Adjuvant and composition thereof

Abstract

To provide the use of a CBR1 agonist as an adjuvant and an adjuvant composition.SOLUTION: A compound represented by formula (1): Wherein R represents a cyclopropyl group or a 2-chloroethyl group. The antibody induction-promoting agent contains a compound represented by general formula (I) as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of cannabinoid receptor type 1 agonists as adjuvants and compositions thereof. [Background technology]

[0002] Cannabis, derived from the cannabis plant and commonly known as marijuana, has long been used to treat pain, anxiety, and loss of appetite. This effect is due to the active compounds contained in cannabis called cannabinoids. In the 1960s, the main active compound, Δ 9 Tetrahydrocannabinol (THC) and cannabidiol, which do not exhibit psychotic symptoms such as euphoria, were discovered. In the 1990s, two types of G protein-coupled receptors were identified as the biological target molecules of THC and named cannabinoid receptor type 1 (CBR1) and type 2 (CBR2). Furthermore, the endogenous ligands for these receptors, anandamide in 1992 and 2-arachidonoylglycerol in 1995, were discovered, and cannabinoid receptors are now thought to play important physiological roles.

[0003] These cannabinoid receptors are widely distributed in the central nervous system, cardiovascular system, digestive system, liver, muscle, bone, and spleen / immune system. CBR1, in particular, is known to be expressed primarily in the central nervous system, including the brain, while CBR2 is primarily expressed in the spleen / immune system. Therefore, CBR1 suppresses neurotransmitter release upon stimulation by agonists, eliciting various pharmacological activities such as analgesia, orthostatic hypotension, and euphoria, while CBR2 has been reported to be involved in the differentiation and regulation of inflammatory cells and immune cells. Because cannabinoid receptors have such diverse physiological activities, they are a target for drug development.

[0004] For example, CBR1 agonists approved in Europe and the United States from the 1990s to the early 2000s include dronabinol, a capsule containing the natural agonist THC dissolved in sesame oil, which is used as an appetite stimulant for AIDS patients and as an antiemetic for cancer chemotherapy; nabilone, a synthetic derivative of THC, which is used as an antiemetic for cancer chemotherapy; and nabiximols, an oral spray containing THC as its main ingredient, which is used to treat spasticity and pain in multiple sclerosis and cancer pain. It has also been reported that cannabinoid agonists can be used as agents for promoting the induction of cellular immunity (Patent Document 1). However, this technology inhibits differentiation into Th2 cells, thereby indirectly promoting Th1 cell differentiation and inducing cellular immunity, and cannabinoid agonists are disclosed as substances that suppress differentiation into Th2 cells, which are involved in humoral immunity.

[0005] In general, vaccines sometimes require adjuvants to improve immunogenicity. In particular, inactivated vaccines that have lost their infectivity and subunit vaccines such as recombinant proteins have low immunogenicity, and adjuvants are sometimes used in combination to enhance the immune response to a level that can protect against infection. Vaccines distributed in Japan also use aluminum gel, monophosphoryl lipid (MPL), and AS01B, a mixed adjuvant that combines MPL and QS-21 with liposomes, as adjuvants. However, it is not known at all whether CBR1 can be used as an adjuvant to induce humoral immunity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6473292 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to the use of cannabinoid receptor type 1 (CBR1) agonists as adjuvants and provides adjuvant compositions. [Means for solving the problem]

[0008] The present inventors have conducted extensive research into substances that enhance vaccine efficacy and have found that a specific arachidonic acid derivative, a CBR1 agonist, has an immunostimulatory effect that enhances antibody induction, and that its combined use with a vaccine can enhance the strength of vaccine antigen-specific immunity. This is completely unexpected given previous reports that use cannabinoid receptor agonists as Th2 cell differentiation inhibitors (see Patent Document 1).

[0009] That is, the present invention relates to the following 1) to 4). 1) An antibody induction enhancer containing a compound represented by the following formula (1) as an active ingredient: 2) An adjuvant composition comprising a compound represented by the following formula (1) and a pharmaceutically acceptable carrier: 3) A vaccine composition comprising a compound represented by the following formula (1), a pharmaceutically acceptable carrier, and a vaccine antigen: 4) The vaccine composition of 3), wherein the vaccine antigen is an influenza virus.

