Mucosal adjuvant

Abstract

The present invention provides a mucosal adjuvant comprising a compound represented by formula (I): A-L-B (In the formula, A represents a structure having TLR7 activity, L represents a linker, and B represents a structure having TLR2 activity.), or a pharmaceutically acceptable salt thereof.

Description

Mucosal adjuvants

[0001] The present invention relates to mucosal adjuvants, compositions containing the same, and novel compounds for use in the same.

[0002] Vaccines are the most effective means of preventing infectious diseases, including influenza. However, when it comes to influenza vaccines, while the current subcutaneously injected vaccine induces IgG antibodies in the blood, which are involved in systemic immunity, it is unable to induce secretory IgA antibodies on the mucosa, which play an important role in mucosal immunity and defense against viral infection. Therefore, the expected effect of current vaccines is not to prevent infection, but to prevent the disease from becoming severe.

[0003] Mucosal immunity is important for host defense against natural viral infections, and the mechanisms of mucosal virus recognition, interferon induction, and mucosal secretory IgA antibody production are becoming increasingly clear. In particular, secretory IgA antibodies secreted in mucosal tissues are known to play a crucial role in defense against respiratory infections, such as influenza. This is because secretory IgA antibodies can neutralize pathogens in the mucosa before they can infect the host. IgA antibodies also possess cross-protective properties, enabling protection against viruses of different subtypes. Influenza vaccines are developed based on the predicted circulating strains of each year. However, current vaccines are significantly less effective, or even ineffective, when the circulating strain differs from the vaccine strain. Given these factors, there is a growing demand for vaccines that can prevent infection by efficiently inducing secretory IgA antibody production through mucosal immune activation in addition to eliciting a systemic immune response via IgG antibodies. Mucosal vaccines, which can efficiently induce IgA antibody production in mucosal tissues, are gaining attention. However, even if the same components as those used in subcutaneous vaccines are administered mucosally, mucosal immunity is hardly induced, and secretory IgA antibodies are hardly induced. Therefore, in addition to conventional vaccine components, substances that induce mucosal immunity, i.e., adjuvants for vaccines that induce mucosal immunity, are needed, and their development is desired.

[0004] Known examples of subcutaneously administered vaccine adjuvants include those containing Toll-like receptor (TLR) agonists, and the following reports have been published. Patent documents 1 and 2 describe the synthesis of compounds that are covalent conjugates of TLR7 and / or TLR8 with a TLR2 agonist and their use in inducing innate immune responses. Examples demonstrate the antitumor effects of the compounds in mouse models. However, the examples of use of the compounds in Patent documents 1 and 2 involve administration into subcutaneously inoculated tumors, and do not teach administration to mucosal membranes. Furthermore, there is no description of mucosal IgA antibody production. The CL553 described in Patent document 1 is used in its racemic form.

[0005] Non-Patent Document 1 describes that a dual ligand for TLR2 and TLR7 induces humoral and cellular immune responses. Experiments have shown that administering a dual ligand for TLR2 and TLR7 together with a protein antigen to mice resulted in an increase in serum IgG antibody titers. However, the example of use of the compound in Non-Patent Document 1 is subcutaneous administration, and no teaching is given about mucosal administration. Furthermore, it is stated that subcutaneous administration does not induce mucosal IgA antibody production. Incidentally, PamadiFectin (registered trademark) described in Non-Patent Document 1 is used in its racemic form.

[0006] EP2769738EP2732825

[0007] J Immunol 2017;198:4205. A Dual TLR2 and TLR7 Ligand Induces Highly Potent Humoral and Cell-Mediated Immune Responses

[0008] In view of the above circumstances, an objective of the present invention is to provide a mucosal adjuvant capable of inducing IgA antibodies in the mucosa, a composition containing the adjuvant, and a novel compound used in the adjuvant.

[0009] The present inventors have conducted extensive research in search of a substance that can be effective as a mucosal adjuvant, and as a result have discovered a specific compound that can act as a mucosal adjuvant and induce IgA antibodies in the mucosa, thereby completing the present invention.

[0010] That is, the gist of the present invention is as follows. [1] A mucosal adjuvant comprising a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof: ALB (I) wherein A represents a structure having TLR7 activity, L represents a linker, and B represents a structure having TLR2 activity. Another embodiment of the present invention includes a mucosal adjuvant preparation containing a compound represented by formula (I) or a pharmacologically acceptable salt thereof. Another embodiment of the present invention includes use of a compound represented by formula (I) or a pharmacologically acceptable salt thereof in the production of a mucosal adjuvant preparation. Another embodiment of the present invention includes a compound represented by formula (I) or a pharmacologically acceptable salt thereof used as a mucosal adjuvant preparation. [2] The adjuvant according to [1], wherein L has a structure represented by the following formula (L-1):

[0011]

[0012] In the formula, m represents an integer of 0 to 4, n represents an integer of 0 to 4, o represents an integer of 0 to 4, p represents an integer of 1 to 6, and * represents a bond to -NH-. [3] The adjuvant according to [1] or [2], wherein the compound represented by formula (I) is compound TY001 represented by the following formula:

[0013]

[0014] [4] The adjuvant according to [3], wherein TY001 is a racemate, S-form, or R-form. [5] A vaccine composition comprising at least one antigen and the adjuvant according to any one of [1] to [4]. Another embodiment of the present invention includes use of at least one antigen and the adjuvant according to any one of [1] to [4] in the production of a vaccine composition. Another embodiment of the present invention includes at least one antigen used in a vaccine composition and the adjuvant according to any one of [1] to [4]. Another embodiment of the present invention includes a method for enhancing immunity in a subject, comprising administering to the subject at least one antigen and the adjuvant according to any one of [1] to [4]. [6] The composition according to [5], further comprising chondroitin sulfate and / or a salt thereof. [7] The composition according to [5] or [6], which is a liquid, spray, semisolid, or solid formulation, wherein the semisolid and solid formulations dissolve in body fluids and / or body temperature. [8] The composition according to any one of [5] to [7], which is for inducing humoral immunity. [9] The composition according to any one of [5] to [8], which is for inducing IgA antibody production in the mucosa.

[10] The composition according to [9], which is for further inducing IgG antibody production in the blood.

[11] The composition according to any one of [5] to

[10] , which is a vaccine for infectious diseases.

[12] The composition according to

[11] , wherein the antigen of the infectious disease vaccine is derived from a pathogenic virus, pathogenic bacteria, pathogenic fungus, or parasite.

[13] The composition according to

[12] , wherein the antigen of the infectious disease vaccine is derived from an influenza virus or a coronavirus.

[14] The composition according to any one of [5] to

[10] , which is a vaccine for non-infectious diseases.

[15] The composition according to

[14] , wherein the antigen of the non-infectious disease vaccine is an antigen derived from amyloid beta, alpha-synucletin, prion, cholesterol ester transfer protein, ApoB100, oxidized LDL, angiotensin I / II, glatiramer acetate, myelin basic protein, a T cell receptor for MBP-specific T cells, insulin, GAD, a T cell receptor for acetylcholine receptor-specific T cells, an allergen, IL-5, a cancer antigen, a neoantigen, a toxic substance, TNFα, HCG, GnRH, Ghrelin, or TRANCE / RANKL.

[16] A compound represented by the following formula, or a pharmaceutically acceptable salt thereof:

[0015]

[0016]

[17] A compound represented by the following formula, or a pharmaceutically acceptable salt thereof:

[0017]

[0018] The present invention provides a mucosal adjuvant capable of inducing IgA antibodies in the mucosa, a composition containing the adjuvant, and a novel compound used in the adjuvant.

[0019] Figure 1 shows the virus infectivity titer in nasal washes when TY001 was used as a mucosal adjuvant (Example 1). Figure 2 shows the virus infectivity titer in nasal washes (2 2 3 shows the amount of antigen-specific IgA antibody in serum (2 dilutions) when TY001 was used as a mucosal adjuvant (Example 1). 12 Figure 4 shows the amount of antigen-specific IgA antibody in nasal washes and the amount of antigen-specific IgG antibody in plasma when CL401, CL413, or CL531 was used as a mucosal adjuvant (Example 2). Figure 5 shows the amount of antigen-specific IgA antibody in nasal washes (×2 dilution) when TY001 S-body was used as a mucosal adjuvant. 3 6 shows the amount of antigen-specific IgA antibody in serum (×2 dilution) when TY001 S-body was used as a mucosal adjuvant (Example 3). 8Fig. 7 is a graph showing the amount of antigen-specific IgG antibody in nasal washes and serum when the TY001 S-body was used as a mucosal adjuvant (Example 4). Fig. 8 is a graph showing the virus infectivity titer in nasal washes when the TY001 S-body was used as a mucosal adjuvant (Example 5). Fig. 9 is a graph showing the amount of antigen-specific IgA antibody in nasal washes and serum when the TY001 S-body was used as a mucosal adjuvant (Example 5). Fig. 10 is a graph showing the amount of antigen-specific IgA antibody in nasal washes and bronchoalveolar lavage fluid, and the amount of antigen-specific IgG antibody in serum and bronchoalveolar lavage fluid when the TY001 S-body was used as a mucosal adjuvant (Example 6).

[0020] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In this specification, when a numerical range is expressed as "lower limit to upper limit," the upper limit may be "equal to or less than" or "less than," and the lower limit may be "equal to or greater than" or "more than."

[0021] <Definitions> As used herein, the terms "vaccine" and "vaccine composition" are used interchangeably and refer to a composition comprising an antigen and an adjuvant that generates an immune response when administered to the body of an animal, such as a human. A vaccine may be a pharmaceutical product for preventing or treating an infectious or non-infectious disease in an animal, such as a human. A vaccine for preventing an infectious or non-infectious disease in an animal, such as a human, is sometimes called a "prophylactic vaccine," and a vaccine for treating an infectious or non-infectious disease in an animal, such as a human, is sometimes called a "therapeutic vaccine."

