2. Further, details on the release behavior of the artificial antigen peptide from the microparticles are shown in Example 4. Further, the in vivo behavior of the fine particles is shown in Example 5. However, the following specific examples do not limit the present invention, and it is needless to say that the polymer can be appropriately changed, for example, by replacing the polymer with another known biodegradable polymer or adjusting the amount or concentration of the solution.

Example 1 Fine particles prepared using an acetic acid solution were prepared by a single emulsion method (O / W). The artificial antigen peptide (FIG. 3) is hardly soluble in distilled water, but could be dissolved in a small amount in acetic acid. In the single emulsion method, since an organic acid solution is used instead of the aqueous phase W ₁ shown in the double emulsion method of FIG. 4, the artificial antigen peptide is uniformly dissolved in the biodegradable polymer solution O. The obtained O / W emulsion was ice-cooled to form more uniform fine particles. This is probably because the viscosity of the emulsion has increased. Also, the organic solvent was quickly removed by blowing nitrogen gas. As a result, the microparticles containing a relatively uniform artificial antigen peptide having an average particle diameter of 0.6 μm were successfully produced. Details of the experimental operation are shown below, and a scanning electron micrograph is shown in FIG.

  To a 200 mL eggplant flask, 1 mg of the antigen peptide and 1 mL of acetic acid were added and dissolved. After adding an organic phase (O) in which 240 mg of Poly (lactic acid-co-glycolic acid) (Wako Pure Chemicals, PLGA7520, molecular weight 20,000) was dissolved in 6 mL of distilled dichloromethane under ice cooling, Ultrasonic Homogenizer (SNT Company, UH-50 model, 4 mm probe was used) and mixed and stirred for 15 minutes to obtain organic phase O.

  180 mL (W) of an ice-cooled 0.5% PVA aqueous solution was added to the organic phase O which was ultrasonically stirred with an ultrasonic homogenizer. Furthermore, ultrasonic stirring was continued for 15 minutes under ice cooling to prepare an O / W emulsion. Thereafter, in order to volatilize dichloromethane, stirring by a magnetic stirrer and blowing of nitrogen gas were performed for 5 hours.

  Pass the O / W emulsion through a coarse glass filter (G1) to remove large suspended solids, transfer to four 50 mL centrifuge tubes, and centrifuge (130 G, 30 minutes, 4 ° C) to remove large precipitates first. did. Centrifugation (2000 G, 100 minutes, 4 ° C.) was performed again to precipitate fine particles. Washing was performed by repeating resuspension in 40 mL distilled water three times. After adding 5 mL of distilled water and freezing in a freezer, freeze drying was performed to obtain fine particles.

Example 2 Fine particles prepared using a formic acid solution were prepared by a single emulsion method (O / W). A solution could be prepared using formic acid in the same manner as in Example 1. As a result, they succeeded in producing fine particles containing a relatively uniform artificial antigenic peptide having an average particle diameter of 0.8 μm. Details of the experimental operation are shown below, and a scanning electron micrograph is shown in FIG.

  To a 200 mL eggplant flask, 12 mg of antigen peptide and 0.6 mL of formic acid were added and dissolved. After adding an organic phase (O) in which 240 mg of Poly (lactic acid-co-glycolic acid) (Wako Pure Chemicals, PLGA7520, molecular weight 20,000) was dissolved in 6 mL of distilled dichloromethane under ice cooling, Ultrasonic Homogenizer (SNT Company, UH-50 model, 4 mm probe was used) and mixed and stirred for 15 minutes to obtain organic phase O.

  180 mL (W) of an ice-cooled 0.5% PVA aqueous solution was added to the organic phase O which was ultrasonically stirred with an ultrasonic homogenizer. Furthermore, ultrasonic stirring was continued for 15 minutes under ice cooling to prepare an O / W emulsion. Thereafter, in order to volatilize dichloromethane, stirring by a magnetic stirrer and blowing of nitrogen gas were performed for 5 hours.

  Pass the O / W emulsion through a coarse glass filter (G1) to remove large suspended solids, transfer to four 50 mL centrifuge tubes, and centrifuge (130 G, 30 minutes, 4 ° C) to remove large precipitates first. did. Centrifugation (2000 G, 100 minutes, 4 ° C.) was performed again to precipitate fine particles. Washing was performed by repeating resuspension in 40 mL distilled water three times. After adding 5 mL of distilled water and freezing in a freezer, freeze drying was performed to obtain fine particles.

