Immune tolerance inducer for sublingual administration

Abstract

To provide an immune tolerance inducer for sublingual administration applicable to sublingual immunotherapy.SOLUTION: An immune tolerance inducer for sublingual administration comprises a phosphorylcholine compound (P), represented by the formula (1) below (where, each symbol is as defined in the specifications), as well as an allergen.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an immune tolerance inducer for sublingual administration which comprises a specific phosphorylcholine compound and an allergen. [Background technology]

[0002] Allergen immunotherapy is known as a radical treatment for allergic rhinitis. Among them, sublingual immunotherapy, in which a therapeutic drug containing an allergen is administered sublingually, is simple and easy to use, and has become popular in recent years.

[0003] Sublingual immunotherapy is known to be effective for, for example, cedar pollen allergy and house dust allergic rhinitis (Non-Patent Documents 1 and 2). In addition, for example, Patent Document 1 discloses that sublingual immunotherapy can be performed using hyaluronic acid in combination with an allergen to enhance the effect of the allergen.

[0004] Meanwhile, immune tolerance inducers are also being studied. For example, Patent Document 2 discloses that T cells that specifically respond to microbial digestive products against cedar pollen allergens have cross-reactivity to cedar pollen allergens, and therefore the digestive products can be used as oral immune tolerance inducers that induce immune tolerance. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 189676 [Patent Document 2] JP 2020-33 A [Non-patent literature]

[0006] [Non-Patent Document 1] Kimihiro Okubo, et al., A Randomized Double-Blind Comparative Study of Sublingual Immunotherapy for Cedar Pollinosis, Allergology International. 2008;57:265-75 [Non-Patent Document 2] Bohai Feng, et al., Efficacy of Sublingual Immunotherapy for House Dust Mite-Induced Allergic Rhinitis: A Meta-Analysis of Randomized Controlled Trials, Allergy Asthma Immunol Res. 2017 May;9(3):220-228 Summary of the Invention [Problem to be solved by the invention]

[0007] Sublingual immunotherapy, which is a treatment for allergic rhinitis caused by house dust or cedar as allergens, as described in Patent Document 1 and Non-Patent Documents 1 and 2, requires a long treatment period of at least three years to induce sufficient immune tolerance in the host (sublingual immunotherapy subject: patient). In order to shorten this treatment period, there is hope for an immune tolerance inducer that can induce immune tolerance more quickly. However, an immune tolerance inducer for sublingual administration that can be used in sublingual immunotherapy has not yet been developed.

[0008] The present invention has been made in consideration of the above problems, and an object of the present invention is to provide an immune tolerance inducer for sublingual administration that can be applied to sublingual immunotherapy. [Means for solving the problem]

[0009] As a result of intensive research in light of the above problems, the inventors discovered that an immune tolerance inducer for sublingual administration containing a specific phosphorylcholine compound is capable of inducing immune tolerance in a host, and thus completed the present invention.

[0010] The present invention based on the above findings is as follows. [1] The following formula (1):

[0011] [ka]

[0012] (In the formula, X represents a hydrogen atom, a monovalent cation residue, or a group represented by the following formula (2):

[0013] [ka]

[0014] It is a group represented by the following formula: An immune tolerance inducer for sublingual administration, comprising a phosphorylcholine compound (P) represented by the following formula: Effect of the Invention

[0015] By using the immune tolerance inducer of the present invention for sublingual administration, immune tolerance can be induced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] <Immune tolerance inducer for sublingual administration> The present invention provides an immune tolerance inducer for sublingual administration (hereinafter sometimes abbreviated as "the inducer of the present invention"). Here, "immune tolerance inducer" means an agent that induces immune tolerance. "Inducing immune tolerance" means that an immune response is suppressed. The suppression of an immune response can be confirmed, for example, from the amount of antibody production. "Immune tolerance inducer for sublingual administration" means an immune tolerance inducer used for sublingual administration.