[0022] As used herein, an "antigen" (also referred to as an antigenic substance or immunogen) refers to a substance contained in a vaccine that, when administered to an animal, such as a human, elicits an immune response (this property is referred to as "having antigenicity"). In this specification, unless interpreted as such, the term "antigen" includes not only a substance or a portion thereof that is the direct target of an immune response, but also a nucleic acid that is transcribed / translated in vivo to produce a substance or a portion thereof that is the target of an immune response. Antigens may be either natural or artificial (e.g., those artificially produced by techniques such as genetic recombination), and may be a microorganism (e.g., a pathogenic virus, a pathogenic bacterium, a pathogenic fungus, a parasite, etc.) or a cell itself or a portion thereof. For example, antigens may be antigenic proteins or portions thereof, peptides, polysaccharides, nucleic acids that express antigenic substances in vivo (typically mRNA or DNA containing a base sequence encoding a gene that expresses an antigenic protein or a portion thereof), other biological substances or portions thereof, or modified versions thereof. When the antigen is a microorganism, an infectious microorganism (such as a live vaccine or an attenuated vaccine) or a microorganism that has lost its infectious ability (such as an inactivated vaccine) is used.

[0023] When targeting infectious diseases, the antigen is typically an antigen derived from a pathogenic virus, pathogenic bacterium, pathogenic fungus, or parasite (e.g., a pathogenic virus, bacterium, pathogenic fungus, or parasite that is transmitted mucosally), and when the vaccine of the present invention contains such an antigen, it is used as a vaccine for infectious diseases. Alternatively, when targeting non-infectious diseases, the antigen is typically an antigen other than that derived from a virus, bacterium, fungus, or parasite, such as a substance such as a disease-related protein, lipid, or allergen, and when the vaccine of the present invention contains such an antigen, it is used as a vaccine for non-infectious diseases.

[0024] As used herein, an antigen derived from a certain substance or bacterium / virus / pathogenic fungus / parasite may be the substance or bacterium / virus / pathogenic fungus / parasite itself, an antigenic portion of the substance or bacterium / virus / pathogenic fungus / parasite, or a substance containing the portion. Here, the substance or bacterium / virus / pathogenic fungus / parasite itself and the antigenic portion of the substance or bacterium / virus / pathogenic fungus / parasite may have a natural structure or may be artificially modified. For example, they may contain a non-natural structure or be artificially modified to accommodate multiple virus variants or to enhance antigenicity.

[0025] Examples of pathogenic bacteria include, but are not limited to, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholerae, Corynebacterium diphtheriae, and the like.

[0026] Examples of pathogenic viruses include, but are not limited to, chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, respiratory syncytial virus, coronavirus, human immunodeficiency virus (HIV), human papillomavirus, rubella virus, and hepatitis virus. Of these, influenza virus, coronavirus, or human immunodeficiency virus is preferred, and influenza virus or coronavirus is most preferred.

[0027] For example, preferred influenza virus antigens are molecules present on the particle surface, such as hemagglutinin (HA: HA1, HA2), neuraminidase (NA), matrix (M1, M2), nonstructural (NS), polymerase (PB1, PB2: basic polymerase 1 and 2, acidic polymerase (PA)), and nucleoprotein (NP). Currently, 16 types of HA and 9 types of NA are known, and any of these can be used as antigens in the vaccines of the present invention. Additionally, composite antigens of HA and NA, universal antigens, etc. can also be preferably used.

[0028] Coronavirus antigens include, for example, spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins, as well as proteins encoded by ORF1a, ORF1b, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10. For specific sequences, see, for example, the genome sequence of SARS coronavirus 2 (accession number LC522975). Coronaviruses include any virus belonging to the Coronaviridae family, preferably severe acute respiratory syndrome (SARS) coronavirus or Middle East respiratory syndrome (MERS) coronavirus, and more preferably SARS coronavirus, particularly SARS coronavirus type 1 (SARS-CoV-1) and SARS coronavirus type 2 (SARS-CoV-2), and coronaviruses derived therefrom.

[0029] When the target disease is a non-infectious disease, the antigen can be appropriately selected from substances whose expression is involved in the onset or exacerbation of the target disease, and examples include, but are not limited to, antigens derived from amyloid beta, alpha-synucletin, prion, cholesterol ester transfer protein, ApoB100, oxidized LDL, angiotensin I / II, glatiramer acetate, myelin basic protein, T cell receptors of MBP-specific T cells, insulin, GAD, T cell receptors of acetylcholine receptor-specific T cells, allergens, IL-5, cancer antigens, neoantigens, toxic substances, TNFα, HCG, GnRH, Ghrelin, or TRANCE / RANKL.

[0030] As used herein, the term "inactivated antigen" refers to an antigen used as an antigen that retains antigenicity but has lost its infectivity, and includes, but is not limited to, virions (complete virus particles), incomplete virus particles, virion structural particles, viral structural proteins or nonstructural proteins, proteins or glycoproteins derived from microorganisms, protective antigens, and substances containing parts of microorganisms that serve as epitopes for neutralization reactions. As used herein, a vaccine containing an inactivated antigen as an antigen is referred to as an "inactivated vaccine." Examples of inactivated antigens that can be used include, but are not limited to, those obtained by inactivating microorganisms by physical (e.g., X-ray irradiation, heat, ultrasound) or chemical (formalin, mercury, alcohol, chlorine) manipulations.

[0031] Antigens can be produced by physically and / or chemically treating and then purifying naturally occurring microorganisms such as pathogenic viruses, pathogenic bacteria, pathogenic fungi, or parasites, by artificial synthesis, by producing them in microorganisms or cells using genetic recombination technology, or by synthesis in a cell-free system. These methods can be carried out using well-known conventional techniques and commercially available equipment, reagents, vectors, etc.

[0032] When the antigen is a nucleic acid that expresses a protein or a portion thereof that is the target of an immune response, the form is not limited and may be DNA or RNA. The nucleotides or nucleosides that make up the nucleic acid may be modified in whole or in part as needed. For example, nudeuridine or 2-thiouridine may be used instead of uridine, 6-methyladenosine or inosine may be used instead of adenosine, 5-methylcytidine may be used instead of cytidine, or a nucleoside in which the 2' hydroxyl group is substituted with a methoxy group (2'-O-nucleoside) may be used. Furthermore, the nucleic acid may have the ability to replicate and may be self-replicating (e.g., self-replicating mRNA) or trans-replicating (e.g., trans-replicating mRNA). The nucleic acid may be a desired vector, such as naked nucleic acid, a plasmid, a viral vector, or a non-viral vector (e.g., liposome). In the case of DNA, DNA containing a nucleic acid encoding a protein or a portion thereof that is the target of an immune response under the control of any promoter that is active in the host may be used. Examples of such promoters that can be used include, but are not limited to, CMV promoter, CAG promoter, SV40 promoter, RSV promoter, EF1α promoter, and SRα promoter.

[0033] In the case of antigens derived from pathogenic viruses, pathogenic bacteria, pathogenic fungi, or parasites, from the viewpoint of safety, it is desirable that the antigen be an inactivated antigen or a nucleic acid containing a base sequence encoding a gene of interest that expresses the antigen.

[0034] As used herein, "mucosal administration" refers to an administration form via a mucosa. As used herein, "mucosa" refers to the inner wall of an externally connected hollow organ, such as the digestive, respiratory, or urogenital organs, in a vertebrate. Therefore, such mucosal administration includes, but is not limited to, nasal administration (intranasal administration), oral administration, intravaginal administration, upper respiratory tract administration, and alveolar administration (pulmonary administration). Nasal administration or oral administration is preferred. This is because the nasal cavity is also a route of infection for respiratory infectious diseases such as influenza virus and coronavirus, and thus provides protection at the early stage of infection.

[0035] As used herein, the term "adjuvant" refers to a substance that activates immunity, and includes substances used to increase or generate an immune response to an administered antigen. The term "mucosal adjuvant" refers to a substance that activates local mucosal and / or systemic immunity when administered mucosally. Preferred mucosal adjuvants are substances that enhance local mucosal IgA production and / or increase the amount of IgG in the blood.

[0036] <Mucosal adjuvant and vaccine> One aspect of the present invention relates to a mucosal adjuvant (hereinafter, sometimes referred to as "the adjuvant of the present invention") comprising a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof: ALB (I) wherein A represents a structure having TLR7 agonist activity, L represents a linker, and B represents a structure having TLR2 agonist activity.

[0037] The present inventors have discovered that a dual agonist of TLR2 and TLR7, i.e., the compound represented by formula (I), is an excellent mucosal adjuvant capable of inducing secretory IgA antibodies on the mucosa. Based on this finding, they have discovered that a vaccine containing the compound represented by formula (I) or a pharmacologically acceptable salt thereof as a mucosal adjuvant is excellent at inducing immunity against a desired antigen. The present invention was thus completed. The adjuvant of the present invention, when administered mucosally together with an antigen, can induce IgA antibodies against the antigen in the mucosa, and preferably can also induce IgG antibodies against the antigen in the blood. One aspect of the present invention is an adjuvant for inducing mucosal immunity. Another aspect of the present invention is an adjuvant for mucosal administration.