Example 3 Fine particles prepared using an aqueous formic acid solution were prepared by the double emulsion method (W ₁ / O / W ₂ ). Artificial antigen peptide (FIG. 3) Aqueous phase W ₁ was prepared using formic acid and distilled water. Mixing was carried out according to the double emulsion method of FIG. 4, and more uniform fine particles were formed by making each emulsion while cooling with ice. This is probably because the viscosity of the emulsion has increased. Also, the organic solvent was quickly removed by blowing nitrogen gas. As a result, fine particles containing an artificial antigen peptide having an average particle diameter of about 3 to 10 μm were successfully produced. Details of the experimental operation are shown below, and a scanning electron micrograph is shown in FIG.

Antigen peptide 3.2 mg, formic acid 0.32 mL, gelatin aqueous solution 3.2 mg / 0.32 mL, and water 0.64 mL were added and dissolved in this order in a 200 mL eggplant flask (aqueous phase W ₁ ). After adding an organic phase (O) obtained by dissolving 240 mg of Poly (lactic acid-co-glycolic acid) (Wako Pure Chemicals, PLGA7520, molecular weight 20,000) in 6 mL of distilled dichloromethane under ice-cooling, Ultrasonic Homogenizer (SNT Company, UH-50 model, using 4 mm probe) and mixed and stirred for 15 minutes to obtain a W ₁ / O emulsion.

180 mL (W ₂ ) of an ice-cooled 0.5% aqueous PVA solution was added to the ice-cooled W ₁ / O emulsion. Using a mechanical homogenizer (main body IKA-T25 Ultra Turrax, probe S25N-18G), stirring was performed at 3200 rpm for 10 minutes. By gently stirring the emulsion using a mechanical probe, droplets in the inner aqueous phase remained, resulting in the formation of double-structured fine particles (W ₁ / O / W ₂ emulsion). The double structure could be confirmed with a confocal laser microscope or a scanning electron microscope. Thereafter, in order to volatilize dichloromethane, stirring by a magnetic stirrer and blowing of nitrogen gas were performed for 5 hours.

The prepared W ₁ / O / W ₂ emulsion was passed through a coarse glass filter (G1) to remove large suspended solids, and then transferred to four 50 mL centrifuge tubes and allowed to stand to remove precipitated substances. Centrifugation (130 G, 30 minutes, 4 ° C.) was performed again to precipitate the fine particles. Washing was performed by repeating resuspension in 40 mL distilled water three times. After adding 5 mL of distilled water and freezing in a freezer, freeze drying was performed to obtain fine particles.

(Comparative Example 1) A glycine buffer solution (150 mM, pH 8.5) was used in place of a fine particle formic acid aqueous solution and an acetic acid aqueous solution prepared using an alkaline glycine buffer solution (150 mM, pH 8.5) for the inner aqueous phase. The formation of fine particles was not observed at all. It was the same even when the pH was 9.2. However, the solubility of the artificial antigen was as good as that of the acetic acid aqueous solution and the formic acid aqueous solution. The operation method is shown below.

Add 10 mg of antigenic peptide, 4 mg of gelatin (PolyScience, USA) and 100 mL of glycine buffer to a 200 mL eggplant flask, and ultrasonically stir with an Ultrasonic homogenizer (SNT Company, UH-50, 4 mm probe) Performed for 30 minutes (W ₁ ). Poly (lactic acid-co-glycolic acid) and 375 mg was dissolved in 2 mL of dichloromethane the organic phase (O) was added to the W _1. Ultrasonic stirring was performed for 3 minutes with an ultrasonic homogenizer to prepare a W ₁ / O emulsion.

Pass the W ₁ / O / W ₂ emulsion through a coarse glass filter (G1) to remove large suspended solids, then remove the large precipitate by decantation, transfer to a 50 mL centrifuge tube, and centrifuge (3000 rpm, 10-10 20 minutes) and allowed to settle. All W ₁ / O / W ₂ emulsions were precipitated and washed with 50 mL distilled water. After adding 5 mL of distilled water and freezing in a freezer, lyophilization was performed.

25 mL (W ₂ ) of an ice-cooled 0.5% PVA aqueous solution was added to the W ₁ / O emulsion that was ultrasonically stirred with an ultrasonic homogenizer. Further, ultrasonic stirring was continued for 3 minutes under ice cooling to prepare a W ₁ / O / W ₂ emulsion. Thereafter, in order to volatilize dichloromethane, stirring by a magnetic stirrer and blowing of nitrogen gas were performed for 6 hours.

(Comparative example 2) Since the polymer fine particle artificial antigen peptide produced using distilled water for the inner aqueous phase does not dissolve in distilled water (10 mg / 100 mL), the fine particles cannot be produced. In addition, when only the distilled water was used for the inner aqueous phase, polymer fine particles having a large diameter and a wide particle size distribution (5 to 30 μm) and micrometer size were formed. The operation method is shown below.

