WO2023277051A1

WO2023277051A1 - Adjuvant

Info

Publication number: WO2023277051A1
Application number: PCT/JP2022/025905
Authority: WO
Inventors: 晶山▲崎▼; 茂宜石塚; デンジャーフィールド，エマ・マリア; ストッカー，ブリジット・ルイス; ティマー，マテウス・サイモン・マリア; タケル松尾; 暢次朗江口
Original assignee: 国立大学法人大阪大学; ヴィクトリアリンクリミテッド; 株式会社 SENTAN Pharma
Priority date: 2021-06-30
Filing date: 2022-06-29
Publication date: 2023-01-05
Also published as: JPWO2023277051A1

Abstract

According to the present invention, an adjuvant includes biocompatible particles that contain the compound of formula 1.

Description

Immunostimulant

The present invention relates to an immunostimulant.

Immunostimulants (adjuvants) are used to enhance the immune response to vaccines. Humoral immunity conferred by vaccines is insufficient to protect against some pathogens. Therefore, there is a need for immunostimulants that enhance acquired cellular (Th1) immunity. Th1 cells secrete cytokines that activate macrophages and induce the production of opsonizing antibodies by B cells. Cell-mediated immunity activates cytotoxic T lymphocytes (CTLs), a subgroup of T cells that induce the death of pathogen-infected cells. In cell-mediated immunity, natural killer (NK) cells are also activated and play a major role in apoptosis in tumors and virus-infected cells.

Effective Th1-stimulating immunostimulants often work with the innate immune system by activating pattern recognition receptors (PRRs) on professional antigen-presenting cells (APCs). Recently, a macrophage-inducible C-type lectin (Mincle) was identified as a PRR on the surface of macrophages and dendritic cells (DC). Mincle has been identified as a promising target for vaccine development.

Pathogen-associated molecular patterns (PAMPs) are molecules associated with pathogen groups recognized by cells that contribute to the innate immune system. Many molecules can act as PAMPs, including glycans and glycoconjugates. PAMPs bind to PRRs and the specificity of the resulting immune response is directed by the type of PRR activated and the structure of each PAMP.

Mincle is activated by several PAMPs such as the Mycobacterium tuberculosis cell wall glycolipid trehalose dimycolate (TDM). Activation of Mincle induces the FcRγ-Syk-Card9-Bcl10-Malt1 signaling axis and immune response. However, TDM is highly toxic and difficult to synthesize as it is a complex mixture of compounds.

A derivative of TDM, trehalose dibehenate (TDB), also binds to Mincle and activates Mincle. Patent Document 1 discloses a vaccine adjuvant comprising a liposomal formulation incorporating TDB as a glycolipid within the liposome to improve stability in aqueous formulations.

In addition, Patent Document 2 shows that a bralutemicin analogue containing a long-chain lipophilic tail has potent Mincle agonist activity and is a Th1-stimulating immunostimulator.

Japanese Patent Publication No. 2008-505131 Japanese Patent Publication No. 2021-501791

The degree of enhancement of acquired cellular immunity depends on the efficiency of PAMP binding to PRR. The brartemicin analogue disclosed in Patent Document 2 has not been examined for transport to PRR in vivo, and there is room for improvement in order to further enhance the enhancement of acquired cell-mediated immunity.

The present invention has been made in view of the above circumstances, and aims to provide an immunostimulant capable of inducing a high immune response.

The immunostimulant according to the present invention is
Compounds of Formula 1

[wherein X _a and X _b are each independently selected from O or NH;
Y _a and Y _b are each independently selected from the group comprising —I, —Br, —Cl, —F, —OH, —R ¹ and OR ¹ , wherein R ¹ is alkyl having 1 to 6 carbon atoms, carbon selected from alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms, wherein alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms are each —OH or 1 carbon atom; optionally substituted with ~6 alkoxy,
n and m are each independently 0 to 4, and Z _a and Z _b are each independently selected from R ² , —OR ² _, —NHR ² , —NHC(O)—R ² and SR ² and R ² is selected from alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms, and alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and each alkynyl of 5 to 26 may be substituted with -OH or alkoxy having 1 to 6 carbon atoms,
r and s are each independently 1 to 3;
alk _a and alk _b are each independently selected from alkylene of 1 to 4 carbon atoms, alkenylene of 2 to 4 carbon atoms and alkynylene of 2 to 4 carbon atoms, or alk _a and alk _b are each absent; the aryl ring may be directly attached to the carbon of C=O,
n+r=1-5 and m+s=1-5. ]
including biocompatible particles containing

X _a and X _b are O;
You can do it.

the aryl ring is attached directly to the carbon of C═O in the absence of alk _a and alk _b respectively;
You can do it.

n and m are 1;
Y _a and Y _b are —OH;
You can do it.

Z _a and Z _b are —OR ² ,
R ² is selected from alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms, and alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and 5 carbon atoms. Each alkynyl of -26 may be substituted with -OH or alkoxy of 1-6 carbon atoms.

Z _a and Z _b are —OR ² ,
R ² is alkyl having 18 carbon atoms,
r and s are 1;
You can do it.

The biocompatible particles are lactic acid/glycolic acid copolymer particles,
You can do it.

The 50% diameter of the biocompatible particles is
is 200 nm or less,
You can do it.

The immunostimulant according to the present invention is
used in combination with vaccines
You can do it.

The vaccine is
is a vaccine against SARS-CoV-2,
You can do it.

