Inhibitors of trimethylamine-responsive receptors, deodorants for trimethylamine odor, methods for deodorizing trimethylamine odor

Abstract

The present invention provides an inhibitor of trimethylamine-responsive receptors that is suitable for deodorizing trimethylamine odor by suppressing the response of trimethylamine-responsive receptors. [Solution] In a preferred example, the inhibitor of the trimethylamine-responsive receptor includes at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, and 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal.

Description

Technical Field

[0001] The present invention relates to an inhibitor of trimethylamine-responsive receptor, a deodorant for trimethylamine odor, and a method for deodorizing trimethylamine odor.

Background Art

[0002] Odors in the living space include excrement odor of humans or animals, sweat odor, aging odor, garbage odor, tobacco odor, etc., and there are also complex odors in which these odors are mixed. Trimethylamine is a major causative substance of excrement odor and garbage odor. Regarding the deodorization of these odors, physical deodorization using an adsorption process and chemical deodorization using a neutralization reaction have been proposed. For example, Patent Document 1 discloses a chemical deodorant fragrance composition for trimethylamine odor, which contains a fragrance component such as aliphatic aldehydes having 6 to 12 carbon atoms as an active ingredient.

[0003] However, in chemical deodorization such as that of Patent Document 1, the odor is eliminated by converting trimethylamine, which causes the odor, into another compound by a deodorant or an adsorbent. Therefore, molecular contact between the malodorous molecules and the deodorant etc. is necessary. Considering this point, for example, in a closed space, the deodorizing effect of trimethylamine odor can be exhibited over time, but in an open space or a large space, it is difficult to obtain the deodorizing effect.

[0004] In order to obtain a deodorizing effect, controlling the response of olfactory receptors may be effective. When a human recognizes trimethylamine odor, it is considered that one type of trace amine-related receptor (TAAR), TAAR5, present in olfactory nerve cells responds. Patent Document 2 discloses α-damascone and β-damascone as fragrance components that inhibit the activation of TAAR5. Non-Patent Document 1 discloses Timberol (registered trademark, chemical name: 2,2,6-trimethyl-α-propylcyclohexane-1-propanol) as a fragrance component that inhibits the activation of TAAR5.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2001-303090 [Patent Document 2] Japanese Patent Publication No. 2023-174084 [Non-patent literature]

[0006] [Non-Patent Document 1] PLOS ONE,2015, DOI:10.1371 / journal.pone.0144704. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] However, the vast majority of candidate ligands for trimethylamine-responsive receptors remain unknown, and the number of candidates is enormous. Furthermore, there is room for improvement in the deodorizing performance of known ligand molecules.

[0008] The present invention provides an inhibitor of trimethylamine-responsive receptors that is suitable for deodorizing trimethylamine odor by suppressing the response of trimethylamine-responsive receptors. [Means for solving the problem]

[0009] The present invention has the following aspects. [1] An inhibitor that suppresses the response of at least one trimethylamine-responsive receptor selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5, trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, 4-[(octahydro-4,7-methano-5H-indene)-5 A trimethylamine-responsive receptor inhibitor comprising at least one selected from the group consisting of -ylidene]butanal, 3-(3-isopropylphenyl)butanal, p-cymene, nopyruacetate, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, γ-dodecanolactone, and 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol. [2] The trimethylamine-responsive receptor inhibitor according to claim 1, comprising at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, and 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal. [3] An inhibitor of trimethylamine-responsive receptors according to [1], comprising at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, and citral. A deodorant for trimethylamine odor, comprising an inhibitor of the trimethylamine-responsive receptor described in any of [4][1] to [3]. A method for deodorizing trimethylamine odor using a trimethylamine-responsive receptor inhibitor described in any of [5][1] to [3]. [Effects of the Invention]

[0010] According to the present invention, an inhibitor of trimethylamine-responsive receptors is provided that is suitable for deodorizing trimethylamine odor by suppressing the response of trimethylamine-responsive receptors. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results of measuring the response intensity of trace amine-related receptors to trimethylamine (normalized lumen) in experimental examples. [Figure 2] Figure 2 shows the results of measuring the concentration dependence of the response intensity of mouse TAAR5 to trimethylamine in experimental examples. [Modes for carrying out the invention]

[0012] [Explanation of terms] A polypeptide having the same function as TAAR5 refers to a polypeptide that can be expressed on the cell membrane and which, upon ligand binding, induces the production of cAMP within the cell, or which, upon ligand binding, promotes the influx of calcium ions from outside the cell into the cell. An "agonist" is a compound that activates the response of a receptor by binding to it. An "antagonist" is a compound that binds to a receptor and inhibits the activation of that receptor by an agonist. The "~" symbol, which indicates a numerical range, means that the numbers before and after it are included as the lower and upper limits, respectively.