Gelatin 4 mg (PolyScience, USA) and distilled water 100 mL were added to a 200 mL eggplant flask, and ultrasonic agitation was performed for 30 minutes with an Ultrasonic homogenizer (SNT company, UH-50 type, using a 4 mm probe) (W ₁ ). Poly (lactic acid-co-glycolic acid) and 375 mg was dissolved in 2 mL of dichloromethane the organic phase (O) was added to the W _1. Ultrasonic stirring was performed for 3 minutes with an ultrasonic homogenizer to prepare a W ₁ / O emulsion.

Pass the W ₁ / O / W ₂ emulsion through a coarse glass filter (G1) to remove large suspended solids, then remove the large precipitate by decantation, transfer to a 50 mL centrifuge tube, and centrifuge (3000 rpm, 10-10 20 minutes) and allowed to settle. All W ₁ / O / W ₂ emulsions were precipitated and washed with 50 mL distilled water. After adding 5 mL of distilled water and freezing in a freezer, lyophilization was performed.

25 mL (W ₂ ) of an ice-cooled 0.5% PVA aqueous solution was added to the W ₁ / O emulsion that was ultrasonically stirred with an ultrasonic homogenizer. Further, ultrasonic stirring was continued for 3 minutes under ice cooling to prepare a W ₁ / O / W ₂ emulsion. Thereafter, in order to volatilize dichloromethane, stirring by a magnetic stirrer and blowing of nitrogen gas were performed for 6 hours.

Example 4 Release Behavior of Artificial Antigen Peptide from Fine Particles Using Fluorescent Labels The release of artificial antigen peptide from fine particles produced in the same manner as in Example 1 was observed. At this time, the antigen peptide labeled with carboxyfluorescein was used. From 1.0 mg of fine particles placed on the bottom of a container containing 40 mL of 0.2% SDS-phosphate buffered saline at 37 ° C (plastic container for pharmaceuticals, CZ vial and fluororesin laminated rubber stopper made by Daikyo Seiko Co., Ltd.) The release behavior (◯) of the fluorescence-labeled artificial antigen peptide was followed by the fluorescence intensity (FIG. 8). As a comparison, the process (x) in which 4.0 μg of the fluorescence-labeled artificial antigen peptide that was allowed to stand in the same manner was dispersed and dissolved was also followed.

  For fluorescence spectroscopic measurement, 50 μL of solution was collected from the vial container using an apparatus (FP-6200 manufactured by JASCO Corporation) and a microcell, and the fluorescence of fluorescein was observed. The collected solution can be returned to the vial container again. According to this method, since the fine particles are not suspended, it is possible to observe the state of release of the artificial antigenic peptide from the fine particles over a long period of several weeks to several months without being dispersed in the container. Even when the microparticles are suspended in the solution, the fluorescence is measured after separating the solution and the microparticle components by the centrifugal separation method, and the release of the same antigen peptide from the microparticles can be observed.

Example 5 Release Behavior of Artificial Antigen Peptide from Fine Particles in Mouse Subcutaneous Release of the artificial antigen peptide from fine particles prepared in the same manner as in Example 1 was observed in the mouse subcutaneous. At this time, the antigen peptide labeled with carboxyfluorescein was microparticulated by the single emulsion method, and disappearance from the subcutaneous layer was observed by the fluorescence imaging method.

  2.0 mg of fine particles were dispersed in 0.20 mL of physiological saline and inoculated subcutaneously into two nude mice (one for each 0.5 mL on the back side and the other one on the back side 0.08 mL). By observing fluorescence of fluorescein every few days with a fluorescence imager (Maestro FL500 manufactured by CRi, USA), it was possible to actually follow the disappearance process of the artificial antigenic peptide under the skin (FIG. 10).

Fluorescence images on days 0, 2 and 5 are 5 nm in the excitation wavelength range 445-490 nm and detection wavelength range 500-720 nm.
Observed every time. The obtained fluorescence image was subjected to image processing by spectral decomposition. As a result, the intensity of fluorescent fine particles administered subcutaneously on the 0th day became 24 to 48% after 2 days and 17 to 49% after 5 days. This was because some of the administered microparticles diffused subcutaneously, and the fluorescence intensity rapidly decreased over the second day (0 to 2 days), and the remaining microparticles were slowly decomposed without much diffusion ( 2-10 days). Further, in the observation after the 10th day, carboxyfluorescein accumulates in subcutaneous adipose tissue, so it is not very suitable to analyze the decomposition behavior of the fine particles from the fluorescence intensity.