[0038] In the above formula (I), A is not limited as long as it has a structure that exhibits TLR7 agonist activity. A may be, but is not limited to, a structure derived from a purine base (e.g., adenine, guanine, etc.), an analog thereof, or a derivative thereof; a pyrimidine base (e.g., cytosine, thymine, uracil, etc.), an analog thereof, or a derivative thereof; or an imidazoquinoline, an analog thereof, or a derivative thereof. A may also include structures derived from multiple compounds. Furthermore, A may be a structure included in specific examples of the compound represented by the general formula (I) described below. Derivatives include, but are not limited to, nucleosides (e.g., guanosine), structures having a substituent on the purine ring of a purine base, the pyrimidine ring of a pyrimidine base, or the imidazoquinoline ring of an imidazoquinoline. Substituents include, but are not limited to, hydroxy, amino, alkylamino having 1 to 6 carbon atoms, alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, and acyl having 1 to 6 carbon atoms. Any carbon atom contained in the alkylamino, alkyl, alkoxy, or acyl may be substituted with one or more heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom. The alkylamino, alkyl, alkoxy, or acyl may be substituted with hydroxy, amino, or the like. Preferred substituents include hydroxy, amino, alkylamino having 1 to 4 carbon atoms which may be substituted with a heteroatom, and alkyl having 1 to 4 carbon atoms which may be substituted with a heteroatom. A may further have a substituent within a range that does not affect the effects of the present invention. B is not limited as long as it has a structure that has TLR2 agonist activity. B is not limited to, and may be, for example, a structure derived from a lipopeptide (such as a monoacylated lipopeptide such as PamCys (palmitoyl-S-glycerylcysteine) or a diacylated lipopeptide such as Pam2Cys (dipalmitoyl-S-glycerylcysteine) or a triacylated lipopeptide such as Pam3Cys (tripalmitoyl-S-glycerylcysteine)) or a derivative thereof (such as PamCSK4, Pam2CSK4, or Pam3CSK4). B may also include structures derived from multiple compounds.Furthermore, B may have a structure included in specific examples of compounds represented by general formula (I) described below. B may have a substituent that does not affect the effects of the present invention. Specific examples of the substituent are the same as those described in A. L represents a linker that connects A and B and is not limited as long as it achieves the effects of the present invention. The linker connects A and B and may be absent if the structure derived from A and / or B can sufficiently ensure the distance between A and B required to achieve the effects of the present invention. The linker generally includes any structure required for bonding A and B. L may have a substituent that does not affect the effects of the present invention. Specific examples of the substituent are the same as those described in A. The compound represented by formula (I) used as an adjuvant of the present invention may include structures other than A, B, and L in its structure that do not affect the effects of the present invention.

[0039] Specific examples of the compound represented by the above general formula (I) include the compounds listed below, but the present invention is not limited thereto: (20R)-20-amino-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-1,4,19-trioxo-22-thia-2,5,9,14,18-pentaazapentacosane-24,25-diyl dipalmitate; (20R)-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-1,4,19-trioxo-20-palmitamido-22-thia-2,5,9,14,18-pentaazapentacosane-24,25-diyl dipalmitate; (6R,9S)-6,23-diamino-9-((4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl) methyl)benzoyloxy)methyl)-7,10-dioxo-4-thia-8,11,15,20-tetraazatricosane-1,2-diyl dipalmitate; (6R,13S)-6,27-diamino-13-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl) methyl)benzamido)-7,14-dioxo-4-thia-8,15,19,24-tetraazaheptacosane-1,2-diyl dipalmitate; (6R,13S)-6,27-diamino-13-(2-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) benzamido)acetamido)-7,14-dioxo-4-thia-8,15,19,24-tetraazaheptacosane-1,2-diyl dipalmitate;(6R,9S)-6,23-diamino-9-(4-(2-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) benzamido)acetamido)butyl)-7,10-dioxo-4-thia-8,11,15,20-tetraazatricosane-1,2-diyl dipalmitate; (6R,9S)-6,25-diamino-9-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl) methyl)phenyl carbamoyl)-7,12-dioxo-4-thia-8,13,17,22-tetraazapentacosane-1,2-diyl dipalmitate; (6S,9S,12S,15S,18S,21R)-6,25-diamino-21-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylcarbamoyl)-12,15,18-tris(4-aminobutyl)-9-(hydroxy methyl)-7,10, 13,16,19-pentaoxo-4-thia-8,11,14,17,20-pentaazapentacosane-1,2-diyl dipalmitate; (2S,5S,8S,11S,14S,17R)-17-amino-5-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin -9-yl)methyl)benzamido)butyl)-2,8,11-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13, 16,24-hexaoxo-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-pentaazanona triacontan-1-oic acid;(2S,5S,8S,11S,14S,17R)-17-amino-5-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin -9-yl)methyl)benzamido)butyl)-2,8,11-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13, 16,24-hexaoxo-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-pentaazanona triacontan-1-oic acid; (2S,5S,8S,11S,14S,17R)-17-amino-11-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)benzamido)butyl)-2,5,8-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13,16,24-hexaoxo-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-pentaazanona triacontan-1-oic acid; ((6R,9S,12S,15S,18S,21S)-25-amino-21-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylcarbamoyl)-12,15,18-tris(4-aminobutyl)-9-(hydroxymethyl)-7,10,13,16,19-pentaoxo-6-palmitamido-4-thia-8,11,14,17,20-pentaazapentacosane-1,2-diyl dipalmitate;(2S,5S,8S,11S,14S,17R)-5-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) benzamido)butyl)-2,8,11-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13, 16,24-hexaoxo-17-palmitamido-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-penta azanonatriacontan-1-oic acid; (2S,5S,8S,11S,14S,17R)-8-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) benzamido)butyl)-2,5,11-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13, 16,24-hexaoxo-17-palmitamido-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-penta azanonatriacontan-1-oic acid; (2S,5S,8S,11S,14S,17R)-11-(4-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl) methyl)benzamido)butyl)-2,5,8-tris(4-aminobutyl)-14-(hydroxymethyl)-4,7,10,13,16,24-hexaoxo-17-palmitamido-21-(palmitoyloxy)-23-oxa-19-thia-3,6,9,12,15-penta azanonatria contan-1-oic acid;(S)-methyl 1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-22-((R)-2-amino-3-(2-(tetradecanoyloxy)ethylthio)propanamido)-1,4,19-trioxo-2,5,9,14,18-pentaazatricosan-23-oate. (R)-3-(2-amino-3-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl amino)-3-oxopropylthio)propane-1,2-diyl dipalmitate; (R)-3-(2-amino-3-(3-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)propyl amino)-3-oxopropylthio)propane-1,2-diyl dipalmitate; (R)-3-(2-amino-3-(3-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)propylamino)-3-oxopropylthio) propane-1,2-diyl dipalmitate; (R)-3-(2-amino-3-(3-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)propyl amino)-3-oxopropylthio)propane-1,2-diyl dipalmitate; (R)-3-(2-amino-3-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)phenyl amino)-3-oxopropylthio)propane-1,2-diyl dipalmitate;3-((R)-2-amino-3-((S)-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) phenylamino)-3-hydroxy-1-oxopropan-2-ylamino)-3-oxopropylthio)propane-1,2-diyl dipalmitate; (R)-3-(3-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-oxo-2-palmitamidopropylthio)propane-1,2-diyl dipalmitate; (R)-2-(2-amino-3-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-oxopropylthio)ethyl tetradecanoate; (R)-2-(2-amino-3-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-oxopropylthio)ethyl palmitate; (R)-2-(2-amino-3-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-oxopropylthio)ethyl stearate; 2-((S)-2-amino-3-((S)-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-hydroxy-1-oxopropan-2-ylamino)-3-oxopropylthio)ethyl tetradecanoate;2-((S)-2-amino-3-((S)-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-hydroxy-1-oxopropan-2-ylamino)-3-oxopropylthio)ethyl palmitate; 2-((S)-2-amino-3-((S)-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-3-hydroxy-1-oxopropan-2-ylamino)-3-oxopropylthio)ethyl stearate; 2-((R)-2-amino-3-((S)-5-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenylamino)-1-methoxy-1,5-dioxopentan-2-ylamino)-3-oxopropylthio)ethyl tetradecanoate; (R)-2-(2-amino-3-(3-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)propyl amino)-3-oxopropylthio)ethyl stearate; (R)-2-(2-amino-3-(3-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)propylamino)-3-oxopropylthio)ethyl stearate.;

[0040] Examples of one embodiment of A include, but are not limited to, structures derived from N1-glycinyl[4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)benzoyl]spermine (CL307; InvivoGen) (which contains a spermine structure, and this structure can also serve as the linker (L)), 2-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)benzamido)acetic acid (CL264; InvivoGen), imiquimod (CAS: 99011-02-6), R-848 (CAS: 144875-48-9), etc.

[0041] Examples of one embodiment of B include, but are not limited to, S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 CF3COOH (Pam2CSK4; InvivoGen), N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine (Pam3CSK4; InvivoGen), and structures derived from PamCSK4, Pam2Cys, Pam3Cys, PamCys, etc.

[0042] An embodiment of L is not limited to, but includes, for example, a cationic linker, etc. Furthermore, a specific embodiment of L includes, for example, a structure represented by the following formula (L-1):

[0043]

[0044] In the formula, m represents an integer of 0 to 4, n represents an integer of 0 to 4, o represents an integer of 0 to 4, p represents an integer of 1 to 6, and * represents a bond to -NH-.

[0045] Examples of L include structures derived from spermine (m=3, n=4, o=3, p=1), putrescine (m=0, n=4, o=0, p=1), and spermidine (m=0, n=4, o=1, p=3), with spermine being preferred, but the present invention is not limited thereto. Note that the compound represented by formula (I) used as an adjuvant of the present invention may have a structure preferably used as L, such as a structure derived from spermine, not as a linker linking A and B, but as a part of A or B, or as a moiety having no activity other than A and B.

[0046] A preferred embodiment of the compound represented by general formula (I) is the compound represented by the following formula: (20R)-20-amino-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-1,4,19-trioxo-22-thia-2,5,9,14,18-pentaazapentacosane-24,25-diyl dipalmitate. This compound is designated "TY001."

[0047]

[0048] Furthermore, the R-isomer of TY001 ((20R,24R)-20-amino-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-1,4,19-trioxo-22-thia-2,5,9,14,18-pentaazapentacosane-24,25-diyl dipalmitate) and the S-isomer ((20R,24S)-20-amino-1-(4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)phenyl)-1,4,19-trioxo-22-thia-2,5,9,14,18-pentaazapentacosane-24,25-diyl dipalmitate) are novel compounds and are the compounds of the present invention. That is, a further aspect of the present invention relates to compounds represented by the following formula or a pharmacologically acceptable salt thereof (hereinafter, these may be collectively referred to as "compounds of the present invention").