The immunostimulant according to the present invention can induce a high immune response.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the structure of the thin-film rotary dispersion machine which concerns on embodiment of this invention. 1 is a diagram showing the particle size distribution of compound-containing nanoparticles according to Example 1. FIG. 1 is a diagram showing GFP positive rates according to Test Example 1. FIG. FIG. 10 is a graph showing changes over time in body weight of mice according to Test Example 2. FIG. (A), (B), (C) and (D) show the body weight of mice dosed with spike protein alone, compound-free nanoparticles, compound-containing nanoparticles and compound, respectively. FIG. 5 is a graph showing changes over time in the average body weight of the mice shown in FIG. 4; FIG. 10 is a graph showing temporal changes in the amount of antibody produced in mice according to Test Example 2. FIG. (A), (B), (C) and (D) show the body weight of mice dosed with spike protein alone, compound-free nanoparticles, compound-containing nanoparticles and compound, respectively. FIG. 7 is a graph showing changes over time in average antibody production levels in the mice shown in FIG. 6. FIG. (A) shows the average amount of spike protein-specific IgG during the test period. (B) shows the average amount of IgG from 0 to 7 weeks shown in (A).

An embodiment according to the present invention will be described with reference to the drawings. In addition, the present invention is not limited to the following embodiments.

The immunostimulant according to the present embodiment contains biocompatible particles containing the compound of Formula 1 below.

In Formula 1, X _a and X _b are each independently selected from O or NH. X _a may be O and X _b may be NH, X _a may be NH and X _b may be O, or X _a and X _b may be NH. Preferably X _a and X _b are O.

Y _a and Y _b are each independently selected from the group comprising —I, —Br, —Cl, —F, —OH, —R ¹ and OR ¹ . Here, R ¹ is selected from alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms. Each of alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms may be substituted with —OH or alkoxy having 1 to 6 carbon atoms. Preferably Y _a and Y _b are each —OH or —CH ₃ , preferably Y _a and Y _b are —OH. n and m are each independently 0-4. Preferably n and m are one.

Z _a and Z _b are each independently selected from R ² , —OR ² , —NHR ² , —NHC(O)—R ² and SR ² . Here, R ² is selected from alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms. Each of alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms may be substituted with —OH or alkoxy having 1 to 6 carbon atoms. Preferably, Z _a and Z _b are —OR ² and R ² is 18 carbon alkyl. r and s are each independently 1 to 3; Preferably r and s are one.

alk _a and alk _b may each be independently selected from alkylene having ₁ to 4 carbon atoms, alkenylene having 2 to 4 carbon atoms and alkynylene having 2 to 4 carbon atoms _; The aryl ring may be attached directly to the carbon of C=O without

　n+r=1 to 5 and m+s=1 to 5. For example, if n and m are each 1, r and S are each selected from 0-4. Preferably, n and m are each one and r and S are each one.

"Alkyl" means any saturated hydrocarbon group having any number of carbon atoms, for example up to 30 carbon atoms. Alkyl includes cyclic (including fused bicyclic) alkyl groups, straight and branched chain alkyl groups, and straight or branched chain alkyl groups substituted with cyclic alkyl groups. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl. group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, n-hexyl group, cyclohexyl group, cyclooctyl group, and 1-methyl-2-ethylpropyl groups.

"Alkenyl" means any hydrocarbon group having at least one double bond and up to 30 carbon atoms. Alkenyl includes both straight-chain and branched-chain alkenyl groups. Examples of alkenyl include ethenyl, n-propenyl, iso-propenyl, n-butenyl, iso-butenyl, sec-butenyl, t-butenyl, n-pentenyl, 1,1-dimethylpropenyl. 1,2-dimethylpropenyl, 2,2-dimethylpropenyl, 1-ethylpropenyl, 2-ethylpropenyl, n-hexenyl and 1-methyl-2-ethylpropenyl groups.

"Alkynyl" means any hydrocarbon group having at least one triple bond and up to 30 carbon atoms. Alkynyl includes both straight-chain and branched-chain alkynyl groups. Examples of alkynyl include ethynyl, n-propynyl, iso-propynyl, n-butynyl, iso-butynyl, sec-butynyl, t-butynyl, n-pentynyl and the like.

"Alkoxy" means an O-alkyl group. "Acyl" means a C(=O)R' group, where R' is alkyl as defined above.

"Alkylene" means a divalent group corresponding to an alkyl group. Examples of alkylene include methylene, cyclohexylene and ethylene groups. An alkylene can contain one or more cyclic alkylene groups in the alkylene chain. Alkylene may include a cyclohexylene group attached to a methylene group. Alkylene may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, alkyl and aryl. An alkylene may include one or more arylene moieties within the alkylene chain, eg, a phenylene group within the alkylene chain.

"Alkenylene" means a divalent group corresponding to an alkene group. Alkenylene may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, alkyl and aryl. An alkenylene may contain one or more arylene moieties within the alkenylene chain, eg, a phenylene group may be contained within the alkenylene chain.

"Alkynylene" means a divalent group corresponding to an alkynyl group. Alkynylene may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, alkyl and aryl. An alkynylene may optionally include one or more arylene moieties within the alkynylene chain, for example, a phenylene group may be included within the alkynylene chain.

"Aryl" means an aromatic group having 4 to 18 carbon atoms, including heteroaromatic groups. Aryl includes fused groups such as monocyclic, bicyclic and tricyclic groups. Examples of aryl include phenyl, indenyl, 1-naphthyl, 2-naphthyl, azulenyl, heptalenyl, biphenyl, inrdacenyl, acenaphthyl, fluorenyl, phenalenyl, phenanthrenyl, anthracenyl, cyclopenta Cyclooctenyl group, benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group (1-H-1,2,3-triazol-1-yl and 1-H-1 , 2,3-triazol-4-yl group), tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group , pyranyl, benzofuryl, isobenzofuryl, thienyl, thiazolyl, isothiazolyl, benzothiazolyl, oxazolyl and isoxazolyl.

"Substituted" means that one or more hydrogen atoms of an indicated group have been replaced with one or more independently selected suitable substituents. provided that the normal valence of each atom to which the substituent is attached is not exceeded, and preferably the substitution results in a stable compound. Suitable substituents include optional substituents set forth herein.

Compounds 2a to 2p are exemplified below as compounds of formula 1 above.