[0017] The inducer of the present invention has the following formula (1):

[0018] [ka]

[0019] The phosphorylcholine compound (P) represented by the formula (hereinafter may be abbreviated as "phosphorylcholine compound (P)") and an allergen. The phosphorylcholine compound (P) may be used alone or in combination of two or more kinds. The allergen may be used alone or in combination of two or more kinds.

[0020] In formula (1), X is a hydrogen atom, a monovalent cationic residue, or a group represented by the following formula (2).

[0021] [ka]

[0022] In the present specification, the term "monovalent cation residue" refers to a monovalent cation, X + (excluding protons) is -COO - This refers to X when it bonds with to form -COOX in formula (1).

[0023] The monovalent cation residue is not particularly limited, but examples thereof include alkali metals such as lithium, sodium, and potassium; and protonated nitrogen-containing compounds such as ammonia, imidazole, dimethylaminopyridine, triethylamine, diazabicyclooctane, diazabicyclononene (DBN), and diazabicycloundecene (DBU).

[0024] The -COOX group in formula (1) is bonded to any carbon atom on the benzene ring, but is preferably bonded at the para position to the phosphorylcholine group.

[0025] X in formula (1) is preferably a group represented by formula (2) because it has high reactivity with allergens (particularly ovalbumin).The phosphorylcholine compound (P) is more preferably a compound represented by formula (5) described in the Examples below.

[0026] The content of the phosphorylcholine compound (P) in the inducer of the present invention is not particularly limited, but is preferably 0.2% by mass or more and 20% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, based on the total amount of the inducer of the present invention.

[0027] Allergens are classified into, for example, inhalant allergens, food allergens, and contact allergens. Inhalant allergens include, for example, pollen, house dust, and mold. Food allergens include, for example, egg, milk, wheat, buckwheat, peanut, shrimp, and crab. Contact allergens include, for example, metals and cosmetics. The allergen contained in the inducer of the present invention is preferably pollen or food allergen, more preferably food allergen, even more preferably egg allergen, and particularly preferably ovalbumin.

[0028] The content of the allergen in the inducer of the present invention is not particularly limited, but is preferably 0.1 parts by mass or more and 10 parts by mass or less, more preferably 0.3 parts by mass or more and 3 parts by mass or less, per part by mass of the phosphorylcholine compound (P).

[0029] In the inducer of the present invention, the phosphorylcholine compound (P) and the allergen may be bound to each other, or they may exist separately without being bound to each other. "An immune tolerance inducer for sublingual administration comprising a conjugate of a phosphorylcholine compound (P) and an allergen" is also included in the "immune tolerance inducer for sublingual administration comprising a phosphorylcholine compound (P) and an allergen" of the present invention. The inducer of the present invention preferably comprises a conjugate of a phosphorylcholine compound (P) and an allergen. By using the conjugate, immune tolerance can be efficiently induced.

[0030] The inducer of the present invention may contain a solvent. Only one type of solvent may be used, or two or more types may be used in combination. Examples of the solvent include water, physiological saline, glycerol (a solvent currently used in injectable preparations), and ethanol. Considering the solubility of the raw material and the reaction rate, the solvent is preferably water, and among them, purified water (e.g., distilled water) is more preferable.

[0031] The content of the solvent in the inducer of the present invention is not particularly limited, but is preferably 1 part by mass or more and 500 parts by mass or less, more preferably 20 parts by mass or more and 200 parts by mass or less, per part by mass of the phosphorylcholine compound (P).

[0032] The form of the inducer of the present invention is not particularly limited, but examples include tablets, sheets, powders, liquids (e.g., viscous liquids), gels, emulsions, and dispersions.

[0033] The inducer of the present invention may contain components other than the phosphorylcholine compound (P) and the solvent that are blended as necessary (hereinafter, may be abbreviated as "other components"). Only one type of other component may be used, or two or more types may be used in combination. The other components are not particularly limited as long as they do not interfere with the effect of the inducer of the present invention (induction of immune tolerance), and examples thereof include wetting agents, surfactants, buffers, preservatives and germicides, anti-inflammatory agents, sweeteners, flavorings, thickening agents, drying agents, mucosal adhesion agents, and dyes.