[0049]

[0050]

[0051] As used herein, the term "pharmacologically acceptable salt" refers to a salt formed by combining with an inorganic or organic base or acid, which is acceptable for administration to the body as a pharmaceutical. Such salts are described, for example, in Berge et al., J. Pharm. Sci. 66:1-19 (1977). When the compound of formula (I) has an acidic group such as a carboxylic acid group, salts with the acidic group include alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, and calcium; salts with amines such as ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, and L-glucamine; or salts with basic amino acids such as lysine, δ-hydroxylysine, and arginine. When the compound of formula (I) has a basic group such as an amino group, examples of salts with the basic group include salts with hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, boric acid, etc. (inorganic acid salts); salts with methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, formic acid, propionate, acetic acid, trifluoroacetic acid, lactic acid, fumaric acid, malic acid, oxalic acid, benzoic acid, mandelic acid, cinnamic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, tosylic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid, etc. (organic acid salts); and salts with acidic amino acids such as aspartic acid and glutamic acid. These salts can be prepared by conventional means. Note that the above examples should not be used to limit the interpretation of "pharmacologically acceptable salt." In other words, "pharmacologically acceptable salt" should be interpreted broadly and is a term that includes various salts. The compounds herein also include hydrates or solvates of the compounds or salts thereof, unless expressly indicated otherwise, unless clearly inappropriate.

[0052] The adjuvant formulations and vaccines of the present invention may contain one type of adjuvant of the present invention, or may contain a combination of two or more types of adjuvants of the present invention. Furthermore, the adjuvant formulations and vaccines of the present invention may contain adjuvant components other than the adjuvant of the present invention.

[0053] The adjuvant of the present invention may be a commercially available compound if it is available, or may be produced or purified by a known production or purification method, for example, based on the method described in the Examples below.

[0054] Induction of mucosal immunity using the adjuvant of the present invention (or administration of the adjuvant of the present invention) is usually carried out by administering to a subject simultaneously with the antigen as a vaccine composition containing the adjuvant of the present invention together with the antigen. However, if necessary, the adjuvant of the present invention may be administered separately from the antigen. For example, the adjuvant of the present invention or a formulation containing the adjuvant (not including the antigen; sometimes referred to herein as the "adjuvant formulation of the present invention") may be administered mucosally at a timing that allows immune induction before or after antigen administration. In this case, the adjuvant and the antigen may be administered via different administration routes. When the adjuvant and the antibody are administered via different administration routes, examples of the administration route for the antigen include conventional administration methods such as intradermal administration and intramuscular administration. The adjuvant formulation of the present invention can also be administered for purposes such as activating natural immunity and suppressing allergies. The adjuvants of the present invention can be formulated as vaccine compositions containing an antigen or as compositions not containing the antigen (adjuvant formulations), typically in the form of liquids (suspensions, solutions, etc.), sprays (suspensions, solutions, etc.), powders (semi-solid preparations, solid preparations, etc.), solids, semi-solids (pastes, creams), films, sheets, gels (preferably those that dissolve in body fluids and / or body temperature), etc. The adjuvant formulations and vaccine compositions of the present invention are administered, for example, by dropping, spraying, spraying, applying, or pasting onto mucous membranes such as the nasal or oral cavity.

[0055] Examples of liquids and sprays (solutions) include those in which an adjuvant and / or antigen is dissolved in purified water, a buffer solution, etc. Examples of liquids and sprays (suspensions) include those in which an adjuvant and / or antigen is suspended in purified water, a buffer solution, etc. together with methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone (PVP), gelatin, casein, etc. Solids and semi-solids include those in which an adjuvant and / or antigen is mixed with a water / oil-based base such as hydrophilic ointment or vanishing cream; or an oil / water-based base such as hydrophilic petrolatum, purified lanolin, aquaphor, eucerin, neoserin, hydrous lanolin, cold cream, or hydrophilic plastibase, which is stirred in an oil-based solvent or water. Examples of powders include those in which an adjuvant and / or antigen is thoroughly mixed with methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, etc. Films and sheets include molded articles having a laminated structure including a mucoadhesive layer containing a water-soluble matrix material (e.g., pullulan, polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), gelatin, starch, etc.). Commonly used absorption enhancers, surfactants, preservatives, stabilizers, moisture-proofing agents, humectants, solubilizers, etc. can be added to these preparations as needed. Known formulation methods can be used to produce the vaccine compositions and adjuvant preparations of the present invention.

[0056] The vaccine composition and adjuvant formulation of the present invention preferably further contain chondroitin sulfate and / or a salt thereof. This can further enhance the antibody-inducing effect. As the chondroitin sulfate salt, alkali metal salts of chondroitin sulfate are preferred, with sodium chondroitin sulfate being more preferred. The amount of chondroitin sulfate and / or a salt thereof added can be adjusted appropriately depending on the type of antigen, etc., but is preferably contained in an adjuvant formulation containing a compound represented by formula (I) or a pharmacologically acceptable salt thereof at about 0.001 to 10% by mass, about 0.008 to 3% by mass, or about 0.01 to 1% by mass. Furthermore, the amount of chondroitin sulfate and / or a salt thereof added is preferably contained in a vaccine formulation containing a compound represented by formula (I) or a pharmacologically acceptable salt thereof and an antigen at about 0.001 to 10% by mass, about 0.008 to 3% by mass, or about 0.01 to 1% by mass.

[0057] The content of the compound of formula (I) or a pharmacologically acceptable salt thereof in the vaccine and adjuvant formulations of the present invention is not particularly limited as long as it is an amount sufficient to exhibit the immune-inducing effect of the present invention, and the content of the compound of formula (I) or a pharmacologically acceptable salt thereof relative to the total formulation may be 0.00001 to 100% by mass, 0.0001 to 90% by mass, 0.001 to 50% by mass, 0.01 to 10% by mass, or 0.1 to 2.5% by mass. Such a content varies depending on the type of adjuvant, etc., but can be routinely determined by one of ordinary skill in the art.

[0058] The dose of the adjuvant of the present invention can be appropriately determined depending on the antigen, the type of adjuvant, the subject of administration, the administration method, the administration form, etc., but for example, taking an adult weighing 60 kg as an example, in the case of parenteral administration, the daily dose may be about 0.0001 mg to about 5 g, about 0.001 to about 500 mg, about 0.01 to about 50 mg, or about 0.1 to about 5 mg, in terms of the amount of the compound represented by formula (I) or a pharmacologically acceptable salt thereof. When administered to other animals, the above dose is converted to a dose per unit body weight, and then multiplied by the body weight of the subject animal to obtain the dose.

[0059] A further aspect of the present invention relates to a vaccine composition (sometimes referred to herein as the "vaccine composition of the present invention" or the "vaccine of the present invention") comprising at least one antigen and the adjuvant of the present invention. The vaccine of the present invention is typically a mucosal vaccine that can induce IgA antibodies against the antigen contained in the vaccine in the mucosa by mucosal administration. One aspect of the present invention is a vaccine for inducing mucosal immunity. Another aspect of the present invention is a vaccine for mucosal administration.

[0060] The content of the antigen in the vaccine composition of the present invention is not particularly limited as long as it is an amount sufficient to exhibit the immune-inducing effect of the present invention. The content of the antigen relative to the total composition may be 0.00001 to 70% by mass, 0.0001 to 60% by mass, 0.001 to 50% by mass, 0.01 to 30% by mass, 0.05 to 10% by mass, 0.1 to 5% by mass, or 1 to 2.5% by mass. Such a content varies depending on the type of antigen, etc., but can be routinely determined by one of ordinary skill in the art. The mass ratio of antigen to adjuvant contained in the vaccine composition of the present invention is not limited, but is recommended to be 500:1 to 1:50.

[0061] The dose of the vaccine composition of the present invention can be determined appropriately depending on the type of antigen and adjuvant, the recipient, the administration method, the administration form, etc. For example, taking an adult weighing 60 kg as an example, the daily dose may be about 0.0001 to about 1000 mg, about 0.001 to about 500 mg, about 0.01 to about 100 mg, about 0.1 to about 50 mg, or about 1 to about 10 mg of antigen. When administering to other animals, the above dose is converted to a dose per unit body weight and then multiplied by the body weight of the recipient animal to obtain the dose.

[0062] The timing of administration is not particularly limited as long as a therapeutic or preventive effect can be achieved, but in the case of a preventive vaccine, it is preferable to administer it at least two weeks before the time of expected infection.

[0063] The subjects of administration can be appropriately determined depending on the type of antigen contained in the vaccine composition, and include, for example, humans and mammals other than humans, such as mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, dogs, cats, monkeys, orangutans, and chimpanzees.

[0064] Diseases targeted by infectious disease vaccines include infectious diseases caused by pathogenic viruses, pathogenic bacteria, pathogenic fungi, or parasites from which the antigens contained in the vaccines are derived. Diseases and conditions targeted by non-infectious disease vaccines include, but are not limited to, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, arteriosclerosis, hypertension, multiple sclerosis, type 1 diabetes, myasthenia gravis, allergies such as hay fever, bronchial asthma, cancer, addiction to nicotine, cocaine, phencyclidine, methamphetamine, heroin, morphine, and the like, rheumatoid arthritis, contraception, obesity, osteoporosis, and the like.

[0065] Furthermore, as shown in the Examples below, the adjuvant formulation and vaccine composition of the present invention can induce humoral immunity and therefore can be used for inducing humoral immunity. Furthermore, as shown in the Examples below, the adjuvant formulation and vaccine composition of the present invention can induce IgA antibody production in the mucosa and therefore can be used for inducing IgA antibody production in the mucosa. Furthermore, as shown in the Examples below, the adjuvant formulation and vaccine composition of the present invention can induce IgG antibody in the blood in addition to IgA antibody production in the mucosa and therefore can be used for inducing IgG antibody production in the blood.

[0066] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as it does not depart from the gist of the invention.