[0013] The sequence identity (homology) of amino acid sequences can be determined as follows, based on the sequence identity of the target amino acid sequence with respect to a reference amino acid sequence. First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, in the reference amino acid sequence and the target amino acid sequence, the number of amino acid residues of the matching amino acids is calculated, and the sequence identity can be determined according to the following formula (1).

[0014] Sequence identity (%) = (number of matching amino acid residues / total number of amino acid residues in the target amino acid sequence) × 100 ··· Formula (1)

[0015] Hereinafter, embodiments of the present invention will be described in detail. However, the following description relates to some examples of the embodiments of the present invention and is not limited to these contents.

[0016] [Inhibitor of trimethylamine-responsive receptor] The inhibitor of the trimethylamine-responsive receptor suppresses the response of at least one or more trimethylamine-responsive receptors selected from the group consisting of TAAR5 and polypeptides having the same function as TAAR5.

[0017] Hereinafter, in this specification, at least one or more trimethylamine-responsive receptors selected from the group consisting of TAAR5 and polypeptides having the same function as TAAR5 may be referred to as "specific trimethylamine-responsive receptors".

[0018] TAAR5 is a trimethylamine-responsive receptor whose expression has been confirmed in human olfactory receptor neurons. Human TAAR5 is registered in GenBank (NCBI) as Gene ID: 9038. Human TAAR5 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0019] The trimethylamine-responsive receptor may be mouse TAAR5. Mouse TAAR5 is registered in GenBank (NCBI) as Gene ID: 215854. Mouse TAAR5 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0020] Certain trimethylamine-responsive receptors, such as TAAR5, exhibit a specific response intensity to trimethylamine. TAAR5 can be replaced by polypeptides having equivalent function to TAAR5. The amino acid sequence of the polypeptide having equivalent function to TAAR5 preferably shows 80% or more homology to the amino acid sequence of TAAR5, more preferably 85% or more homology, even more preferably 90% or more homology, even more preferably 95% or more homology, particularly preferably 98% or more homology, and most preferably 99% or more homology.

[0021] In addition to TAAR5, any polypeptide with equivalent function can be used as a trimethylamine-responsive receptor. Examples include homologous trimethylamine-responsive receptors derived from animals other than humans and mice. Examples of animals other than humans and mice include rats and other experimental model organisms.

[0022] The inventors tested various substances and found that trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene] We found that at least one substance selected from the group consisting of butanal, 3-(3-isopropylphenyl)butanal, p-cymene, nopyruacetate, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, γ-dodecanolactone, and 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol suppresses the response of certain trimethylamine-responsive receptors, such as TAAR5, to trimethylamine odor.

[0023] In particular, at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, and 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal is preferred as an inhibitor of trimethylamine-responsive receptors because it further improves the inhibitory performance of trimethylamine-responsive receptors.

[0024] In addition, at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, and citral is more preferred as an inhibitor of trimethylamine-responsive receptors because it significantly improves the inhibitory performance of trimethylamine-responsive receptors.

[0025] As inhibitors of trimethylamine-responsive receptors, trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, and 3-(4-tert-butylphenyl)propanal are more preferred, trans-cinnamaldehyde and 3-(1,3-benzodioxol-5-yl-2-methylpropanal are particularly preferred, and trans-cinnamaldehyde is most preferred.

[0026] Trimethylamine-responsive receptor inhibitors may be used alone or in combination of two or more.

[0027] Trimethylamine-responsive receptor inhibitors are thought to function as antagonists of specific trimethylamine-responsive receptors because they satisfy the following two conditions. - In trimethylamine-responsive receptors after mixing with an inhibitor, when the trimethylamine-responsive receptors come into contact with trimethylamine, they become less responsive to trimethylamine compared to when the inhibitor is not mixed. • Trimethylamine-responsive receptors do not respond to the inhibitor after being mixed with the inhibitor.

[0028] Trimethylamine-responsive receptors can be experimentally used to verify the inhibitory effect of inhibitors. Trimethylamine-responsive receptors can be used in any manner as long as they do not lose their responsiveness to trimethylamine. For example, trimethylamine-responsive receptors can be used in the form of cells or tissues that naturally express trimethylamine-responsive receptors and their cultures; membranes of olfactory receptor cells carrying trimethylamine-responsive receptors; genetically modified cells expressing trimethylamine-responsive receptors and their cultures; membranes of genetically modified cells expressing trimethylamine-responsive receptors; and lipid bilayers expressing trimethylamine-responsive receptors. In addition, tissues containing olfactory mucus (olfactory epithelium, olfactory mucosa, etc.) may be used as trimethylamine-responsive receptors.