  The above-mentioned fluorescence imaging method is limited in finding the disappearance amount of the artificial antigenic peptide with an approximate distribution in the body. In order to obtain more precise quantitative data, it is preferable to label a radioisotope with an artificial antigen peptide (for example, label to a tyrosine residue with radioiodine), and to strictly quantify and track the biodistribution and metabolic processes. This is a method that is generally used in practice, and can be easily implemented in this embodiment.

(Example 6) Immunization experiment 1 to mice with microparticles
Immunization experiments on mice subcutaneously with microparticles prepared by the same method as in Example 1 and changes in antibody titer were examined. As shown in FIG. 11, antibody titers against artificial antigenic peptides were plotted from 0 to 15 weeks. The antibody titer is expressed by the dilution ratio of the serum, and the dilution rate when the "absorbance at 405 nm of the sample serum measured by ELISA method" exceeds "absorbance in pre-immune serum + twice the standard deviation" Is defined as the antibody titer. Each experimental group used 5 Balb / c mice (7-week old mice). Experimental group 1 has an antigen amount of 1.6 μg / animal (0.4 mg microparticles / animal), experimental group 2 has an antigen amount of 16 μg / animal (4.0 mg microparticles / animal), and experimental group 3 has an antigen amount of 16 μg / animal (antigen alone and no microparticles) ), Experimental group 4 is administered with an antigen amount of 0.0 μg / animal (without antigen, only 4.0 mg microparticles / animal). From the transition of the antibody titer in Experimental Group 2, it was found that the microparticles of the present invention can produce antibodies by immunization of mice at the dose in this experiment. It was also revealed that the antibody titer increased continuously.

(Example 7) Immunization experiment 2 to mice with microparticles
Here, 4 mice in experimental group 2 described in Example 6 and FIG. 11 were re-administered with fine particles (antigen amount 16 μg / animal, 4.0 mg microparticle / animal) at 2 weeks (mouse 3 and 5). The remaining 2 animals (mouse 1 and 4) were observed again for up to 27 weeks without re-administering anything. As shown in FIG. 12, since the antibody titers of two re-administered mice (mouses 3 and 5) were significantly increased compared to those before administration, the microparticles of the present invention may exhibit a booster effect in immunizing mice. It was revealed.

  For example, the anti-serum of mice obtained in Examples 6 and 7 can be examined for the growth inhibitory effect by adding 10-fold, 20-fold, and 200-fold dilutions to the culture system of Plasmodium falciparum. At this time, it is expected that the growth rate can be suppressed by increasing the dilution rate of serum. This is a method that is generally used in practice, and can be easily implemented in this embodiment.

  At this time, for example, as described in the Examples of Patent Application 2001-176044 (Patent Publication 2002-371098), when the morphology of the protozoa in the culture system is observed with Giemsa staining under a microscope, matured schizonts with antiserum added It is not possible to expect to die in red blood cells. Schizonts are called mitotic bodies and are a form of growth cycle in the bloodstream. Trophozoites (nutrients, rings) in erythrocytes mature into schizonts (mitotics), becoming merozoites that cause fever and infection of new erythrocytes. That is, when the microparticles of the present invention are administered to humans, it is considered that fever and seriousness (infection with new erythrocytes) during P. falciparum infection can be directly suppressed by stopping maturation into schizonts. .

Claims (11)

A microparticle comprising a peptide and a biodegradable polymer derived from Plasmodium, particles formed by the sheet ring Le emulsion method using a solution of the peptide acetate and / or formic acid from the malaria parasite. The fine particles according to claim 1, wherein the biodegradable polymer is a polylactic acid-glycolic acid copolymer. The microparticle according to claim 1 , wherein the biodegradable polymer is polylactic acid. The microparticle according to claim 1 , wherein the biodegradable polymer is a polydepsipeptide. The P. falciparum-derived peptide is enolase or its partial peptide derived from Plasmodium falciparum, fine particles according to any one of claims 1 to 4. The microparticle according to claim 5 , wherein the partial peptide comprises a structural difference between SEQ ID NO: 1 and SEQ ID NO: 2, and is a peptide capable of inducing a cellular or humoral immunological response against Plasmodium falciparum. . The microparticle according to claim 6 , wherein the partial peptide is a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 6. A pharmaceutical composition comprising the microparticles according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing malaria parasite infection comprising the microparticles according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier. As an active ingredient a particle according to any one of claims 1 to 7, an antigen for immunization for suppressing the growth of malaria parasites. Acetic acid and / or formic acid solution of the peptide derived from the malaria parasite, a volatile organic solvent and mixed match that step comprising a biodegradable polymer, and a step of mixing an aqueous solution of a polymer having a negative charge of the mixture And a method for producing microparticles containing malaria parasite-derived peptides .