[0034] The humectant is not particularly limited, but examples thereof include polyhydric alcohols such as glycerin, propylene glycol, butylene glycol, pentylene glycol, dipropylene glycol, polyethylene glycol, xylitol, sorbitol, mannitol, and erythritol, and polysaccharides such as hyaluronic acid, alginic acid, and gellan gum.

[0035] The surfactant is not particularly limited, but examples thereof include polyoxyethylene hydrogenated castor oil, sorbitan fatty acid ethylene adduct, polyglycerin fatty acid ester, acylamino acid salt, fatty acid aminopropyl betaine, and fatty acid amide betaine.

[0036] The buffering agent is not particularly limited, but examples thereof include citric acid, phosphoric acid, malic acid, gluconic acid, and salts thereof.

[0037] The antiseptic and disinfectant is not particularly limited, but examples thereof include cetylpyridinium chloride, benzethonium chloride, benzalkonium chloride, chlorhexidine gluconate, polyhexanide hydrochloride, isopropylmethylphenol, triclosan, hinokitiol, benzoic acid and its salts, parabens, and alkyldiaminoethylglycine chloride.

[0038] Anti-inflammatory agents include, but are not limited to, glycyrrhizic acid and its salts, glycyrrhetin, azulene, epsilon-aminoaproic acid, and allantoin.

[0039] Sweeteners include, but are not limited to, saccharin, stevioside, sucrose, aspartame, and licorice extract.

[0040] The thickening agent is not particularly limited, but examples thereof include cellulose-based thickening agents such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and carboxymethyl cellulose; synthetic polymer-based thickening agents such as polyvinyl alcohol and polyvinylpyrrolidone; and polysaccharides such as xanthan gum.

[0041] The inducer of the present invention can be prepared by mixing an allergen with a phosphorylcholine compound (P). The mixing is preferably carried out in the presence of the above-mentioned solvent.

[0042] The temperature at which the allergen and the phosphorylcholine compound (P) are mixed is not particularly limited, but is usually 0 to 100° C., and preferably 20 to 50° C. The time for which the allergen and the phosphorylcholine compound (P) are mixed is not particularly limited, but is usually 1 to 120 hours.

[0043] The inducer of the present invention can be sublingually administered to mammals, such as humans, cows, pigs, horses, dogs, cats, rabbits, mice, rats, and guinea pigs, with humans being preferred. EXAMPLES

[0044] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0045] <Synthesis of phosphorylcholine compound (P1)> A phosphorylcholine compound (P1), which is a type of phosphorylcholine compound (P), was synthesized by the following production method.

[0046] <Synthesis of compound represented by formula (3)> 20 g of methyl 4-hydroxybenzoate was dissolved by adding 160 g of acetonitrile and 15 g of triethylamine, and cooled to 0°C. Then, 21 g of 2-chloro-2-oxo-1,3,2-dioxaphosphorane was added dropwise. After the completion of the dropwise addition, the mixture was reacted at 0°C for 5 hours, and the generated triethylamine hydrochloride was removed by filtration. 200 g of acetonitrile and 12 g of trimethylamine were added to the obtained filtrate, and the mixture was reacted at 75°C for 15 hours. Then, the mixture was cooled to 60°C, and concentrated until the amount of the solution became about 200 ml while blowing in nitrogen. After the concentration, the solution was cooled to 25°C, and the precipitated crystals were filtered off and dried under reduced pressure to obtain 35.2 g of a white solid.