The compounds according to the present embodiment are 6,6'-di-O-(2-hydroxy-4-butoxybenzoyl)-α,α'-D-trehalose, 6,6'-di-O-(2- hydroxy-4-methoxybenzoyl)-α,α'-D-trehalose and 6,6'-di-O-(2,4-dihydroxybenzoyl)-α,α'-D-trehalose.

　Biocompatible particles are particles whose main component is a biocompatible polymer. Biocompatible polymers have varying average chain lengths that lead to differences in internal viscosity and polymer properties. The polymer used in the present embodiment is preferably a biodegradable polymer that is biocompatible with low irritation and toxicity to living bodies and is degraded and metabolized after administration. Biodegradable polymers include, for example, macromolecules produced by microorganisms such as polyhydroxybutyrate and polyhydroxyvalerate, and natural high-molecular weight compounds such as collagen, cellulose acetate, bacterial cellulose, high amylose corn starch, starch and chitosan. molecules and the like. The molecular weight of the biocompatible polymer is for example 5000-200000 or 15000-25000.

The biocompatible polymer is preferably biocompatible polyester. Biocompatible polyesters include, for example, D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone, ε-hydroxyhexanoic acid, It is a polyester synthesized by polymerizing one or more monomers selected from γ-butyrolactone, γ-hydroxybutyric acid, δ-valerolactone, δ-hydroxyvaleric acid, hydroxybutyric acid, malic acid, and the like. Preferably, the biocompatible polymer is polylactic acid, polyglycolic acid, lactic acid-aspartic acid copolymer, lactic acid-glycolic acid copolymer (PLGA) or polyethylene glycol/chitosan modified-PLGA (PEG/CS-PLGA). be.

PLGA is a copolymer consisting of lactic acid (or lactide) and glycolic acid (or glycolide) in a ratio of, for example, 1:99 to 99:1, preferably 3:1. PLGA may be synthesized from arbitrary monomers by a general method, or commercially available products may be used. Examples of commercially available PLGA include PLGA7520 (lactic acid:glycolic acid=75:25, average weight molecular weight: 20,000, manufactured by Wako Pure Chemical Industries, Ltd.). PLGA with a lactic acid and glycolic acid content of 25% to 65% by weight is preferred because it is amorphous and soluble in an organic solvent such as acetone.

The biocompatible particles according to the embodiment are preferably nano-sized and have a particle diameter of, for example, 1 to 800 nm. The particle size of the biocompatible particles is 500 nm or less, 400 nm or less, 300 nm or less, or 200 nm or less. For example, the particle size of the biocompatible particles is 10-500 nm, 25-300 nm, 30-250 nm, more preferably 30-300 nm or 40-200 nm.

The particle size of biocompatible particles is measured by sieving method, sedimentation method, microscopic method, light scattering method, laser diffraction/scattering method, electrical resistance test, observation by transmission electron microscope, observation by scanning electron microscope, etc. can. The particle size may be measured with a known particle size distribution meter. The particle diameter can be represented by an equivalent stalk diameter, an equivalent circle diameter, or an equivalent sphere diameter depending on the measurement method. In addition, the particle size of the biocompatible particles may be an average particle size, a volume average particle size, an area average particle size, or the like, with a plurality of particles being measured.

For example, the particle size of the biocompatible particles described above may be an average particle size calculated from a volume distribution or the like based on a measurement such as a laser diffraction/scattering method. Specifically, when the cumulative curve is obtained with the total volume of the group of particles as 100%, the volume average particle diameter (50% diameter; D ₅₀ ), which is the particle diameter at the point where the cumulative curve reaches 50%, is the particle diameter. It may be the diameter. Cumulative curves and _D50 can be determined using a commercially available particle size analyzer.

The biocompatible particles have a particle diameter span value of 3.0 or less. A span value is obtained by (D ₉₀ -D ₁₀ )/D ₅₀ . Here, D90 is the ₉₀ % diameter, which is the particle diameter at the point where the cumulative curve reaches 90%. D10 is the ₁₀ % diameter, which is the particle diameter at the point where the cumulative curve reaches 10%. Preferably, the biocompatible particles have a particle diameter span value of 5.0 or less, 4.0 or less, preferably 3.0 or less, and more preferably 2.5 or less.

The content of the compound in the biocompatible particles is not particularly limited, but is 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, relative to the mass of the biocompatible particles. 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, It may be 60% by mass or less, 50% by mass or less, or 40% by mass or less. Preferably, the compound content in the biocompatible particles is 10-80%, 12-50% or 15-40% by weight. Here, the content rate is the mass of the compound relative to the mass of the biocompatible particles calculated based on the quantified value of the concentration of the compound extracted from the biocompatible particles.

The biocompatible particles may contain excipients. Excipients enhance the fluidity of the biocompatible particles and enhance the stability of the compound. Any pharmacologically acceptable excipient can be used. For example, excipients include water, saline, oils such as animal oils, vegetable oils, synthetic oils and petroleum oils, aqueous dextrose, glycerol, starch glucose, lactose, mannitol, trehalose, inositol, erythritol, lactose, sucrose, sucrose. , pullulan, sorbitol, starches, dextrin, dextran, sodium alginate, sugars such as crystalline cellulose, methylcellulose, carmellose sodium, polyvinyl alcohol (PVA) and PVP, natural polymers, synthetic polymers, glycine, leucine, isoleucine, arginine and amino acids such as histidine, gelatin, sodium stearate, glyceryl monostearate, sodium chloride, propylene glycol, ethanol, wetting agents, emulsifiers, binders, dispersants, thickeners, lubricants, pH adjusters, Examples include solubilizers, softeners, surfactants, and the like. More preferably, the excipient is PVA.