[0010] [ka] [wherein R represents a cyclopropyl group or a 2-chloroethyl group.] [Effects of the Invention]

[0011] According to the present invention, it is possible to enhance the effectiveness of vaccines by using CBR1 agonists that can be used as pharmaceuticals, and this can make a significant contribution to the pharmaceutical industry by creating high-quality prophylactic drugs. [Brief explanation of the drawings]

[0012] [Figure 1]IgG antibody titer against the A / Victoria / 1 / 2020 strain (A / H1N1) on Day 21 after a single dose. [Figure 2] IgG antibody titer against B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) on Day 21 after a single dose. [Figure 3] IgG antibody titer against the A / Victoria / 1 / 2020 strain (A / H1N1) on Day 42 after two doses. [Figure 4] IgG antibody titer against B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) on Day 42 after two doses. [Figure 5] IgG subclass antibody titers against the A / Victoria / 1 / 2020 strain (A / H1N1) on Day 42 after two doses. [Figure 6] IgG subclass antibody titer against B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) on Day 42 after two doses. DETAILED DESCRIPTION OF THE INVENTION

[0013] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0014] Among the compounds of the present invention represented by formula (1) (also referred to as "compounds of the present invention"), a compound in which R is a cyclopropyl group is arachidonyl cyclopropylamide (ACPA), and a compound in which R is a 2-chloroethyl group is arachidonyl-2-chloroethylamide (ACEA). Both are known as synthetic agonists of the cannabinoid receptor type 1 (CBR1). CBR1 agonists bind to CBR1 and transmit the signal into cells, thereby exerting physiological activities mediated by CBR1 activation.

[0015] The compounds of the present invention may be in the form of pharmaceutically acceptable salts, and examples of such salts include alkali metal salts such as sodium salts and potassium salts. Furthermore, the compounds of the present invention may exist not only in unsolvated forms but also as hydrates or solvates. Therefore, all crystalline forms and hydrates or solvates thereof are encompassed by the compounds of the present invention. The compounds of the present invention can be produced by known chemical synthesis methods, or can be purchased as commercially available products.

[0016] The compounds of the present invention can be used either alone or in combination in the antibody inducibility enhancer, adjuvant composition or vaccine composition of the present invention.

[0017] As shown in the Examples below, when mice are subcutaneously injected with a vaccine composition prepared by adding the compound of the present invention to an influenza virus split vaccine distributed in Japan, the titer of IgG specific to the antigen is significantly higher than that of a group administered with the vaccine alone. Furthermore, when comparing the geometric mean antibody titers (GMTs) of IgG subclasses, the addition of these CBR1 agonists increases the antibody titer of IgG2a, a Th1-restricted subclass, in addition to IgG1. That is, when an immunogen (antigen) is administered, the compound of the present invention serves as an antibody induction enhancer that increases the strength of immunity specific to the antigen, and the compound can be used as an adjuvant. Furthermore, a composition containing the compound of the present invention and a pharmaceutically acceptable carrier can be used as an adjuvant composition. Furthermore, the compound of the present invention can be used to prepare an antibody induction enhancer or an adjuvant composition. Here, the promotion of antibody inducibility means the effect of increasing the strength of antigen-specific immunity, that is, humoral immunity, specifically the effect of enhancing antigen-specific IgG antibody titer.

[0018] Such an antibody inducibility enhancer can be a pharmaceutical material or preparation for humans or animals that enhances the antibody inducibility of an antigen, and the adjuvant composition can be a pharmaceutical preparation for humans or animals that has adjuvant activity to enhance the antibody inducibility of an antigen, i.e., to enhance the strength of humoral immunity. Such pharmaceutical formulations can be prepared by appropriately combining the compound of the present invention with a pharmaceutically acceptable carrier, such as an excipient, binder, filler, disintegrant, lubricant, dispersant, buffer, flavoring agent, diluent, etc.

[0019] The antibody inducibility enhancer and adjuvant composition of the present invention can be administered in combination with an antigen, and administration may be simultaneous with, before, or after administration of the antigen. The dose of the adjuvant composition of the present invention can be appropriately determined depending on the subject, administration method, administration form, and type of antigen.