[0029] In one embodiment, the use of cells that naturally express trimethylamine-responsive receptors, genetically modified cells that express trimethylamine-responsive receptors, and cultures thereof is preferred as the trimethylamine-responsive receptor.

[0030] When experimentally using trimethylamine-responsive receptors, metal ions may also be used. The manner in which metal ions are used and present is not particularly limited. Examples include: a method of mixing trimethylamine-responsive receptors and a test substance in a metal ion-containing solution; a method of mixing a membrane carrying trimethylamine-responsive receptors or cells or tissues expressing trimethylamine-responsive receptors with the test substance while immersed in a metal ion-containing solution; a method of adding metal ions to a culture medium for cells or tissues expressing trimethylamine-responsive receptors, and then adding the test substance; and a method of mixing a metal ion-containing solution together with the test substance in a culture medium for cells or tissues expressing trimethylamine-responsive receptors. Examples of metal ions include copper ions and silver ions.

[0031] To verify the inhibitory performance, for example, the trimethylamine-responsive receptor and the test substance may be mixed, and then the trimethylamine-responsive receptor and trimethylamine may be brought into contact. In this case, the following reference data 1 and test data 1 can be obtained.

[0032] Reference Data 1: Data measuring the response of trimethylamine-responsive receptors before contact with trimethylamine in the presence of the test substance. Test data 1: Data measuring the response of trimethylamine-responsive receptors after contact with trimethylamine in the presence of the test substance.

[0033] A comparison of reference data 1 and test data 1 allows us to evaluate the response of trimethylamine-responsive receptors in the presence of the test substance before and after contact with trimethylamine. For example, if the response of trimethylamine-responsive receptors is suppressed before and after contact with trimethylamine, the test substance may be useful as a trimethylamine odor inhibitor.

[0034] Alternatively, to verify the inhibitory performance, the olfactory receptor may be brought into contact with trimethylamine before mixing the trimethylamine-responsive receptor with the test substance. In this case, the following reference data 2 and comparative data 2 can be obtained.

[0035] Reference Data 2: Data measuring the response of trimethylamine-responsive receptors after contact with trimethylamine and before the addition of the test substance. Test data 2: Data measuring the response of trimethylamine-responsive receptors when the test substance was added after contact with trimethylamine.

[0036] A comparison of reference data 2 and test data 2 allows for the evaluation of the response of trimethylamine-responsive receptors, which are already activated by trimethylamine, before and after the addition of the test substance. For example, if the response index of trimethylamine-responsive receptors in test data 2 is statistically significantly reduced compared to reference data 2, the test substance may be useful as a trimethylamine odor suppressant.

[0037] (Effects and Benefits) Trimethylamine-responsive receptor inhibitors function as antagonists of specific trimethylamine-responsive receptors that respond specifically to trimethylamine odor. Therefore, a trimethylamine-responsive receptor inhibitor according to one embodiment can suppress the response of these specific trimethylamine-responsive receptors.

[0038] Trimethylamine-responsive receptor inhibitors can suppress the response of specific trimethylamine-responsive receptors to trimethylamine. Therefore, the concentration required to obtain a similar level of deodorizing effect in the target space can likely be set lower compared to conventional physical or chemical deodorization methods. Consequently, it is not necessary to have the trimethylamine-responsive receptor inhibitor in the target space at a higher concentration than trimethylamine itself. For these reasons, trimethylamine-responsive receptor inhibitors are useful as active ingredients in deodorizers for trimethylamine odor.

[0039] [Examples of applications of trimethylamine-responsive receptor inhibitors] According to one embodiment, a deodorant for trimethylamine odor is provided, which includes the above-mentioned trimethylamine-responsive receptor inhibitor. According to the deodorant according to one embodiment, the trimethylamine-responsive receptor inhibitor binds to the trimethylamine-responsive receptor, thereby suppressing the response of the trimethylamine-responsive receptor to trimethylamine. As a result of inhibiting the perception of trimethylamine odor, a deodorant effect is exerted.

[0040] Since trimethylamine-responsive receptor inhibitors are antagonists, they antagonize trimethylamine odor and inhibit the response of trimethylamine-responsive receptors and the perception of malodor. Therefore, there is less need to maintain a diffusion state beforehand, as is the case with conventional physical and chemical deodorization. Thus, trimethylamine-responsive receptor inhibitors are also suitable for instantaneous deodorization applications such as sprays.

[0041] The deodorant may be in the form of a trimethylamine-responsive receptor inhibitor, or in the form of a composition. In the case of a composition, the trimethylamine-responsive receptor inhibitor is included in the composition as an active ingredient for suppressing trimethylamine odor.