The mass ratio of compound to excipient in the biocompatible particles ranges from 100:1 to 1:100. The mass ratio of the compound contained in the biocompatible particles to the excipient is preferably 10:1 to 1:20, more preferably 8:1 to 1:15, still more preferably 6:1. ~1:10 or 4:1 to 1:10.

Next, a method for producing an immunostimulant according to this embodiment will be described. The compounds described above can be prepared according to known chemical synthesis methods. A method for synthesizing the structurally symmetric compound 3 is illustrated with reference to Scheme 1 shown below. Y, Z, n and m in Compound 3 are the same as Y _a , Z _a , n and r defined in Formula 1 above, and p is 0-4. with the proviso that when X is O, n is 0, Z is —OR ² , R ² is C ₁₆ -C ₂₂ and m is 1 or 2, then p is not 3. and

In the synthesis of compound 3, for example, compound 4 (X is O or NH) in which R is protected with a benzyl group, trimethylsilyl group or levulinoyl group or is unprotected and R is H is reacted with a coupling reagent. or an activated form of compound (carboxylic acid) 5, such as the anhydride or acid chloride, is used to condense with compound 5. Subsequent optional deprotection using, for example, hydrogenation, acid or base provides compound 3. Coupling reagents are, for example, N,N'-dicyclohexylcarbodiimide (DCC), BOP reagents, HATU, COMU, triphenylphosphine (Ph ₃ P)/DEAD, or derivatives thereof.

Compound 4 (where X is NH) is also formed by reduction of the corresponding azide using a reducing agent such as hydrogen, hydride, sulfide or phosphine. Further, compound 4 is subjected to an enzyme-, acid- or base-catalyzed reaction together with an ester derivative of compound 5. Subsequent optional deprotection using, for example, hydrogenation, acid or base, can also lead to the formation of compound 3.

Subsequently, a method for synthesizing the structurally symmetric compound 6 is illustrated with reference to Scheme 2 shown below. In compound 6, Y _a , Y _b , Z _a , Z _b , n, m, r and s are each as defined in Formula 1 above. p and q are independently 0-4.

In the synthesis of compound 6, for example, compound 7 in which R ₁ is protected with a benzyl group, trimethylsilyl group or levulinoyl group, or in which unprotected R ₁ is H (X _a is O or NH; Y is OR ₂ ( where R ₂ is H or a protecting group), or Y is NR ₂ R ₃ (wherein R ₂ and R ₃ are H or a protecting group)) under the action of a coupling reagent, or compound (carboxylic acid) 8 is condensed with compound 8 using an activated form such as the anhydride or acid chloride of This is followed by optional deprotection using, for example, hydrogenation, acid or base to form compound 9 ( _Xb is O or NH). Condensation of compound 9 with compound 10 is then carried out under the action of a coupling reagent or using an activated form such as the anhydride or acid chloride of compound (carboxylic acid) 10, optionally e.g. Hydrogenation, deprotection using acid or base gives compound 6.

Alternatively, compound 7 (Xa is NH) or compound 9 ( _Xb is NH) is formed by reduction of the corresponding azide using _a reducing agent such as hydrogen, hydride, sulfide or phosphine. Further, compound 7 or compound 9 is subjected to an enzyme-, acid-, or base-catalyzed reaction along with compound 8 or the ester derivative of compound 10. Subsequent optional deprotection using, for example, hydrogenation, acid or base, can form compound 6.

Next, the method for producing biocompatible particles will be exemplified. A method of making the biocompatible particles includes a mixing step and a precipitation step. In the mixing step, an emulsifier aqueous solution (hereinafter referred to as "A liquid") and a raw material solution (hereinafter referred to as "B liquid") are mixed in the thin film fluid. First, liquid A will be described. The emulsifier dissolved in liquid A is not particularly limited as long as it enhances the dispersion stability of the particles. For example, emulsifiers include surfactants, polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, water-soluble resins, lecithin, saponins, sterols, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, polyglycerin. Examples include fatty acid esters and polysorbates.

Preferably, the emulsifier is polyvinyl alcohol (hereinafter referred to as "PVA"). Commercially available PVA can be used as an emulsifier. PVA may be used for industrial or medical purposes, but PVA containing little residual organic solvent or containing no organic solvent is preferred.

The concentration of the emulsifier in liquid A is, for example, 0.01 to 10.0% by mass, 0.01 to 5.0% by mass, preferably 0.05 to 2% by mass. It is preferable that the pH of the thin film fluid in which the A liquid and the B liquid are mixed is 6-8. Therefore, the pH of liquid A is preferably adjusted to, for example, 7-8. Any pH adjusting agent may be used to adjust the pH of liquid A. For example, alkaline species such as sodium hydrogen carbonate, sodium carbonate, calcium hydroxide, ammonia, sodium hydroxide and potassium hydroxide may be used.

Next, I will explain the B liquid. Liquid B contains an organic solvent in which the compound is dissolved. The organic solvent is not particularly limited, but solvents including acetone and ethanol are preferred. The compound and PLGA are dissolved in the solvent. In the preparation of solution B, for example, the compound and PLGA are added to acetone, dissolved using ultrasonic waves or Clearmix, and then ethanol is added and mixed. Here, the mass ratio of PLGA to the compound is, for example, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 5:1 to 1:5 or 4:1 to 1. :4. The volume ratio of acetone to ethanol is 3:1 to 1:1, preferably 2:1 to 1:1. The drug and PLGA may be added to a mixed solution obtained by mixing acetone and ethanol in advance. If necessary, the solvent for liquid B may be mixed with a solvent other than acetone and ethanol.

In the mixing step, the A liquid and the B liquid are introduced between processing surfaces that are arranged to face each other and at least one of which rotates relative to the other. Thereby, a thin film fluid is formed between the processing surfaces, and the A liquid and the B liquid are mixed.

The mixed A and B liquids react in the thin film fluid to form nanoparticles containing compounds. The deposition step deposits the formed nanoparticles into a thin film fluid.