[0067] <Synthesis of Compounds> Compounds were synthesized as follows.

[0068] (General Procedure) １H NMR spectra were recorded on a Bruker AVANCE III (300 MHz). Chemical shifts are expressed in parts per million (ppm) downfield from tetramethylsilane (δ) as an internal standard for deuterated solvents, and coupling constants (J) are expressed in hertz (Hz). Data are reported as follows: chemical shift, integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, brs = broadened singlet), and coupling constant. All solvents and reagents were commercially available. The optical purity of the compounds was determined by analysis on a UFLC (Shimadzu Corporation).

[0069]

[0070] (Step 1) A mixture of 2-chloroadenine (10.0 g, 59 mmol), butylamine (43 g, 589 mmol), and water (60 mL) was reacted in an autoclave at 180°C for 10 hours. After cooling, the reaction mixture was concentrated under reduced pressure, and water was poured into the residue to precipitate a solid. The resulting precipitate was collected by filtration and washed with water and isopropyl ether (IPE) to obtain Intermediate 1 (10.2 g, 49.5 mmol, 83.9%) as a green solid.

[0071]

[0072] (Step 2) Intermediate 1 (3.50 g, 17.0 mmol) and cesium carbonate (Cs ２ CO ３ To a suspension of 4-(bromomethyl)benzoic acid methyl ester (5.05 g, 22.1 mmol) (6.12 g, 18.7 mmol) in N,N-dimethylformamide (DMF) (70 mL) was added methyl 4-(bromomethyl)benzoate (5.05 g, 22.1 mmol) (6.12 g, 18.7 mmol) at room temperature, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured into ice water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with water and brine, and then cooled to room temperature. ４), and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 30% to 100% ethyl acetate (EtOAc) in n-hexane, followed by 7% methanol (MeOH) in ethyl acetate (EtOAc)) to give Intermediate 2 (3.40 g, 9.59 mmol, 56.5%) as off-white crystals.

[0073]

[0074] (Step 3) A suspension of Intermediate 2 (5.00 g, 14.1 mmol) in N,N-dimethylformamide (DMF) (50 mL) was added with dibromine (Br ２ ) (0.94 mL, 18.3 mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 2 hours. ２ S ２ SO ３ ) aqueous solution (10 mL), water (100 mL) and triethylamine (Et ３ N) (10 mL) was added and the resulting mixture was stirred at room temperature for 1 h. The resulting precipitate was collected by filtration and washed with water to give intermediate 3 (5.10 g, 11.8 mmol, 83.4%) as a yellow solid.

[0075]

[0076] (Step 4) To a suspension of intermediate 3 (2.50 g, 5.77 mmol) in methanol (MeOH) (25 mL), 6N aqueous sodium hydroxide (NaOH) solution (14.4 mL) was added, and the resulting mixture was refluxed overnight. After cooling, 12N aqueous hydrochloric acid (HCl) solution (30.1 g) was added to the mixture, and the resulting mixture was stirred at 90° C. for 3 days. After cooling, the pH was adjusted to 5 with 2N aqueous sodium hydroxide (NaOH) solution, causing precipitation of a solid. The resulting precipitate was collected by filtration, washed with water, and dried under reduced pressure to obtain intermediate 4 (1.90 g, 5.33 mmol, 92.4%) as a pale yellow solid.

[0077]

[0078] (Step 5) To a suspension of intermediate 4 (720 mg, 2.02 mmol) in dry N,N-dimethylformamide (DMF) (20 mL), compound 7 (1.32 g, 2.22 mmol), PyBOP® (tripyrrolidinophosphonium hexafluorophosphate) (1.58 g, 3.03 mmol), and N-methylmorpholine (1.1 g, 10.1 mmol) were added, and the resulting mixture was stirred at room temperature overnight. The mixture was poured into water and extracted with ethyl acetate (EtOAc) and tetrahydrofuran (THF). The organic layer was separated and saturated sodium bicarbonate (NaHCO ３ ) aqueous solution, 0.1N hydrochloric acid (HCl) aqueous solution, water, and brine, and ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 0% to 25% methanol (MeOH) in ethyl acetate (EtOAc)) to give Intermediate 10 (1.50 g, 1.61 mmol, 79.7%) as a pale yellow amorphous solid.

[0079]

[0080] (Step 6) A mixture of intermediate 10 (1.50 g, 1.61 mmol) and 10% palladium on carbon (Pd / C) (PE type) (514 mg) in methanol (MeOH) (30 mL) was hydrogenated under balloon pressure at room temperature for 6 hours. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo to give intermediate 11 (1.20 g, 1.50 mol, 93.4%) as a white amorphous solid.

[0081]

[0082] (Step 7) To a solution of compound 1 (5.00 g, 24.7 mmol) in N,N-dimethylformamide (DMF) (50 mL) was added benzylphenyl carbonate (10.2 mL, 51.9 mmol) at room temperature, and the resulting mixture was stirred at room temperature overnight. 1N aqueous hydrochloric acid (HCl) solution (55 mL) was added to the mixture, and the resulting mixture was stirred at room temperature for 1 hour. The resulting precipitate was collected by filtration and washed with ethyl acetate (EtOAc) to give compound 2 (8.60 g, 16.0 mmol, 64.0%) as white crystals.

[0083]

[0084] (Step 8) Compound 2 (8.60 g, 15.8 mmol) and sodium bicarbonate (NaHCO ３ To a solution of di-tert-butyl dicarbonate (Boc) (1.33 g, 15.8 mmol) in water (27 mL) was added di-tert-butyl dicarbonate (Boc) at room temperature. ２ HCl (11 mL, 47.5 mmol) and tetrahydrofuran (THF) (43 mL) were added, and the resulting mixture was stirred at room temperature for 2 hours. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 10% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 3 (10.6 g, 15.8 mmol, quantitative) as a colorless oil.

[0085]

[0086] (Step 9) A mixture of compound 3 (1.70 g, 2.53 mmol) and 10% palladium on carbon (Pd / C) (PE type) (539 mg) in ethanol (EtOH) (35 mL) was hydrogenated under balloon pressure at room temperature for 2 hours. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo to give compound 4 (1.02 g, 2.53 mmol, quantitative) as a colorless oil.

[0087]

[0088] (Step 10) To a solution of compound 4 (1.02 g, 2.53 mmol) in ethanol (EtOH) (20 mL) was added benzylphenyl carbonate (0.50 mL, 2.53 mmol) at 0° C., and the resulting mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo, and the residue was purified by column chromatography (NH silica gel, eluted with 50% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 5 (0.60 g, 1.10 mmol, 44.0%) as a colorless oil.

[0089]

[0090] (Step 11) A solution of (((9H-fluoren-9-yl)methoxy)carbonyl)glycine (499 mg, 1.68 mmol) in DMF (10 mL) was heated at room temperature with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC.HCl) (321 mg, 1.68 mmol), triethylamine (EtN) (0.39 mL, 2.79 mmol), 1-hydroxybenzotriazole monohydrate (HOBt.H ２ Compound 5 (600 mg, 1.12 mmol) was added, and the resulting mixture was stirred at room temperature overnight. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with 0.1 N aqueous hydrochloric acid (HCl) and brine, and then washed with magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 50% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 6 (710 mg, 0.87 mmol, 77.8%) as a colorless amorphous solid.

[0091]

[0092] (Step 12) To a solution of compound 6 (710 mg, 0.87 mmol) in N,N-dimethylformamide (DMF) (7 mL) was added piperidine (0.86 mL, 8.70 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into a 1N aqueous solution of sodium hydroxide (NaOH) and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with water and brine, and then extracted with magnesium sulfate (MgSO ４ ) and concentrated in vacuo to give compound 7.

[0093]

[0094] (Step 13) L-cystine bis(t-butyl ester) dihydrochloride ((H-Cys-OtBu) ２To a suspension of (compound 8) (3.00 g, 7.05 mmol) and 9-fluorenylmethylsuccinimidyl carbonate (3.81 g, 11.3 mmol) in tetrahydrofuran (THF) (30 mL) was added 4-methylmorpholine (3.1 mL, 28.2 mmol) at 0°C, and the resulting mixture was stirred at 0°C for 3 hours and then at room temperature overnight. After evaporation of the solvent, the residue was dissolved in ethyl acetate (EtOAc) and washed with 0.1 N aqueous hydrochloric acid (HCl), water, and brine. The organic layer was separated and magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 10% to 40% ethyl acetate (EtOAc) in n-hexane) to give compound 9 (3.50 g, 4.39 mmol, 62.3%) as white crystals.

[0095]

[0096] (Step 14: R-isomer) To a solution of compound 9 (1.25 g, 1.57 mmol) in chlorobenzene (10 mL) and tetrahydrofuran (THF) (20 mL) was added zinc powder (718 mg, 11.0 mmol) and freshly prepared methanol (MeOH), concentrated hydrochloric acid (c. HCl), and sulfuric acid (H ２ SO ４ ) (100:7:1, 4 mL) was added at 0°C, and the resulting mixture was stirred at 0°C for 30 minutes. To the mixture was added (R)-oxiran-2-ylmethanol (697 mg, 9.41 mmol). The resulting mixture was stirred at 40°C for 2 hours. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and then washed with magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 20% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 10a (1.21 g, 2.56 mmol, 81.5%) as a colorless amorphous solid.

[0097]

[0098] (Step 14: S-isomer) To a solution of compound 9 (1.25 g, 1.57 mmol) in chlorobenzene (10 mL) and tetrahydrofuran (THF) (20 mL) was added zinc powder (718 mg, 11.0 mmol) and freshly prepared methanol (MeOH), concentrated hydrochloric acid (c. HCl), and sulfuric acid (H ２ SO ４ ) (100:7:1, 4 mL) was added at 0°C, and the resulting mixture was stirred at 0°C for 30 minutes. To the mixture was added (S)-oxiran-2-ylmethanol (697 mg, 9.41 mmol). The resulting mixture was stirred at 40°C for 2 hours. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and then cooled to 100°C. The resulting mixture was stirred at 40°C for 2 hours. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and cooled to 100°C. The resulting mixture was stirred at 40°C for 2 hours. The resulting mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with brine, and then ... resulting mixture was washed with magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 20% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 10b (1.20 g, 2.53 mmol, 84.1%) as a colorless amorphous solid.