[0047] Regarding the above obtained white solid 1 H-NMR measurement, 31 The results of P-NMR measurement and mass spectrometry are as follows, and the above white solid was identified as the compound represented by formula (3). 1 H NMR:δ=8.04ppm(d,2H,J=8.8:d),7.30ppm(d,2H,J=8.8:e),4.41ppm(brs,2H:c),3.91ppm(s,3H:f),3.68ppm(m,2H:b),3.18ppm(s,9H:a) 31 P NMR: -4.59 ppm (t, J = 15.9) MS: [M+H] + =317.91, [M+Na] + =340.01

[0048] [ka]

[0049] <Synthesis of compound represented by formula (4)> 5 g of the compound represented by formula (3) obtained was dissolved in 45 g of distilled water, 3.19 g of triethylamine was added, and the mixture was reacted under reflux for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and 3.4 g of concentrated hydrochloric acid was added to neutralize the reaction solution, which was then concentrated under reduced pressure using a rotary evaporator. 50 g of 2-propanol was added to the residue obtained, and the mixture was concentrated under reduced pressure twice. 50 g of 2-propanol was then added, and the mixture was stirred overnight at -10°C to precipitate crystals. The solution was filtered under pressure, and the obtained crystals were dried under reduced pressure to obtain 4.0 g of a white solid.

[0050] Regarding the above obtained white solid 1 H NMR measurement, 31 The results of P NMR measurement and mass spectrometry are as follows, and the above white solid was identified as the compound represented by formula (4). The compound represented by formula (4) is also a type of phosphorylcholine compound (P). 1 The results of H NMR measurement are as follows: 1 H NMR: δ=8.06ppm(d,2H,J=8.8:d),7.33ppm(d,2H,J=8.8:e),4.43ppm(brs,2H:b),3.70ppm(m,2H:c),3.19ppm(s,9H:a) 31 P NMR: -4.56 ppm (t, J = 15.9) MS: [M+H] + =304.12

[0051] [ka]

[0052] <Synthesis of compound represented by formula (5) (i.e., phosphorylcholine compound (P1))> 5.0 g of the compound represented by formula (4) and 3.0 g of N-hydroxysuccinimide were suspended in 35 g of N,N-dimethylformamide. The solution was kept at 25° C., and 6.8 g of 1,3-dicyclohexylcarbodiimide dissolved in 10 g of N,N-dimethylformamide was added, followed by reaction at 25° C. for 72 hours. After the reaction, the solution was filtered to recover the solid, which was then recrystallized with 160 g of acetonitrile to obtain 4.5 g of a white solid.

[0053] Regarding the above obtained white solid 1 H-NMR measurement, 31 The results of P-NMR measurement and mass spectrometry are as follows, and the above white solid was identified as the compound represented by formula (5) (i.e., phosphorylcholine compound (P1)). 1 H NMR:δ=8.23ppm(d,2H,J=8.8:e),7.42ppm(d,2H,J=8.3:d),4.45ppm(brs,2H:b),3.72ppm(m,2H:c),3.22ppm(s,9H:a),3.04ppm(s,4H:f) 31 P NMR: -4.96 ppm (t, J = 15.9) MS: [M+H] + =400.99, [M+Na] + =423.03

[0054] [ka]

[0055] The above 1 H NMR measurement, 31 The measurement conditions for P NMR measurement and mass spectrometry are shown below. [ 1 H NMR measurement] Measurements were performed using a JEOL "JNM-AL400" under the following conditions: solvent: D2O, standard substance: HOD, sample concentration: 10 mg / g, and number of accumulations: 32. [ 31 P NMR measurement] Measurements were performed using a JEOL "JNM-AL400" under the following conditions: solvent: D2O, standard substance: H3PO4, sample concentration: 10 mg / g, and number of accumulations: 32. [Mass spectrometry] The mass spectrometer used was a Waters product, trade name "Q-micro2695," and measurements were performed under the following conditions: sample concentration: 100 ppm, detection mode: ESI+, capillary voltage: 3.54 V, cone voltage: 30 V, ion source heater: 120°C, desolvation gas: 350°C.

[0056] <Ovalbumin> As the allergen in Example 1, commercially available ovalbumin (Sigma-Aldrich, A5503-5G) was used.

[0057] [Example 1 and Comparative Example 1] (Preparation of the Inducer of the Present Invention) The inducer of the present invention was prepared by dissolving 10 mg of ovalbumin (hereinafter abbreviated as "OVA") in 1 mL of distilled water, adding 10 mg of phosphorylcholine compound (P1), and stirring at 40°C for 18 hours.