[0042] The composition may further contain other components besides trimethylamine-responsive receptor inhibitors, provided that the trimethylamine odor-suppressing effect of the trimethylamine-responsive receptor inhibitor is not impaired. Examples of other components include fragrances and additives.

[0043] The fragrance may be a natural fragrance, a single fragrance isolated from a natural fragrance, a synthetic single fragrance, or a blend of these. Conventional oily fragrances can be used without restriction as the fragrance.

[0044] The fragrances used are not particularly limited, but examples include animal-derived fragrances, plant-derived fragrances, synthetic fragrances, and extracted fragrances. One type of fragrance may be used alone, or two or more types may be used in combination.

[0045] While there are no particular limitations on animal-derived fragrances, examples include musk, spirit incense, and lilac. While not particularly limited, examples of plant-derived fragrances include abies oil, aquatica oil, almond oil, angelica root oil, pail oil, bergamot oil, perch oil, boa rose oil, sedge oil, gananga oil, capsicum oil, caraway oil, cardamom oil, cassia oil, celery oil, cinnamon oil, citronella oil, cognac oil, coriander oil, cumin oil, camphor oil, zill oil, estgolan oil, eucalyptus oil, fennel oil, garlic oil, ginger oil, grapefruit oil, hop oil, lemon oil, lemongrass oil, nutmeg oil, mandarin oil, peppermint oil, orange oil, sage oil, star anise oil, and turpentine oil. Animal-derived fragrances and plant-derived fragrances may be used individually or in combination of two or more.

[0046] Artificial fragrances such as synthetic fragrances and extracted fragrances are not particularly limited, but examples include: hydrocarbon fragrances such as pinene and limonene; alcohol fragrances such as linalool, tetrahydrolinalool, geraniol, citronellol, 2-isobutyl-4-methyltetrahydro-2H-pyran-4-ol, menthol, borneol, benzyl alcohol, anise alcohol, and β-phenethyl alcohol; phenol fragrances such as anethole and eugenol; aldehyde fragrances such as n-butyraldehyde, isobutyraldehyde, hexylaldehyde, citronellal, benzaldehyde, cinnamic aldehyde, and cumin aldehyde; 1-(2,3,8 Examples of fragrances include ketone-based fragrances such as ,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-2-yl)ethane-1-one, carvone, menthone, camphor, acetophenone, and ionone; lactone-based fragrances such as γ-butyllactone, coumarin, and cineole; ester-based fragrances such as methyl dihydrojasmonate, 1,1-dimethyl-2-phenylethyl acetate, 2,2,2-trichloro-1-phenylethyl acetate, hexyl acetate, octyl acetate, benzyl acetate, styraryl acetate, cinnamyl acetate, linalyl acetate, butyl propionate, and methyl benzoate; and benzopyran-based fragrances such as galaxolide. Artificial fragrances may be used individually or in combination of two or more.

[0047] Examples of additives include nonionic and anionic surfactants, insecticides, antibacterial agents, insect repellents, deodorizers, stabilizers, UV absorbers, antioxidants, pH adjusters, and dyes.

[0048] In the case of a composition, the content of the trimethylamine-responsive receptor inhibitor is preferably 0.002% by mass or more of the total amount, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. If the content of the trimethylamine-responsive receptor inhibitor is above the lower limit, it is considered sufficient to deodorize the trimethylamine odor by suppressing the response of the trimethylamine-responsive receptor when used as a deodorant.

[0049] The deodorant may be carried with the object that is to be suppressed for trimethylamine odor, left standing in a space where trimethylamine odor may be generated, or mixed with a substance that may generate trimethylamine odor.

[0050] According to one embodiment, a method for deodorizing trimethylamine odor is provided, using the above-mentioned trimethylamine-responsive receptor inhibitor. In the deodorizing method according to one embodiment, the trimethylamine-responsive receptor inhibitor binds to the trimethylamine-responsive receptor, thereby suppressing the response of the trimethylamine-responsive receptor to the trimethylamine odor. As a result of inhibiting the perception of the trimethylamine odor, a deodorizing effect is exerted.

[0051] According to another embodiment, a method for suppressing trimethylamine odor using a trimethylamine-responsive receptor inhibitor is provided. In this method for suppressing trimethylamine odor, the trimethylamine-responsive receptor inhibitor is applied to a target (individual) whose perception of trimethylamine odor is to be suppressed. The trimethylamine-responsive receptor inhibitor may be applied before the target is exposed to trimethylamine, after the target is exposed to trimethylamine, or simultaneously with the target being exposed to trimethylamine.