[0099]

[0100] (Step 15: R-isomer) To a solution of compound 10a (1.00 g, 2.11 mmol) and palmitic acid (1.62 g, 6.33 mmol) in tetrahydrofuran (THF) (20 mL) was added N,N'-diisopropylcarbodiimide (DIC) (0.98 mL, 6.33 mmol) and 4-dimethylaminopyridine (DMAP) (103 mg, 0.85 mmol) at room temperature, and the resulting mixture was stirred at room temperature overnight. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with 10% aqueous citric acid and brine, and then extracted with magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 0% to 30% ethyl acetate (EtOAc) in n-hexane) to give compound 11a (2.01 g, 2.11 mmol, quantitative) as a white solid.

[0101]

[0102] (Step 15: S-form) To a solution of compound 10b (1.00 g, 2.11 mmol) and palmitic acid (1.62 g, 6.33 mmol) in tetrahydrofuran (THF) (20 mL) was added N,N'-diisopropylcarbodiimide (DIC) (0.98 mL, 6.33 mmol) and 4-dimethylaminopyridine (DMAP) (103 mg, 0.85 mmol) at room temperature, and the resulting mixture was stirred at room temperature overnight. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, washed with 10% aqueous citric acid and brine, and then extracted with magnesium sulfate (MgSO ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluted with 0% to 30% ethyl acetate (EtOAc) in n-hexane) to give compound 11b (2.01 g, 2.11 mmol, quantitative) as a white solid.

[0103]

[0104] (Step 16: R-configuration) A mixture of compound 11a (2.00 g, 2.10 mmol) and trifluoroacetic acid (TFA) (16.2 mL, 210 mmol) was stirred at room temperature for 1 hour. After evaporation of trifluoroacetic acid, the residue was purified by column chromatography (silica gel, eluted with 10% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 12a (1.60 g, 1.79 mol, 85.0%) as a white solid.

[0105]

[0106] (Step 16: S-configuration) A mixture of compound 11b (2.01 g, 2.11 mmol) and trifluoroacetic acid (TFA) (16.2 mL, 211 mmol) was stirred at room temperature for 1 hour. After evaporation of trifluoroacetic acid (TFA), the residue was purified by column chromatography (silica gel, eluted with 10% to 100% ethyl acetate (EtOAc) in n-hexane) to give compound 12b (1.56 g, 1.74 mmol, 82.5%) as a white solid.

[0107]

[0108] (Step 17: R-isomer) To a solution of intermediate 11 (1.34 g, 1.68 mmol) and compound 12a (1.50 g, 1.68 mmol) in N,N-dimethylformamide (DMF) (30 mL) was added PyBOP® (tripyrrolidinophosphonium hexafluorophosphate) (1.75 g, 3.35 mmol) and 2,4,6-trimethylpyridine (0.44 mL, 3.35 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate (EtOAc) and tetrahydrofuran (THF). The organic layer was separated and washed with 0.1 N aqueous hydrochloric acid (HCl), saturated sodium bicarbonate (NaHCO ３ ) aqueous solution and brine, and ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 0% to 20% ethyl acetate (EtOAc) in methanol (MeOH)) to give compound 13a (2.40 g, 143 mol, 85.5%) as a colorless amorphous solid.

[0109]

[0110] (Step 17: S-form) To a solution of intermediate 11 (1.34 g, 1.68 mmol) and compound 12b (1.50 g, 1.68 mmol) in N,N-dimethylformamide (DMF) (30 mL) was added PyBOP® (tripyrrolidinophosphonium hexafluorophosphate) (1.75 g, 3.35 mmol) and 2,4,6-trimethylpyridine (0.44 mL, 3.35 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate (EtOAc) and tetrahydrofuran (THF). The organic layer was separated and washed with 0.1 N aqueous hydrochloric acid (HCl), saturated sodium bicarbonate (NaHCO ３ ) aqueous solution and brine, and ４), and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 0% to 20% ethyl acetate (EtOAc) in methanol (MeOH)) to give compound 13b (2.20 g, 1.31 mol, 78.3%) as a colorless amorphous solid.

[0111]

[0112] (Step 18: R-configuration) Piperidine (1.42 mL, 14.3 mmol) was added to a solution of compound 13a (2.40 g, 1.43 mmol) in N,N-dimethylformamide (DMF) (25 mL) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate (EtOAc) and tetrahydrofuran (THF). The organic layer was separated, washed with water and brine, and then filtered. The resulting mixture was purified by filtration using magnesium sulfate (MgSO ). ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 0% to 25% ethyl acetate (EtOAc) in methanol (MeOH)) to give compound 14a (1.90 g, 1.31 mol, 91.3%) as a colorless amorphous solid.

[0113]

[0114] (Step 18: S-form) Piperidine (1.30 mL, 13.1 mmol) was added to a solution of compound 13b (2.20 g, 1.31 mmol) in N,N-dimethylformamide (DMF) (25 mL) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate (EtOAc) and tetrahydrofuran (THF). The organic layer was separated, washed with water and brine, and then filtered. The resulting mixture was filtered. The resulting mixture was then ... ４ ) and concentrated in vacuo. The residue was purified by column chromatography (silica gel, eluting with 0% to 25% ethyl acetate (EtOAc) in methanol (MeOH)) to give compound 14b (1.70 g, 1.17 mmol, 89.1%) as a colorless amorphous solid.

[0115]

[0116] (Step 19: R-isomer) To a solution of compound 14a (1.90 g, 1.31 mmol) in cyclopentyl methyl ether (CPME) (10 mL) was added 4 N hydrochloric acid (HCl) in cyclopentyl methyl ether (16.4 mL, 65.4 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure. Isopropyl ether (IPE) (50 mL) was added to the residue, and the resulting mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with isopropyl ether (IPE) to give the tetrahydrochloride salt of TY001 (R-isomer) (1.78 g, 1.27 mol, 97.3%) as a pale yellow amorphous solid. The NMR spectrum of the synthesized compound is shown below.

[0117] 1 H NMR (400 MHz, DMSO-d6, 298 K) δ 0.79-0.92 (9H, m), 1.14-1.39 (54H, m), 1.43-1.59 (7H, m), 1.64-1.75 (4H, m), 1.75-1.93 (4H, m), 2.22-2.34 (4H, m), 2.69-2.81 (1H, m), 2.82-3.06 (11H, m), 3.08-3.24 (3H, m), 3.25-3.38 (3H, m), 3.78-3.92 (2H, m), 3.94-4.06 (1H, m), 4.10-4.22 (1H, m), 4.26-4.38 (1H, m), 4.84-4.98 (2H, m), 5.07-5.22 (1H, m), 7.32-7.46 (2H, m), 7.82-7.90 (2H, m), 7.95-8.08 (1H, m), 8.12-8.18 (1H, m), 8.20-8.30 (2H, m), 8.41-8.56 (3H, m), 8.78-8.87 (1H, m), 8.89-9.02 (2H, m), 9 02-9.17 (3H, m), 11.00-11.18 (1H, m).

[0118]

[0119] (Step 19: S-isomer) To a solution of compound 14b (1.70 g, 1.17 mmol) in cyclopentyl methyl ether (CPME) (10 mL) was added 4N hydrochloric acid (HCl) in cyclopentyl methyl ether (14.6 mL, 58.5 mmol) at room temperature, and the resulting mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure. Isopropyl ether (IPE) (50 mL) was added to the residue, and the resulting mixture was stirred at room temperature for 30 minutes. The resulting precipitate was collected by filtration and washed with isopropyl ether (IPE) to give the tetrahydrochloride salt of TY001 (S-isomer) (1.53 g, 1.09 mol, 93.5%) as a pale yellow amorphous solid. The NMR spectrum of the synthesized compound is shown below.

[0120] 1 H NMR (400 MHz, DMSO-d6, 298 K) δ0.80-0.90 (10H, m), 1.15-1.38 (62H, m), 1.42-1.56 (8H, m), 1.61-1.73 (5H, m), 1.73-1.87 (5H, m), 2.25-2.31 (4H, m), 2.70-2.81 (2H, m), 2.81-2.99 (14H, m), 3.11-3.23 (5H, m), 3.23-3.33 (3H, m), 3.80-3.87 (3H, m), 3.89-4.00 (2H, m), 4.10-4.16 (1H, m), 4.26-4.36 (1H, m), 4.85-4.97 (2H, m), 5.07-5.21 (1H, m), 7.34-7.44 (2H, m), 7.79-7.90 (2H, m), 8.06-8.19 (1H, m), 8.32-8.49 (3H, m), 8.72-8.84 (3H, m), 8.85-9.02 (4H, m), 10.52-10.89 (1H, m).

[0121] In the Examples described later, TY001 R-4HCl salt was used as the TY001 R-isomer, and TY001 S-4HCl salt was used as the TY001 S-isomer.

[0122] Example 1: Test to confirm the nasal adjuvant effect of the R- and S-type TY001. Test to test the protective effect of influenza HA antigen vaccine combined with TY001 by nasal administration to mice.

[0123] (Method) For the intranasal administration study, 7-week-old female BALB / c mice (nine to ten mice per group) were used. Influenza virus HA protein antigen (A / Singapore / GP1908 / 2015(IVR-180)(H1N1)pdm09) and influenza virus (A / Singapore / GP1908 / 2015(IVR-180)(H1N1)pdm09) (Research Foundation for Microbial Diseases, Osaka University) were used. Vaccines were divided into groups containing 0.1 μg of HA alone and groups containing 3 μg of TY001 R-isomer and 3 μg of TY001 S-isomer adjuvants. The vaccine solution was administered intranasally twice, 3 weeks apart, in 5 μL increments (10 μL / mouse) through the left and right nostrils. Two weeks after the second administration (day 35), 2 μL of virus solution was administered into each nostril (1 × 10 4 TCID 50 Three days later (day 38), serum and nasal washes were collected. The mice were anesthetized with ketamine (10 mg / mL) and xylazine (1 mg / mL) administered intraperitoneally (0.2 mL / mouse).