[0020] The antibody induction enhancer and adjuvant composition of the present invention can be combined with an antigen to form a vaccine composition for humans or animals, and the above-mentioned pharmaceutically acceptable carrier can be added as needed to form an appropriate formulation. In the vaccine composition of the present invention, the compound of the present invention may be chemically bound to an antigen or other component, or may exist in a free molecular state.

[0021] Antigens include natural products purified from pathogens, or proteins or peptides artificially produced by techniques such as genetic recombination. Specific examples include complete virus particles (virions), incomplete virus particles, viral structural proteins, viral nonstructural proteins, whole pathogen cells, pathogen-derived proteins, glycoproteins, capsular polysaccharides, protective antigens, and neutralizing epitopes. These antigens include both infectious and non-infectious antigens (inactivated antigens). Inactivated antigens include, but are not limited to, antigens inactivated by physical (X-ray irradiation, UV irradiation, heat, ultrasound) or chemical (formalin, beta-propiolactone, binary ethyleneimine, mercury, alcohol, chlorine) procedures. From the perspective of safety, pathogen-derived antigens are preferably inactivated antigens derived from the viruses or pathogens listed above.

[0022] Examples of viruses include measles virus, rubella virus, mumps virus, poliovirus, rotavirus, norovirus, adenovirus, enterovirus, herpes virus, varicella virus, severe acute respiratory syndrome (SARS) virus, novel coronavirus (SARS-CoV-2), human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV-1), human papillomavirus, Japanese encephalitis virus, West Nile virus, yellow fever virus, dengue virus, Zika virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, respiratory syncytial virus, and influenza virus, with influenza virus being preferred.

[0023] Pathogenic bacteria include Corynebacterium diphtheriae, Corynebacterium tetani, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, Vibrio cholerae, Mycobacterium tuberculosis, and periodontal disease bacteria.

[0024] Examples of dosage forms of the vaccine composition include liquids, suspensions, freeze-dried preparations, etc. Liquids include those dissolved in purified water or buffer solutions, etc. Suspensions include those suspended in purified water or buffer solutions together with methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, gelatin, casein, etc. These preparations may contain commonly used absorption enhancers, surfactants, preservatives, stabilizers, moisture-proofing agents, solubilizing agents, etc., as needed.

[0025] The vaccine composition of the present invention may contain adjuvant substances other than the compounds of the present invention, as long as they do not impair the immunogenicity and safety of the vaccine. Examples of such adjuvant substances include aluminum salts, squalene emulsion, 3-O-deacylated-4'-monophosphoryl lipid A (MPL), CpG-ODN, and QS-21, which are used in already approved vaccines.

[0026] The amount of antigen contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to induce a specific antibody response, and can be appropriately determined taking into account the ratio with the adjuvant composition used in combination. For example, when an influenza virus split antigen is used as the antigen, the content should be within the range of 1 to 60 μg HA (equivalent to HA), which is the single dose administered, with 9 to 15 μg HA (equivalent to HA) being more preferable. The above concentration is a value obtained by measuring the concentration of HA protein using a test method established by WHO or national standards, such as a single radial immunodiffusion test.

[0027] Furthermore, the content of the compound of the present invention in the vaccine composition can be adjusted appropriately taking into consideration the antibody titer, but it may be contained within the range of, for example, 150 to 1600 μg, which is the single administration dose, and 300 to 800 μg is more preferable.

[0028] The route of administration of the vaccine composition of the present invention is not particularly limited, and may be any of injection (subcutaneous administration, intramuscular administration, intradermal administration, intravenous administration), oral administration, and parenteral administration (e.g., nasal administration, ocular administration, vaginal administration, sublingual administration, transdermal administration), and may be administered, for example, by dropping, spraying, or instilling into the nasal cavity.

[0029] The adjuvant composition or vaccine composition of the present invention can be administered to humans and mammals other than humans, preferably humans, including mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, horses, goats, sheep, dogs, cats, rhesus monkeys, cynomolgus monkeys, orangutans, and chimpanzees. [Example]

[0030] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0031] <Test Compound> In the following examples, the test compounds used are shown below. ACPA Chemical name:Arachidonylcyclopropylamide Sold by:abcam ACEA Chemical name:Arachidonyl-2-chloroethylamide Distributor: Merck Ltd.