[0052] In one embodiment, a trimethylamine-responsive receptor inhibitor is applied to the target of trimethylamine odor suppression before it is exposed to trimethylamine. The applied trimethylamine-responsive receptor inhibitor inhibits the response of the trimethylamine-responsive receptors in the target of suppression. As a result, even when the target of suppression is exposed to trimethylamine, the response of the trimethylamine-responsive receptors to trimethylamine is suppressed, and the perception of trimethylamine odor is suppressed.

[0053] Examples of applicable items include, for example, toilets or waste disposal for humans and animals; waste disposal in medical and nursing care facilities; waste disposal; disposable diapers and sanitary products; odor treatment in fishing facilities, seafood processing facilities, medical facilities, and nursing care facilities; odor treatment in garbage cans, kitchen spaces, bathrooms, and nursing care spaces, especially odor treatment for food waste; clothing such as underwear, masks, face shields, and linens; laundry detergents and fabric softeners; topical agents such as permanent wave agents, cosmetics, cleaning agents, and deodorants; pharmaceuticals; food products, etc.; and manufacturing facilities for products that generate trimethylamine odor. However, the application of trimethylamine-responsive receptor inhibitors is not limited to these examples.

[0054] Although several embodiments have been described above, each embodiment is presented as an example and does not limit the scope of the present invention. Each embodiment described herein can be modified in various ways within the scope that the effects of the invention are achieved, and can be combined with features described in other embodiments to the extent that is feasible.

[0055] The embodiments will be described in more detail below with reference to the experimental results of the inventors. However, the present invention is not limited to the following experimental results.

[0056] [Preparation of trimethylamine-responsive receptor-expressing cells] Based on sequence information registered in GenBank, the genes encoding olfactory receptors listed in Table 1 were cloned. Each gene was cloned using PCR with human genomic DNA Human mixed (G3041:Promega) and genomic DNA extracted from mouse tails as templates. Each gene amplified by PCR was incorporated into a pCI vector (Promega) according to the product protocol. Specifically, the Rho tag sequence was incorporated using the NheI restriction enzyme site and BamHI restriction enzyme site present on the pCI vector. The receptor gene was then incorporated downstream of the Rho tag sequence using the MluI restriction enzyme site and NotI restriction enzyme site downstream of the Rho tag sequence. Next, the gene encoding human RTP1S or mouse RTP1S was incorporated into the MluI restriction enzyme site and NotI restriction enzyme site of the pCI vector. Hereafter, cells in which the gene encoding the olfactory receptor was not incorporated will be referred to as "Rho".

[0057] [Table 1]

[0058] Hana3A cells were cultured in 96-well plates (Corning, BioCoat) to 50% confluence. Reaction solutions were prepared with the compositions shown in Table 2. After standing the reaction solutions in a clean bench for 15 minutes, 50 μL was added to each well of the 96-well plate (Corning, BioCoat). Subsequently, Hana3A cells expressing each of the 18 human and mouse receptors shown in Table 1 were prepared by culturing for 24 hours in an incubator maintained at 37°C and a 5% CO2 atmosphere. The amount of TAAR gene added was varied from 1 to 50 μg depending on the ease of expression of each gene.

[0059] [Table 2]

[0060] [Glo Sensor Assay] The Glo Sensor assay was used to measure the response of trimethylamine-responsive receptors. Trimethylamine (TMA) was used as the odor molecule. Trimethylamine-responsive receptors expressed in Hana3A cells couple with intrinsic Gαs and Gαolf, activating adenylyl cyclase and increasing intracellular cAMP levels. The increase in intracellular cAMP levels was measured as a luminescence value derived from the firefly luciferase gene, and the response intensity of the trimethylamine-responsive receptor was determined.

[0061] Luciferase activity was measured using the Glo Sensor cAMP Reagent (Promega) according to its product protocol. For each olfactory receptor, the luminescence value derived from luciferase after trimethylamine stimulation was divided by the luminescence value derived from luciferase before trimethylamine stimulation. That is, (luminescence value after stimulation) / (luminescence value before stimulation) was calculated and used as the measured response intensity.

[0062] [Searching for receptors that respond to trimethylamine] (Identification of TAAR5) As described in Patent Document 2, TAAR5 is known. In this test system, TAAR5 was identified as follows: First, the culture medium was removed from the culture of receptor-expressing cells. Then, 25 μL of Glo Sensor cAMP Reagent diluted in HBSS buffer containing 10 mM HEPES was added to each well of a 96-well plate. The cells were cultured in a light-shielded environment for 2-3 hours to introduce cAMP Reagent into the cells.