[0124] [Method for collecting serum and nasal wash] Three days after influenza virus inoculation, blood was collected under isoflurane anesthesia. The blood was incubated at 37°C for at least one hour and then at 4°C for at least one hour. The supernatant obtained by centrifugation was used as serum. After euthanasia by whole blood collection, 1 mL of MEM medium containing 10% FBS was infused through a catheter inserted into the trachea, and the fluid drained from the nasal opening was collected three times. 1 mL of medium was added to the collected nasal wash, and the entire volume was centrifuged through a 0.22 μm filter (Ultrafree-CL-GV, 0.22 μm, Merck), and the filtrate was collected.

[0125] [Measurement of viral titer TCID 50Median tissue culture infectious dose (50% infectious dose) MDCK cells (CCL-34, ATCC) were seeded in a 96-well microplate (flat bottom) (1 x 10 4 After culturing (37°C, 5% CO2) for 3 days, the cell plates were used when they were nearly confluent. The samples were diluted to 10 mL with MEM medium containing acetyltrypsin at a final concentration of 10 μg / mL. 0.5 A 2-fold dilution series was prepared. After washing the cell plate with PBS, 100 μL of the prepared sample was added per well and cultured at 37°C and 5% CO2 for 3 days. After culture, 10% formalin / PBS was added and the plate was left to stand at room temperature for 10 minutes or more, after which the liquid in the wells was removed, NB staining solution was added, and the plate was left to stand at room temperature for 30 minutes or more. After washing the plate with tap water, it was dried at room temperature, and 50 μL / well of 0.1N NaOH aqueous solution was added and stirred, and the absorbance at 630 nm was measured using a plate reader. The OD of the control cells cultured in MEM medium was 630 The detection limit is the average value of 0.85 × OD 630 <When the detection limit was reached, it was judged as positive. Based on this, the virus titer of the sample was measured by the Reed-Münch method: TCID 50 was calculated.

[0126] [ELISA] HA antigen (A / Singapore / GP1908 / 2015(IVR-180)(H1N1)pdm 09) was diluted with 100 mM carbonate-bicarbonate buffer (pH 9.6) to a concentration of 1 μg / mL and added to a 96-well plate. After overnight incubation at 4°C, the plate was washed three times with PBS containing 0.05% polysorbate 20. 100 μL / well of 1% BSA-PBS was added, incubated at room temperature for 2 hours, and then washed three times with PBS containing 0.05% polysorbate 20. Samples diluted with PBS containing 0.05% polysorbate 20 and 1% BSA (4-fold dilution series) were added to the plate at 50 μL / well and incubated overnight at 4°C. After washing with PBS containing 0.05% polysorbate 20, horseradish peroxidase-conjugated goat anti-mouse IgA antibody (Bethyl) or anti-mouse IgG antibody (Bethyl) diluted in PBS containing 0.05% polysorbate 20 and 1% BSA was added and incubated at room temperature for 2 hours. After washing five times with PBS containing 0.05% polysorbate 20, TMB chromogenic substrate solution was added and the plate was left to stand at room temperature for 20 minutes in the dark. The reaction was then stopped by adding 2N aqueous sulfuric acid. The absorbance (450 nm) was immediately measured using a microplate reader.

[0127] [HI antibody titer: Hemagglutination Inhibition Test] Serum samples were mixed with 3x the volume of RDE(II) "Seiken" (Denka Seiken) and incubated at 37°C for 18-20 hours, then heated at 56°C for 30 min. After returning to room temperature, 6x the volume of PBS was added to prepare the test sample. For the test, 50% chicken red blood cells (preserved chicken blood, Japan Bioserum) (1 / 10 the volume of the test sample) were added, left at room temperature for 1 hour, and then centrifuged at 1,200 x g for 5 min to obtain the supernatant. A 2x dilution series was prepared, and A / Singapore / GP1908 / 2015(IVR-180)(H1N1)pdm09 (Denka Seiken) was added as the HI test antigen at 4HA / 25μL and incubated for 60 min at room temperature. The reciprocal of the highest dilution ratio of the sample that completely inhibited agglutination was defined as the HI antibody titer. For HI antibody titers ≦10, the HI antibody titer was set at 5 to calculate the geometric mean.

[0128] (Results) Figure 1 shows the viral titers (mean + standard error) in nasal washes. Nasal administration of the HA antigen vaccine did not provide sufficient protection against viral infection, but the group receiving the HA antigen vaccine plus the TY001 R or S subunit suppressed viral infection. Figure 2 shows the IgA levels (mean + standard error) in nasal washes. Nasal administration of the HA antigen vaccine did not induce antigen-specific IgA antibodies in the nasal cavity, but the group receiving the HA antigen vaccine plus the TY001 R or S subunit induced antigen-specific IgA antibodies in the nasal cavity. Figure 3 shows the serum IgG levels (mean + standard error), and Table 1 shows the serum HI antibody titers (geometric mean antibody titers). Nasal administration of the HA antigen vaccine did not result in an increase in antigen-specific IgG antibodies or HI antibody titers in the blood, but the group receiving the HA antigen vaccine plus the TY001 R or S subunit did.

[0129]

[0130] Example 2: Test to confirm the adjuvant effect of intranasal administration of compounds Test to evaluate the IgA and IgG antibody induction ability of intranasal administration of influenza HA antigen vaccine in combination with CL401, CL413, or CL531

[0131] (Method) For the intranasal administration study, 7-week-old female BALB / c mice (8-13 mice per group) were used. The influenza HA protein antigen (A / California / 7 / 2009(H1N1)pdm) (Research Foundation for Microbial Diseases, Osaka University) was used. The vaccine was administered in groups containing 1 μg of HA alone or 0.662 or 6.62 nmol of CL401, CL413, or CL531 (InvivoGen) as the test compound. Mice were intranasally administered 6.5 μL of the vaccine solution (13 μL / mouse) twice, 2 weeks apart (days 0 and 14), into the left and right nostrils. Two weeks after the booster immunization, plasma (day 27) and nasal washes (day 28) were collected. Antigen-specific IgA antibody levels in the nasal washes and antigen-specific IgG antibody levels in the plasma were measured by ELISA. At the same time, the antibody titer of the measurement sample was expressed as a relative value (unit) using a calibration curve (1.2 - 0.005 unit) obtained by measuring two-fold serial dilutions of mouse nasal wash or plasma stored as an internal standard.

[0132]

[0133] (Results) The results of three experiments conducted for each compound are shown in Figure 4 (top row: IgA amount in nasal washes, bottom row: IgG amount in plasma, mean ± standard error). Nasal administration of the HA antigen vaccine alone did not induce sufficient antigen-specific IgA in nasal washes or antigen and IgG antibodies in plasma. In the group administered in combination with the test compounds, the induction of antigen-specific IgA and antigen-specific IgG antibodies in plasma was observed in the following order: CL413 > CL531 > CL401.

[0134] Example 3: Test to confirm the adjuvant effect of TY001 S-enantiomer administered intranasally with a novel coronavirus antigen. Performance evaluation test in mice of a nasal vaccine containing TY001 S-enantiomer, which uses the novel coronavirus SARS-CoV-2 spike protein as an antigen.

[0135] (Method) For the nasal administration study, six 9-week-old female BALB / c mice were used per group. The vaccine was administered with the novel coronavirus S protein (ACROBiosystems). Two groups received either 3 μg of S protein alone or 0.3 μg of TY001 S-form as an adjuvant. Mice were administered 5 μL of the vaccine solution intranasally (10 μL / mouse) through the left and right nostrils twice (days 0 and 21) at three-week intervals. Serum and nasal washes were collected two weeks after the booster immunization (day 35). Administration was performed under anesthesia with ketamine (10 mg / mL) and xylazine (1 mg / mL) administered intraperitoneally at 0.2 mL / mouse.

[0136] [Method for collecting serum and nasal lavage fluid] Under isoflurane anesthesia, blood was collected from the inferior vena cava into a serum separator tube (Sansho) and mixed by inversion. After leaving the tube at room temperature for 30 minutes, the supernatant obtained by centrifugation was used as serum. After euthanasia by whole blood collection, 200 μL of MEM medium was infused into the trachea, and the fluid discharged from the nasal opening was collected twice. The collected solution was centrifuged at 4°C for 5 minutes at 13,000 × g, and the supernatant was used as nasal lavage fluid.

[0137] [ELISA] A solution containing 1 μg / mL of novel coronavirus S protein trimer prepared in 100 mM carbonate-bicarbonate buffer (pH 9.6) was added to a 96-well plate at 50 μL / well, and the plate was left to stand overnight at 4°C, followed by washing with PBS containing 0.05% polysorbate 20. 100 μL / well of 1% BSA-PBS was added and the plate was left to stand at room temperature for 2 hours, followed by washing with PBS containing 0.05% polysorbate 20. 50 μL / well of samples diluted with PBS containing 0.05% polysorbate 20 and 1% BSA (4-fold dilution series) were added and allowed to react overnight at 4°C. After washing with PBS containing 0.05% polysorbate 20, 50 μL / well of Horseradish Peroxidase-conjugated goat anti-mouse IgA antibody (Bethyl) or anti-mouse IgG antibody (Bethyl) diluted 20,000-fold in PBS containing 0.05% polysorbate 20 and 1% BSA was added and incubated at room temperature for 2 hours. After washing with PBS containing 0.05% polysorbate 20, 50 μL / well of TMB chromogenic substrate solution was added. The plate was then incubated at room temperature for 20 minutes in the dark, and the reaction was stopped by adding 50 μL / well of Stop Solution for TMB Substrate (Abcam). The absorbance (OD450 / 630) was immediately measured using a microplate reader.