Here, an example of an apparatus suitable for the above manufacturing method will be described. FIG. 1 shows a schematic diagram of a cross-section of a thin film rotary disperser 100 . The thin film rotary disperser 100 includes a holder 10 , a holder 20 , an introduction section 30 , a fluid pressure application section 40 , an introduction section 50 , a fluid supply section 60 and a case 70 .

The holder 10 is arranged below the holder 20 . The holder 10 and the holder 20 hold the processing section 11 and the processing section 21, respectively. The processing section 11 and the processing section 21 are each annular (ring-shaped). The processing section 11 has a processing surface 1 . The processing section 21 has a processing surface 2 facing the processing surface 1 . A contact surface pressure applying portion 22 is arranged between the holder 20 and the processing portion 21 . The contact surface pressure applying unit 22 applies pressure (hereinafter, simply referred to as “back pressure”) to the processing unit 21 to bring the processing unit 21 closer to the processing unit 11 below. Therefore, the processing surface 1 and the processing surface 2 can move toward and away from each other.

The holder 10 is rotated relative to the holder 20 around the axis of the holder 10 by a motor, as shown in FIG. As a result, the processing surfaces 1 arranged to face each other rotate relative to the processing surface 2 .

A fluid pressure application section 40 is connected to the introduction section 30 . A liquid is supplied between the processing surface 1 and the processing surface 2 by pressurizing the A liquid by the fluid pressure applying unit 40 having a compressor. More specifically, the A liquid is introduced from the introducing section 30 into the space inside the holder 10 and the holder 20 by being pressurized by the fluid pressure applying section 40 . Furthermore, the A liquid passes between the processing surface 1 and the processing surface 2 and tries to escape to the outside of the holder 10 and the holder 20 . At this time, the holder 20 receiving the pressure of the A liquid moves away from the holder 10 against the back pressure. As a result, a minute gap is formed between the processing surface 1 and the processing surface 2 . Alternatively, the A liquid may be directly supplied between the processing surface 1 and the processing surface 2 from the introduction part 30 .

The introduction part 50 is a passage for liquid B provided inside the processing part 21 . A fluid supply section 60 having a compressor is connected to one end of the introduction section 50 . The fluid supply unit 60 supplies the B liquid between the processing surface 1 and the processing surface 2 via the introduction unit 50 . Liquid B is supplied from the introduction part 50 between the processing surface 1 and the processing surface 2 and joins with the A liquid.

Due to relative rotation of the processing surface 1 with respect to the processing surface 2, the A liquid and the B liquid join between the processing surface 1 and the processing surface 2 maintaining a minute gap to form a thin film fluid. Mixing and reaction of the A liquid and the B liquid are promoted in the thin film fluid. The product produced by the reaction of liquid A and liquid B contains a medicinal component and precipitates in the thin film fluid as uniform fine nanoparticles. The precipitated nanoparticles are discharged outside the holder 10 and the holder 20 while being suspended in the liquid.

The case 70 is arranged outside the outer peripheral surfaces of the holder 10 and the holder 20 . The case 70 accommodates the nanoparticle dispersion discharged outside the holder 10 and the holder 20 .

Apparatus suitable for the above manufacturing method includes, for example, the forced thin film microreactor ULREA SS-11 (manufactured by M Technic). When using ULREA SS-11, the number of rotations of the holder 10, the back pressure, the flow rates and temperatures of the A and B liquids, etc. can be appropriately set.

In addition, in order to increase the mixing of the A liquid and the B liquid and obtain finer nanoparticles with good reproducibility by rapid phase separation, a vibrator or an ultrasonic device is attached to the thin film rotary disperser 100, or a mixing channel You may use the micro waterway which deform|transformed more complicatedly.

The immunostimulant according to the present embodiment is produced by a known method, and contains 0.000001 to 99.9% by weight, 0.00001 to 99.8% by weight, and 0.0001 to 99.7% by weight of the active ingredient. , 0.001-99.6% by weight, 0.01-99.5% by weight, 0.1-99% by weight, 0.5-60% by weight, 1-50% by weight, or 1-20% by weight of living organisms Contains compatible particles.

The administration route of the immunostimulant according to the present embodiment to humans and animals other than humans is not particularly limited. As an administration route, intradermal, subcutaneous or intramuscular injection is particularly preferable, and in this case, one aspect of the immunostimulant is an injection. The immunostimulant may be, for example, a combination drug containing a pharmacologically acceptable carrier. Pharmaceutically acceptable carriers are various organic or inorganic carrier substances used as pharmaceutical ingredients. Pharmaceutically acceptable carriers are, for example, excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, tonicity agents and buffers in liquid formulations. and as an analgesic, etc., in an immunostimulant. Additives such as preservatives, antioxidants, coloring agents and sweeteners may also be added as necessary.

The dosage of the immunostimulant according to the present embodiment is appropriately determined according to the age, body weight, symptoms, etc. of the administration subject. The immunostimulant is administered in an effective amount of the compound. An effective amount is that amount of the compound necessary to produce the desired result, that amount necessary to slow, inhibit, prevent, reverse or cure the condition being treated or treated.

The dosage of the immunostimulant is, for example, 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.2 mg/kg to 20 mg/kg, and Single or multiple doses can be administered. The pharmaceutical composition may also be administered at different dosing frequencies, such as daily, every other day, once a week, every other week, once a month, and the like. Preferably, the dosing frequency is easily determined by a physician or the like. Amounts outside the above ranges can also be used, if desired.

The immunostimulant according to this embodiment is preferably used together with a vaccine. "Combination" refers to administering an immunostimulant and a vaccine to the same patient for a given period of time. In combination, the immunostimulant is preferably administered simultaneously with the vaccine, but each may be administered separately in chronological order, such as by administering the other while the effect of one remains. In combination, the route of administration of the immunostimulant and vaccine may be the same or different. Immunostimulatory agents may be incorporated into vaccines and administered as vaccine compositions. For example, in a combination, the adjuvant and vaccine are administered over a defined period of time according to a single regimen that defines the dose and regimen of each adjuvant and vaccine. An immunostimulatory agent, when used in combination with a vaccine, accelerates, prolongs or enhances the antigen-specific immune response to the vaccine.