[0063] Next, 25 μL of 0.03% (v / v) trimethylamine was added between the wells of a 96-well plate. The lid of the well plate was closed, and the trimethylamine was allowed to volatilize within the well plate, bringing human and mouse trace amine-related receptors (18 types in total) into contact with trimethylamine in the gas phase. The response intensity (fold increase) of trimethylamine-responsive receptors to trimethylamine was measured over time, and the total response intensity over 20 minutes was calculated. The results are shown in Figure 1.

[0064] The vertical axis in Figure 1 shows the relative response intensity to trimethylamine in cells expressing each receptor (Figure 1). The relative response intensity was calculated by setting the total response intensity of cells not expressing olfactory receptors (Rho) as 0, and then summing the response intensities for each of the 18 types of trace amine receptors expressed in each cell. As a result, mouse TAAR5 was identified as the trimethylamine-responsive receptor that showed the highest responsiveness to trimethylamine (Figure 1).

[0065] (Concentration dependence of TAAR5 response on trimethylamine) 25 μL of trimethylamine was dropped between the wells of a 96-well plate so that the final concentrations of trimethylamine were as shown on the horizontal axis of Figure 2. A GloSensor assay was performed, and mouse TAAR5 was exposed to trimethylamine for 15 minutes. The response intensity (fold increase) of mouse TAAR5 to trimethylamine was measured over time. The total response intensity over 15 minutes was calculated. The results are shown in Figure 2. As a result, mouse TAAR5 showed a concentration-dependent response to trimethylamine, confirming that it is an olfactory receptor that responds specifically to trimethylamine.

[0066] [Test substance] The following compounds were diluted with dimethyl sulfoxide to prepare a final concentration of 100 mM as test substances (compounds). • trans-cinnamaldehyde 3-(1,3-benzodioxol-5-yl-2-methylpropanal) • 3-(4-tert-butylphenyl)propanal • trans-2-hexenal Citral • Litsea cubeba fruit oil 3-methyl-5-phenylpentan-1-ol ·4,8-dimethyl-4,9-decadienal • Undecanal Ethyl dehydrocyclogeranate ·4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal 3-(3-isopropylphenyl)butanal p-cymene • Nopil acetate 3-Methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol • γ-Dodecanolactone ·2-Ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol • 1,1-dimethyl-2-phenylethyl acetate

[0067] [Searching for antagonists of trimethylamine-responsive receptors] The culture medium was removed from the mouse TAAR5-expressing cell culture. Then, 25 μL of GloSensor cAMP Reagent, diluted in HBSS buffer containing 10 mM HEPES, was added to each well of a 96-well plate. The cells were cultured in a light-shielded environment for 2-3 hours to introduce the cAMP Reagent into the cells. Subsequently, a GloSensor assay was performed to measure the luminescence value of mouse TAAR5 before the addition of the test substance.

[0068] Next, the test substance was added to each well of a 96-well plate so that the final concentrations were as shown in Table 3. A GloSensor assay was performed, and the response intensity (fold increase) of mouse TAAR5 to the test substance was measured over a 5-minute period to obtain baseline data.

[0069] Subsequently, 25 μL of trimethylamine was dropped between the wells of a 96-well plate to a final concentration of 0.03% (v / v), and the plate was then covered. A GloSensor assay was then performed, allowing mouse TAAR5 to be in contact with trimethylamine for 15 minutes. The response intensity (fold increase) of mouse TAAR5 to trimethylamine was measured over time to obtain the test data.

[0070] The response intensity to the test substance was determined by dividing the luminescence value derived from luciferase 5 minutes after the addition of the test substance by the luminescence value derived from luciferase before the addition of the test substance. That is, the formula (luminescence value 5 minutes after the addition of the test substance) / (luminescence value before the addition of the test substance) was calculated to obtain the baseline data.

[0071] The response intensity when trimethylamine and the test substance were mixed was calculated by determining the response value induced by trimethylamine stimulation over one minute. The one-minute response value was calculated as (luminescence value after trimethylamine stimulation at each time) / (luminescence value 5 minutes after adding the test substance). The sum of these values ​​over 15 minutes was obtained as the total response intensity. Next, the total response intensity of Rho was set to 0, and the obtained total response intensity was divided by the total response intensity when only trimethylamine was added. That is, ((total response intensity when trimethylamine and test substance are mixed) - (total response intensity of Rho)) / ((total response intensity of trimethylamine alone) - (total response intensity of Rho)) was calculated to obtain the test data. The measurement results of the response intensity are shown in Table 3.

[0072] [Table 3]

[0073] In the test data in Table 3, the lower the test data when the test substance concentration is 100 μM compared to the test data when the test substance concentration is 0 μM, the better the inhibitory performance on trimethylamine-responsive receptors is considered to be.