[0058] (procedure) The evaluation was carried out according to the following procedure. (1) Eighteen 6-week-old male BALB / c mice were prepared in advance.

[0059] (2) In Example 1, after intraperitoneal anesthesia, the inducer of the present invention was sublingually administered to each of 9 mice once a day (amount administered at one time: 5 μL), and the mice were kept in a prone position for 30 minutes. This procedure was carried out for 14 days. Meanwhile, in Comparative Example 1, phosphate buffered saline (hereinafter abbreviated as "PBS") was sublingually administered to each of 9 mice once a day (amount administered at one time: 5 μL), and the mice were treated in the same manner as in Example 1. This procedure was carried out for 14 days.

[0060] (3) On the 21st, 28th, and 35th days after the start date of sublingual administration of the inducer of the present invention or PBS (hereinafter abbreviated as "start date"), OVA was intraperitoneally administered once a day (OVA concentration in the intraperitoneally administered solution: 0.008% by mass, Alum (adjuvant) concentration: 0.33% by mass, single dose of solution: 300 μL). Five minutes after the intraperitoneal administration of OVA on the 35th day after the start date, the number of times the mice scratched their noses and sneezed was measured. The results are shown in Table 1 below. Table 1 shows the values ​​of "mean ± standard deviation".

[0061] (4) Intraperitoneal administration of OVA was terminated on day 35 from the start date, and intranasal administration of OVA was initiated on day 42 from the start date. Intranasal administration of OVA was performed once a day between day 42 and day 49 from the start date (OVA concentration in the intranasally administered solution: 1.6% by mass, single dose of solution: 30 μL).

[0062] (5) Serum was collected from the mice before sublingual administration of the inducer of the present invention or PBS, on the 21st day from the start date (after intraperitoneal administration of OVA), and on the 42nd day from the start date (after intranasal administration of OVA), and the amount of OVA-specific IgE antibody in the collected serum was measured using an ELISA kit (Anti-Ovallbmin IgE (mouse) EIA Kit) manufactured by Cayman Chemical Company. The results are shown in Table 2 below. Table 2 shows the values ​​of "mean ± standard deviation".

[0063] (6) After intranasal administration of OVA on the 49th day from the start date, the nasopharyngeal cavity of the mice was washed with saline, and the saline used for washing (hereinafter referred to as "saline after washing") was collected, and the OD value (optical density value), which is related to the amount of OVA-specific IgE antibody in the saline after washing, was measured with a plate reader using an ELISA kit (Anti-Ovallbmin IgE (mouse) EIA Kit) manufactured by Cayman Chemical Company. The results are shown in Table 3 below. Table 3 shows the values ​​of "mean ± standard deviation".

[0064] [Table 1]

[0065] [Table 2]

[0066] [Table 3]

[0067] (evaluation) As shown in Table 1, the frequency of nose scratching and sneezing was reduced more in Example 1 than in Comparative Example 1, confirming the effectiveness of the inducer of the present invention.

[0068] As shown in Table 2, the amount of OVA-specific IgE antibody in the serum was suppressed more in Example 1 than in Comparative Example 1, confirming the effectiveness of the inducer of the present invention.

[0069] As shown in Table 3, the OD value measured with a plate reader was smaller in Example 1 than in Comparative Example 1, and the amount of OVA-specific IgE antibody in the saline after washing was suppressed, confirming the effectiveness of the inducer of the present invention. [Industrial Applicability]

[0070] The inducing agents of the present invention are useful for sublingual immunotherapy.

Claims

[Claim 1] The following formula (1): 【Chemistry 1】 (In the formula, X represents a hydrogen atom, a monovalent cation residue, or a compound represented by the following formula (2): 【Chemistry 2】 It is a group represented by the following formula: An immune tolerance inducer for sublingual administration, comprising a phosphorylcholine compound (P) represented by the following formula (1):

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