Vaccines are not particularly limited as long as they can be used to prevent or treat various diseases, especially infectious diseases caused by pathogens, cancer and other diseases. Preferably, the vaccine is against the novel coronavirus (SARS-CoV-2) that causes novel coronavirus disease (COVID-19).

As shown in the examples below, the immunostimulant according to the present embodiment can enhance the immune response induced by the above compounds and increase the production of antigen-specific IgG antibodies.

In another embodiment, use of the above compounds for the manufacture of an immunostimulant is provided. In other embodiments, methods are provided for enhancing, reinforcing, promoting, accelerating or prolonging the immune response induced by the compounds. Also provided in another embodiment is a compound as described above for use in the prevention or treatment of an infectious disease or cancer.

The above compounds may have an asymmetric or chiral center. Asymmetric or chiral centers can be designated as (R) or (S), depending on the three-dimensional arrangement of the substituents on the chiral atom. All stereochemically isomeric, d-isomers, l-isomers, including diastereomeric, enantiomeric and epimeric forms of a compound, as well as enantiomerically enriched mixtures and diastereomerically enriched stereochemical isomers These mixtures are included in the above compounds, including mixtures of the above compounds.

It should be noted that individual enantiomers can be synthetically prepared from commercially available enantiopure starting materials. Enantiomers can also be prepared by preparing an enantiomeric mixture and resolving the mixture into the individual enantiomers. Resolution includes conversion of a mixture of enantiomers to a mixture of diastereomers and separation of diastereomers by, for example, recrystallization or chromatography, and other suitable methods known in the art.

The above compounds may also exist as conformational or geometric stereoisomers, including cis, trans, syn, anti, entgegen (E) and zusammen (Z) isomers. All such stereoisomers and any mixtures thereof are included in the above compounds. Any tautomers of the above compounds or mixtures thereof are also included in the above compounds.

The above compounds may also exist as isotopologues and isotopomers, wherein one or more atoms in the compound are replaced with different isotopes. Suitable isotopes include, for example, 1H, ^2H ⁽ D), ^3H (T), ^12C , ^13C , ^14C , ^16O and ^18O . Isotopologues and isotopomers are also included in the above compounds.

A pharmacologically acceptable salt of the above compound may be contained in the biocompatible particles. Salts include acid addition, base addition, and quaternary salts of basic nitrogen-containing groups. Acid addition salts can be prepared by reacting a compound in its free base form with an inorganic or organic acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of organic acids include acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, stearic acid. Acids include, but are not limited to, salicylic acid, methanesulfonic acid, benzenesulfonic acid, isethionic acid, sulfanilic acid, adipic acid, butyric acid, and pivalic acid. Base addition salts can be prepared by reacting the compound in the free acid form with an inorganic or organic base. Examples of inorganic base addition salts include alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal salts such as aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc salts. . Examples of organic base addition salts include amine salts such as salts of trimethylamine, diethylamine, ethanolamine, diethanolamine and ethylenediamine. A quaternary salt of a basic nitrogen-containing group in a compound is, for example, converting the compound to alkyl halides such as methyl, ethyl, propyl, butyl chloride, bromide and iodide, dialkyl sulfates such as dimethyl sulfate, diethyl, dibutyl and diamyl sulfate. and the like.

The compounds also include N-oxides of the above compounds. The compounds described above form solvates with various solvents and can exist as solvates with various solvents. All solvated and unsolvated forms of the compounds are included in the above compounds.

The present invention will be explained more specifically by the following examples, but the present invention is not limited by the examples. In addition, hereinafter, "%" means "% by mass" unless otherwise specified.

Example 1: Preparation of Immunostimulant [Synthesis of C18Brar]
An immunostimulant (C18Brar) was prepared according to Scheme 3 below.

First, partially protected trehalose (compound 12) was obtained from D-trehalose (compound 11) through three steps of reactions a), b) and c). In reaction a), D-trehalose, triphenylmethyl chloride (Ph ₃ CCl) and pyridine were mixed and allowed to react at room temperature for 16 hours. In reaction b), the product of reaction a was mixed with benzyl bromide (BnBr), sodium hydride (NaH) and N,N-dimethylformamide (DMF) and allowed to react at room temperature for 16 hours (yield 85 %). In reaction b), the product of reaction b) was reacted with triethylsilane (Et ₃ SiH) and dichloromethane (CH ₂ Cl ₂ ) in the presence of trifluoroacetic acid (TFA) at room temperature for 30 minutes (yield 80%).

Compound 12 (55 mg, 0.062 mmol) and 2-(benzyloxy)-4-(octadecyloxy)benzoic acid (compound 13, 136 mg, 0.274 mmol) were co-evaporated with toluene (2 mL) and dissolved in dry toluene. . 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 64 mg, 0.33 mmol) and 4-dimethylaminopyridine (DMAP, 11 mg, 0.090 mmol) were added and the reaction mixture was stirred at 60°C. . After 18 hours an additional portion of compound 13 (21 mg, 0.042 mmol) was added and stirred overnight. The resulting residue was purified by gradient silica gel flash column chromatography (petroleum ether:ethyl acetate (EtOAc), 19:1-9:1 (v:v)) to give 2,2′,3,3′,4 ,4′-Hexa-O-benzyl-6,6′-di-O-(2-benzyloxy-4-octadecyloxybenzoyl)-α,α′-D-trehalose (compound 14) was converted to a colorless oil (75 mg, 0.041 mmol, 66% yield).