[0138] (Results) Figure 5 shows the amount of IgA (mean value + standard error) in nasal wash fluid. Nasal administration of S protein antigen did not result in sufficient induction of antigen-specific IgA antibodies, but the group administered S protein antigen in combination with the TY001 S-body induced antigen-specific IgA antibodies in the nasal cavity. Figure 6 shows the amount of IgG in serum (mean value + standard error). Nasal administration of S protein antigen alone did not result in an increase in antigen-specific IgG antibody titers, but an increase in antigen-specific IgG antibodies was confirmed in the group administered S protein antigen and the TY001 S-body.

[0139] Example 4: Test to confirm the nasal adjuvant effect of the TY001 S-enantiomer <Test to confirm the adjuvant effect of an improved influenza HA antigen vaccine formulation combined with the TY001 S-enantiomer by nasal administration to mice>

[0140] (Method) For the intranasal administration study, 7-week-old female BALB / c mice (groups of eight) were used. The influenza virus HA protein antigen A / Guangdong-Maonan / SWL1536 / 2019 (CNIC-1909) (H1N1) (Research Foundation for Microbial Diseases, Osaka University) was used. The vaccine was administered in groups containing 0.1 μg of HA alone, 0.1 μg of TY001 S-enantiomer as an adjuvant, and either with or without 0.01% sodium chondroitin sulfate. Mice were administered 5 μL of the vaccine solution intranasally (10 μL / mouse) twice, three weeks apart, via the left and right nostrils. Two weeks after the second administration (day 35), nasal washes and serum were collected, and the levels of antigen-specific IgA in the nasal washes and IgG in the serum were measured by ELISA.

[0141] (Results) The amounts of IgA in nasal washes and IgG in serum (mean + standard error) are shown in Figure 7. The combined use of the TY001 S-enantiomer with the HA antigen vaccine induced antigen-specific IgA antibodies in the nasal cavity and IgG antibodies in the serum. Furthermore, the IgA and IgG antibody-inducing effect was even stronger in the group in which 0.01% sodium chondroitin sulfate was added to the vaccine solution combined with the TY001 S-enantiomer.

[0142] Example 5: Test to confirm the nasal adjuvant effect of an improved formulation of TY001 S-enantiomer> <Test for the protective effect on mice from infection by nasal administration of an improved formulation of influenza HA antigen vaccine combined with TY001 S-enantiomer>

[0143] (Method) For the intranasal administration study, 7-week-old female BALB / c mice (n = 9-10 per group) were used. Influenza virus HA protein antigen: A / Guangdong-Maonan / SWL1536 / 2019 (CNIC-1909) (H1N1) and influenza virus: A / Guangdong-Maonan / SWL1536 / 2019 (CNIC-1909) (H1N1) (Research Foundation for Microbial Diseases, Osaka University) were used. The vaccine consisted of 0.1 μg of HA alone or 0.01-0.3 μg of TY001 S-isomer as an adjuvant, with 0.01% sodium chondroitin sulfate as an additive. The vaccine solution was administered intranasally twice, 5 μL each (10 μL / mouse) into the left and right nostrils, at 3-week intervals. As a control, a group was administered 0.1 μg of HA subcutaneously. Two weeks after the second administration (day 35), 2 μL of virus solution was administered into each nostril (1 × 10 5 TCID 50 Three days later (day 38), nasal washes and serum samples were collected. Virus titers and antigen-specific IgA levels in the nasal washes and IgG levels in the serum were measured by ELISA.

[0144] (Results) Figure 8 shows the viral titers in nasal washes (mean + standard error). Nasal and subcutaneous administration of the HA antigen vaccine alone did not provide any protective effect against intranasally inoculated virus. In the group receiving the HA antigen vaccine in combination with the TY001 S-enantiomer, a dose-dependent inhibition of viral infection was observed at 0.01 to 0.3 μg of the TY001 S-enantiomer, while no virus was detected in any individual at 0.1 or 0.3 μg. Figure 9 shows the levels of antigen-specific IgA in nasal washes and serum IgG (mean + standard error). Nasal administration of the HA antigen vaccine alone did not induce intranasal IgA or serum IgG, whereas subcutaneous administration of the HA antigen vaccine only induced serum IgG. In the group receiving the HA antigen vaccine in combination with the TY001 S-enantiomer, a dose-dependent induction of intranasal IgA and serum IgG was observed at 0.01 to 0.3 μg of the TY001 S-enantiomer. These results confirmed that the induction of antigen-specific IgA in the nasal cavity plays an important role in protection against inoculated virus infection.

[0145] Example 6: Test to confirm the nasal adjuvant effect of an improved formulation of the TY001 S-enantiomer> <Comparative test of influenza HA antigen vaccine combined with the TY001 S-enantiomer and various adjuvants>

[0146] (Method) For the intranasal administration study, 8-week-old female BALB / c mice (groups of six) were used. The influenza virus HA protein antigen (A / Guangdong-Maonan / SWL1536 / 2019 (CNIC-1909) (H1N1) (Research Foundation for Microbial Diseases, Osaka University) was used. The vaccine was administered intranasally with 0.1 μg of HA alone or in combination with 1 μg of the following adjuvants: 0.3 μg of TY001 S-enantiomer (containing 0.01% sodium chondroitin sulfate), cholera toxin B subunit (CTB) (Fujifilm Wako Pure Chemical Industries), CpG ODN (ODN 1826) (InvivoGen), and polyinosinic-polycytidylic acid (Poly(I:C)) (InvivoGen). Each vaccine solution was administered intranasally to mice twice, 3 weeks apart, at 5 μL each (10 μL / mouse) through the left and right nostrils. A subcutaneous dose of 0.1 μg HA plus 2 mg Alum was also administered. Two weeks after the second dose (day 35), nasal washes, alveolar lavage fluids, and serum were collected, and the levels of antigen-specific IgA in the nasal washes and alveolar lavage fluid, and IgG in the serum and alveolar lavage fluid were measured by ELISA.

[0147] (Results) Figure 10 shows the amounts of antigen-specific IgA in nasal washes and bronchoalveolar lavage fluid, and IgG in serum and bronchoalveolar lavage fluid (mean + standard error). The combination of HA antigen vaccine and TY001 S-enantiomer showed stronger antibody induction ability at a lower dose than either adjuvant. Furthermore, IgA and IgG antibodies were induced in bronchoalveolar lavage fluid, confirming its ability as a mucosal adjuvant.

[0148] As described above, the effect of the dual agonist of TLR2 and TLR7 as a mucosal adjuvant was demonstrated.

[0149] The present invention can be used as a mucosal adjuvant.

Claims

1. A mucosal adjuvant for inducing IgA antibody production in mucosa, comprising the compound shown in formula (I) below, or a pharmacologically acceptable salt thereof: A-L-B (I) During the ceremony, Structure A exhibits TLR7 agonist activity, L indicates the linker. B represents Pam2Cys (dipalmitoyl-S-glycerylcysteine).

2. The adjuvant according to claim 1, wherein L has the structure shown by the following formula (L-1): 【Chemistry 1】 During the ceremony, m represents an integer from 0 to 4. n represents an integer from 0 to 4. o represents an integer from 0 to 4. p represents an integer from 1 to 6. * indicates binding to -NH-.

3. The adjuvant according to claim 1, wherein the compound represented by formula (I) is the compound TY001 represented by the following formula. 【Chemistry 2】

4. The adjuvant according to claim 3, wherein TY001 is a racemic, S-isomer, or R-isomer.

5. A mucosal vaccine composition for inducing the production of IgA antibodies in mucosa, comprising at least one antigen and an adjuvant according to any one of claims 1 to 4.

6. The vaccine composition according to claim 5, further comprising chondroitin sulfate and / or a salt thereof.

7. The composition according to claim 5, wherein the composition is a liquid, a spray, a semi-solid preparation, or a solid preparation, the semi-solid preparation and the solid preparation being dissolved by body fluids and / or body temperature.

8. (delete)

9. (delete)

10. Furthermore, the composition according to claim 5 for inducing the production of IgG antibodies in the blood.

11. The composition according to claim 5, which is a vaccine for infectious diseases.

12. The composition according to claim 11, wherein the antigen of the infectious disease vaccine is an antigen derived from a pathogenic virus, pathogenic bacteria, pathogenic fungus, or parasite.

13. The composition according to claim 12, wherein the antigen of the infectious disease vaccine is an antigen derived from an influenza virus or a coronavirus.

14. The composition according to claim 5, which is a non-infectious vaccine.

15. The composition according to claim 14, wherein the antigen of the non-infectious vaccine is an antigen derived from amyloid β, α-synucletin, prion, cholesterol ester transfer protein, ApoB100, oxidized LDL, angiotensin I / II, glatiramer acetate, myelin basic protein, MBP-specific T cell T cell receptor, insulin, GAD, acetylcholine receptor-specific T cell T cell receptor, allergen, IL-5, cancer antigen, neoantigen, toxic substance, TNFα, hCG, GnRH, Ghrelin, or TRANCE / RANKL.

16. The compound represented by the following formula, or a pharmacologically acceptable salt thereof. 【Transformation 3】

17. The compound represented by the following formula, or a pharmacologically acceptable salt thereof. 【Chemistry 4】

18. The adjuvant according to claim 2, wherein L is a structure derived from spermine, putrescine, or spermidine.

19. The adjuvant according to claim 18, wherein L is a structure derived from spermine.

20. The adjuvant according to claim 2, wherein A is [4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)benzoyl]glycinyl.

21. The adjuvant according to claim 1, wherein the compound represented by formula (I) is a compound selected from TY001, CL413, and CL531, represented by the following formula. 【Transformation 5】 【Transformation 6】

22. L is the adjuvant according to claim 1, wherein L has one of the following structures: 【Transformation 7】

23. The vaccine composition according to claim 6, comprising 0.008 to 3% by mass of chondroitin sulfate and / or a salt thereof.

Images