[0074] From the results of the response intensity measurement shown in Table 3, trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, 3-(3-isopropyl Pyrphenyl)butanal, p-cymene, nopyruacetate, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, γ-dodecanolactone, and 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol suppressed TAAR5's response to trimethylamine, suggesting they function as antagonists of TAAR5. These are considered to be inhibitors that readily exhibit sufficient deodorizing effects against trimethylamine odor.

[0075] In contrast, no suppression of the TAAR5 response to trimethylamine was observed with respect to 1,1-dimethyl-2-phenylethyl acetate.

[0076] [Test substances used in sensory evaluation 1] The deodorizing effect of trimethylamine was tested using the following test substances. • Undecanal • α-Damascon ·4,8-dimethyl-4,9-decadienal 3-methyl-5-phenylpentan-1-ol • 1,1-dimethyl-2-phenylethyl acetate

[0077] [Contents of Sensory Test 1] Sensory evaluation test 1 was conducted by six evaluators. The evaluators consisted of men and women in their 20s or 30s who had passed a separate olfactory test.

[0078] The following fragrance masterbatches were prepared. Test strips of fragrance paper, cut to approximately 4 cm, were prepared. One drop (approximately 0.02 g) of a 0.1% solution of each test substance, diluted with DMSO, was applied to the test strip. The test strips were then sealed in a 3 L bag, filled with odorless air, and left to stand at room temperature overnight.

[0079] The trimethylamine masterbatch was prepared as follows: A 1 g solution of 30% trimethylamine was impregnated into a pleated filter paper with a radius of 5.5 cm. The pleated filter paper was then sealed in a 10 L bag with a fan, and the bag was filled with odorless air. It was left to stand overnight at room temperature to obtain a trimethylamine masterbatch.

[0080] The following evaluation samples were prepared. Each fragrance masterbatch was sealed in a 3L bag, and 0.1ml of trimethylamine masterbatch was injected into it to achieve an odor intensity of 3-4, as defined by the Fragrance Deodorizer and Odor Eliminator Association, thereby obtaining each sample. Furthermore, 0.1ml of trimethylamine masterbatch was injected into another 3L bag filled with odorless air to obtain a sample of trimethylamine alone.

[0081] The evaluation tests were conducted as follows: Each sample was presented to an evaluator, who scored the trimethylamine intensity according to the following criteria. Table 4 shows the average score of the six evaluators, comparing the intensity of the trimethylamine-only sample with the malodor intensity of the trimethylamine-containing sample infused with scented air.

[0082] [Standards for Odor Intensity] The following evaluation criteria were used to rate scores on a 6-point scale from +5 to 0, in increments of 1 point. +5:Intense +4: Strong +3: Easily detectable +2: Weak +1: Finally detectable. 0: Odorless

[0083] [Table 4]

[0084] In Table 4, "Difference in Odor Intensity" refers to the difference between the average odor intensity evaluation score of trimethylamine and the average odor intensity evaluation score when each fragrance masterbatch is added. A value of 1.0 or higher indicates a deodorizing effect.

[0085] This test adopted the sensory deodorization test standards set by the Japan Fragrance and Deodorizing Agents Association. The Japan Fragrance and Deodorizing Agents Association defines "deodorizing effect" as a reduction of one level or more in malodor intensity. The results of sensory test 1 confirmed that undecanal, α-damascone, 4,8-dimethyl-4,9-decadienal, and 3-methyl-5-phenylpentan-1-ol have a sensory deodorizing effect against trimethylamine.

[0086] [Confirmation of the inhibitory effect of formulated fragrances on mouse TAAR5] As shown in Table 5 below, fruity-like and soap-like fragrances were prepared by incorporating the test substances. Each fragrance contains the compound indicated by "+" in Table 5. Both the fruity-like and soap-like fragrances were provided by Takasago International Corporation.

[0087] [Table 5]

[0088] Table 6 shows the results of measuring the response intensity of mouse TAAR5 for the fragrance formulations shown in Table 5. For comparison, Table 6 also shows the results of measuring the response intensity of mouse TAAR5 for each fragrance formulation with all antagonist-effect test substances removed. After adding 5 μL of the fragrance formulation at the concentrations listed in Table 6 to each well, the response intensity was measured. For the response intensity when trimethylamine and the fragrance formulation were mixed, the response value induced by trimethylamine stimulation over 1 minute was calculated per minute. The 1-minute response value was calculated as (luminescence value after trimethylamine stimulation at each time point) / (luminescence value 5 minutes after fragrance formulation addition). The sum of these values ​​over 20 minutes was obtained as the total response intensity. Next, the total Rho response intensity was set to 0, and the obtained total response intensity was divided by the response intensity when only trimethylamine was added. Specifically, the formula ((total response intensity when trimethylamine and blended fragrance are mixed) - (total response intensity of Rho)) / ((total response intensity of trimethylamine alone) - (total response intensity of Rho)) was calculated to obtain the test data.