To a solution of compound 14 (140 mg, 0.076 mmol) in MeOH:CH2Cl2 ₍ ₅ mL, 1:1 (v:v)) was added Pearlman's catalyst (Pd(OH) ₂ /C). _H2 gas was bubbled through the reaction mixture overnight. The resulting residue was subjected to gradient silica gel flash column chromatography (EtOAc:methanol (MeOH), 1:0-9:1 (v:v)) and lipophilic Sephadex (CH ₂ Cl ₂ :MeOH, 1:1 ( v:v)) to convert 6,6′-di-O-(2-hydroxy-4-octabenzyloxybenzoyl)-α,α′-D-trehalose (C18Brar) to an amorphous white solid ( 58 mg, 0.052 mmol, 68%).

[C18Brar nanoparticulation]
Using ULREA SS-11 (manufactured by M Technic), C18Brar nanoparticles (C18Brar-NP) were prepared as follows. First, the A liquid tank was filled with the A liquid, and the A liquid was fed at 40 mL/min at 10°C, and then the B liquid was fed at 10 mL/min at 50°C. Liquid A is an aqueous solution containing PVA (Gosenol EG-05P) at a concentration of 0.1 mg/ml. Liquid B is a solution of acetone:ethanol=2:1 (v:v) containing PLGA7520 (1.25 mg/ml) and C18Brar (0.25 mg/ml). After reacting for 50 minutes, 2500 mL of the discharged liquid was recovered, and the organic solvent was distilled off from the discharged liquid by an evaporator (40° C., 50 hPa). Ultrafiltration (Vivaflow 200 Hydrosart 30K, manufactured by Sartorius) was performed on 2000 mL of the discharged liquid after the solvent was distilled off, and the liquid was concentrated to 300 mL. Then 400 mg of mannitol was added. The obtained C18Brar-NP concentrate was lyophilized to obtain 1.2 g of C18Brar-NP. The obtained C18Brar-NP was dispersed in milli-Q water so as to be 1 mg/ml, and the particle size distribution was measured with EX150 (manufactured by MicrotracBEL).

Using ULREA SS-11 (manufactured by M Technic), nanoparticles (Empty-NP) not containing C18Brar were produced as follows. First, the A liquid tank was filled with the A liquid, and the A liquid was fed at 40 mL/min at 10°C, and then the B liquid was fed at 10 mL/min at 50°C. Liquid A is an aqueous solution containing PVA (Gosenol EG-05P) at a concentration of 0.1 mg/ml. Liquid B is a solution of acetone:ethanol=2:1 (v:v) containing PLGA7520 (1.25 mg/ml). After reacting for 50 minutes, 2500 mL of the discharged liquid was recovered, and the organic solvent was distilled off from the discharged liquid by an evaporator (40° C., 50 hPa). Ultrafiltration (Vivaflow 200 Hydrosart 30K, manufactured by Sartorius) was performed on 2000 mL of the discharged liquid after the solvent was distilled off, and the liquid was concentrated to 300 mL. Then 400 mg of mannitol was added. The resulting Empty-NP concentrate was freeze-dried to obtain 1.1 g of Empty-NP. The obtained Empty-NP was dispersed in milli-Q water so as to be 1 mg/ml, and the particle size distribution was measured with EX150 (manufactured by MicrotracBEL).

FIG. 2 shows the particle size distribution of C18Brar-NP. The _D50 of C18Brar-NP was 125 nm with a span value of 2.0. Empty-NP had a D ₅₀ of 147 nm and a span value of 0.9.

Test Example 1: Evaluation of C18Brar Content Based on Activity by Mincle Reporter Cells Mincle and reporter cells expressing FcRg as an adapter molecule (Mincle reporter cells) were used. Using a 2B4 cell line expressing NFAT (nuclear factor of activated T-cell)-GFP as a parent strain, Mincle reporter cells were produced by transfecting the Mincle and FcRγ CDS sequences with a retroviral vector.

C18Brar stock solution in chloroform:methanol (2:1) was serially diluted with 2-propanol and added to 96-well plates. The solvent was dried and the plates were coated with C18Brar. C18Brar-NP or Empty-NP was suspended in medium (RPMI containing 10% FCS), serially diluted to each concentration, and added to the plate at 100 μl/well. For C18Brar drug substance, medium was added to C18Brar-coated wells at 100 μl/well to match volume and final concentration. A Mincle reporter cell suspension (6×10 ⁵ cells/ml) was added to each plate at 100 μl/well (final concentration 3×10 ⁴ cells/well) for stimulation. Cells were cultured in a _CO2 incubator at 37°C for 20 hours.

In addition, Mincle reporter cells express CD3 in T cell hybridomas. As a positive control for the assay system, it was confirmed that GFP was expressed by stimulation with a monoclonal antibody (2C11, manufactured by MBL) against CD3e contained in the T cell receptor complex. In addition, in order to confirm that GFP is expressed by signals mediated by Mincle, trehalose-6,6'-dimycolate (TDM), a glycolipid that constitutes the cell wall of Mycobacterium tuberculosis, was treated with 2-propanol at 1.5 μg/ It was diluted to ml, added to 20 μl/well, and stimulated in the same manner as the C18Brar drug substance group.

　The GFP expression of the Mincle reporter cells was evaluated with a flow cytometer. A calibration curve was prepared using the GFP positive rate in the C18Brar raw material as a standard, and the C18Brar content was quantified from the GFP positive rate by stimulation with C18Brar-NP.

(result)
FIG. 3 shows the GFP positive rate. The amount of C18Brar-NP corresponding to 0.131 μg/well of C18Brar drug substance was 20 μg/well.