[0032] Example 1 Evaluation of the adjuvant activity of CBR1 ligand The CBR1 ligand ACEA was dissolved in dimethyl sulfoxide (DMSO) at 30 mg / mL, and a commercially available 10 mg / mL ethanol solution of the CBR1 ligand ACPA was used as the adjuvant solution. The antigen solution was a standardized stock solution of influenza HA vaccines for the A / H1N1 (A / Victoria / 1 / 2020 strain) and B / Yamagata lineage (B / Phuket / 3073 / 2013 strain). The adjuvant solution, antigen solution, and phosphate-buffered saline containing 0.1% polysorbate 80 were mixed to prepare a 0.3 mL solution containing 0.4 mg of each CBR1 ligand and 1 μg of HA of each strain. This was used as the test substance. An adjuvant-free test substance was also prepared by adding phosphate-buffered saline containing 0.1% polysorbate 80 instead of the adjuvant solution, and this was used as a control for immunogenicity testing in mice.

[0033] Influenza vaccines containing each CBR1 ligand and a control vaccine were administered subcutaneously to the dorsal skin of BALB / c mice (female, 4 weeks old) at 0.3 mL doses twice, 3 weeks apart (8 mice per group). Partial blood samples were collected 3 weeks after the first dose, and whole blood samples were collected 3 weeks after the second dose. Serum was prepared by centrifugation, and the IgG (total IgG) titers specific for the A / Victoria / 1 / 2020 and B / Phuket / 3073 / 2013 strains were measured. IgG subclass titers specific for each influenza antigen were also measured.

[0034] IgG titers after a single administration of the A / Victoria / 1 / 2020 strain (A / H1N1) are shown in Figure 1. Compared to the control group (non-adjuvanted), the group administered the vaccine containing ACPA and ACEA showed a tendency for IgG titers to be higher, with a significant increase observed in the group administered the vaccine containing ACEA (p<0.01, Mann-Whitney U test). Furthermore, as shown in Figure 2, IgG titers after a single administration of the B / Phuket / 3073 / 2013 strain (B / Yamagata lineage) showed a tendency for IgG titers to be higher in the group administered the vaccine containing ACPA and ACEA, similar to A / H1N1. Again, similar to A / H1N1, ACEA showed significantly higher IgG titers than the control (p<0.05, Mann-Whitney U test).

[0035] The IgG titers after two doses are shown in Figures 3 and 4. As with the single dose, the groups administered the vaccine containing ACPA or ACEA showed higher IgG titers against all strains compared to the control group without adjuvant. Similarly, ACEA showed significantly higher antibody titers against all strains than the group without adjuvant (p<0.01, Mann-Whitney U test). Therefore, adding ACPA or ACEA to the vaccine is thought to enhance the antibody-inducing ability of the vaccine, and these CBR1 ligands are thought to have adjuvant activity.

[0036] The results for each IgG subclass specific to influenza antigens are shown in Figure 5 (A / Victoria / 1 / 2020 strain (A / H1N1)) and Figure 6 (B / Phuket / 3073 / 2013 strain (B / Yamagata lineage)). As shown in Figures 5 and 6, adding ACEA to the vaccine not only increased the induction of IgG1, which is the primary IgG subclass, but also increased the induction of IgG2a, suggesting that these CBR1 ligands enhance the induction of various IgG subclasses. Since Th1 induction is considered important for protection against influenza virus infection, increasing the induction of IgG2a, a Th1-restricted subclass, is expected to improve the vaccine's protective effect against infection. Therefore, adding these CBR1 ligands to the vaccine will increase the induction of both Th1- and Th2-restricted IgG subclasses, potentially leading to the creation of highly effective vaccines.

Claims

1. The following formula (1): 【Chemistry 1】 [wherein R represents a cyclopropyl group or a 2-chloroethyl group.] An antibody induction enhancer comprising, as an active ingredient, a compound represented by the formula:

2. The following formula (1): 【Chemistry 2】 [wherein R represents a cyclopropyl group or a 2-chloroethyl group.] and a pharmaceutically acceptable carrier.

3. The following formula (1): 【Transformation 3】 [wherein R represents a cyclopropyl group or a 2-chloroethyl group.] a pharmaceutically acceptable carrier, and a vaccine antigen.

4. The vaccine composition according to claim 3, wherein the vaccine antigen is an influenza virus.

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