[0089] [Table 6]

[0090] As shown in Table 6, the results of the response intensity measurement showed that the fruity-like fragrance and soap-like fragrance, each containing the test substance, suppressed the response of mouse TAAR5 to trimethylamine, suggesting that they function as antagonists of mouse TAAR5. These are considered to be inhibitors that readily exhibit a sufficient deodorizing effect against trimethylamine.

[0091] [Sensory Test 2] The deodorizing effect of trimethylamine was tested using the fragrance formulations shown in Table 5. For comparison, each fragrance formulation was also tested with all antagonist substances removed. Sensory evaluation 2 was conducted by 8 evaluators for the fruity fragrance formulation and 7 evaluators for the soapy fragrance formulation. The evaluators were women in their 20s or 30s who had passed a separate olfactory test.

[0092] The following fragrance masterbatches were prepared. Each fragrance formulation was dissolved in water using a surfactant. The fruity fragrance formulation was diluted to 1.2%, and the soapy fragrance formulation to 0.1%, and these were added to a container containing the volatile material. The mixture was then sealed in a 10L bag and filled with odorless air. After standing at room temperature for 30 minutes, a masterbatch was obtained.

[0093] The following evaluation samples were prepared. 20 ml of scented air from each fragrance masterbatch was transferred to a 3 L bag. Then, 0.1 ml of trimethylamine masterbatch was injected into the 3 L bag to obtain each test sample. In addition, 0.1 ml of trimethylamine masterbatch was added to another 3 L bag filled with odorless air (a sample of trimethylamine only).

[0094] The evaluation tests were conducted as follows: Each sample was presented to an evaluator, who scored the trimethylamine intensity according to the following criteria. Table 7 shows the average score of the evaluators' results, comparing the malodor intensity of the sample containing only malodor with the malodor intensity of the evaluation sample in which trimethylamine was injected with scented air.

[0095] [Standards for Odor Intensity] The following evaluation criteria were used to rate scores on a 6-point scale from +5 to 0, in increments of 1 point. +5:Intense +4: Strong +3: Easily detectable +2: Weak +1: Finally detectable. 0: Odorless

[0096] [Table 7]

[0097] In Table 7, "Difference in Odor Intensity" refers to the difference between the average odor intensity evaluation score of trimethylamine and the average odor intensity evaluation score when each fragrance masterbatch is added. A value of 1.0 or higher indicates a deodorizing effect.

[0098] In this test, we also adopted the sensory deodorization test standards set by the Japan Fragrance and Deodorizing Agents Association. As previously mentioned, the Japan Fragrance and Deodorizing Agents Association defines a product as having a "deodorizing effect" if the odor intensity is reduced by one level or more. Based on the results of Sensory Test 2, the fruity-like and soap-like fragrances containing the respective test substances demonstrated a sensory deodorizing effect against trimethylamine, suggesting that they have high potential as deodorants for trimethylamine. [Industrial applicability]

[0099] According to the present invention, an inhibitor of trimethylamine-responsive receptors is provided that is suitable for deodorizing trimethylamine odor by suppressing the response of trimethylamine-responsive receptors.

Claims

1. An inhibitor that suppresses the response of at least one trimethylamine-responsive receptor selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5, trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, 4-[(octahydro-4,7-methano-5H-indene)-5 An inhibitor of trimethylamine-responsive receptors comprising at least one selected from the group consisting of -ylidene]butanal, 3-(3-isopropylphenyl)butanal, p-cymene, nopyruacetate, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, γ-dodecanolactone, and 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol.

2. An inhibitor of a trimethylamine-responsive receptor according to claim 1, comprising at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, citral, Litsea cubeba fruit oil, 3-methyl-5-phenylpentan-1-ol, 4,8-dimethyl-4,9-decadienal, undecanal, ethyl dehydrocyclogeranate, and 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal.

3. An inhibitor of a trimethylamine-responsive receptor according to claim 1, comprising at least one selected from the group consisting of trans-cinnamaldehyde, 3-(1,3-benzodioxol-5-yl-2-methylpropanal, 3-(4-tert-butylphenyl)propanal, trans-2-hexenal, and citral.

4. A deodorant for trimethylamine odor, comprising a trimethylamine-responsive receptor inhibitor according to any one of claims 1 to 3.

5. A method for deodorizing trimethylamine odor using a trimethylamine-responsive receptor inhibitor according to any one of claims 1 to 3.

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