Test Example 2: Immunity test A SARS-CoV-2 spike protein (full length, trimer, hereinafter also referred to as "spike protein") was prepared as follows. The extracellular domain of the S protein with a foldon sequence followed by a 9×His tag and a Strep tag at the C-terminus was cloned into the expression vector pCMV. The polybasic cleavage site (RRAR) of the S protein was replaced with alanine and K986P and V987P substitutions were introduced to stabilize the structure. Four days after the introduction of the expression vector into Expi293F cells (manufactured by Gibco), TALON metal affinity resin (manufactured by Clontech) and Amicon Ultra 10K filter device (manufactured by Millipore) were used to extract the secreted protein from the medium supernatant. purified from

The Empty-NP, C18Brar-NP and the spike protein prepared in Example 1 were diluted with PBS or suspended in PBS to prepare a predetermined concentration. C18Brar-oil-in-water (C18Brar-o/w) was prepared by adding 0.5% Tween-80.5% mineral oil to a PBS solution of C18Brar (94.5% PBS). Mice were administered 10 μg of spike protein alone or 10 μg of spike protein and each sample subcutaneously (base of tail, 100 μl/side). The doses per mouse of Empty-NP, C18Brar-NP and C18Brar-o/w were 7.65 mg, 7.65 mg and 50 μg, respectively. Blood was collected from the cheeks of the mice every week from week 0 (immediately before immunization) after immunization, and serum was collected. Also, the body weight of the mice was measured every week.

At the beginning of week 12, a boost was given by administering PBS containing 10 μg of spike protein again to the same site as the first. After the boost, blood was drawn every other week and serum was collected. Body weight was also measured in the same manner.

The amount of spike protein-specific antibody in serum was measured by ELISA. Using a commercially available monoclonal antibody 1A9 (manufactured by GENETEX) against the spike protein as a standard, the spike protein-specific antibody level in the serum was quantified. Serum was diluted with 10% BSA/PBS from 100-fold to 10000-fold and used for measurement.

Specifically, a 96-well plate (manufactured by Themo Fisher Scientific) whose surface was coated with nickel was coated with a His-tagged spike protein (manufactured by R&D). The diluted serum, HRP-labeled anti-mouse IgG (SouthernBiotech) and TMB coloring kit (Sumitomo Bakelite) were allowed to react in order, and OD450 was measured. The amount of antibody in the serum was quantified using the calibration curve obtained by 1A9.

(result)
Figures 4 (A), (B), (C) and (D) show the body weight of mice administered spike protein alone, Empty-NP, C18Brar-NP and C18Brar-o/w, respectively. FIG. 5 shows the average body weight of mice administered each sample. No body weight loss was observed in any of the samples.

Figures 6 (A), (B), (C) and (D) show the amount of IgG in each mouse administered spike protein alone, Empty-NP, C18Brar-NP and C18Brar-o/w, respectively. FIG. 7(A) shows the average amount of spike protein-specific IgG. After boosting, the amount of IgG in the C18Brar-NP administration group increased significantly. According to FIG. 7(B), which shows the average IgG amount from 0 to 7 weeks shown in FIG. 7(A), the amount of IgG in the C18Brar-NP administration group increased even before the boost.

Various embodiments and modifications of the present invention are possible without departing from the broad spirit and scope of the present invention. Moreover, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2021-108679 filed on June 30, 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-108679 are incorporated herein by reference.

The present invention is suitable for pharmaceuticals, especially immunostimulants that enhance the effects of vaccines.

Reference Signs List

1, 2

processing surface

10, 20 holder 11, 21 processing unit 22 contact surface

pressure applying unit

30, 50 introduction unit 40 fluid pressure applying unit 60 fluid supply unit 70 case 100 thin film rotary disperser

Claims

Compounds of Formula 1

[wherein X a and X b are each independently selected from O or NH;
Y a and Y b are each independently selected from the group comprising —I, —Br, —Cl, —F, —OH, —R 1 and OR 1 , wherein R 1 is alkyl having 1 to 6 carbon atoms, carbon selected from alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms, wherein alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms and alkynyl having 2 to 6 carbon atoms are each —OH or 1 carbon atom; optionally substituted with ~6 alkoxy,
n and m are each independently 0 to 4, and Z a and Z b are each independently selected from R 2 , —OR 2 , —NHR 2 , —NHC(O)—R 2 and SR 2 and R 2 is selected from alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms, and alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and each alkynyl of 5 to 26 may be substituted with -OH or alkoxy having 1 to 6 carbon atoms,
r and s are each independently 1 to 3;
alk a and alk b are each independently selected from alkylene of 1 to 4 carbon atoms, alkenylene of 2 to 4 carbon atoms and alkynylene of 2 to 4 carbon atoms, or alk a and alk b are each absent; the aryl ring may be directly attached to the carbon of C=O,
n+r=1-5 and m+s=1-5. ]
An immunostimulatory agent comprising a biocompatible particle containing
X a and X b are O;
The immunostimulant according to claim 1.
the aryl ring is attached directly to the carbon of C═O in the absence of alk a and alk b respectively;
The immunostimulant according to claim 1.
n and m are 1;
Y a and Y b are —OH;
4. The immunostimulant according to any one of claims 1-3.
Z a and Z b are —OR 2 ,
R 2 is selected from alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and alkynyl having 5 to 26 carbon atoms, and alkyl having 5 to 26 carbon atoms, alkenyl having 5 to 26 carbon atoms and 5 carbon atoms. each alkynyl of ~26 may be substituted with -OH or alkoxy having 1 to 6 carbon atoms,
5. The immunostimulant according to any one of claims 1-4.
Z a and Z b are —OR 2 ,
R 2 is alkyl having 18 carbon atoms,
r and s are 1;
6. The immunostimulant according to any one of claims 1-5.
The biocompatible particles are lactic acid/glycolic acid copolymer particles,
7. The immunostimulant according to any one of claims 1-6.
The 50% diameter of the biocompatible particles is
is 200 nm or less,
8. The immunostimulant according to any one of claims 1-7.
used in combination with vaccines
9. The immunostimulant according to any one of claims 1-8.
The vaccine is
is a vaccine against SARS-CoV-2,
The immunostimulant according to claim 9.