Ammonia-responsive receptor inhibitor, deodorizing agent for ammonia odor, and deodorization method for ammonia odor

Abstract

The present invention provides an ammonia-responsive receptor inhibitor suitable for deodorizing an ammonia odor through inhibition of response of trace amine-associated receptors such as TAAR5.　In one example, the ammonia-responsive receptor inhibitor contains at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, a Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, ethyl dehydrocyclogeranate, 2-ethyl-3-hydroxy-γ-pyrone, citral, 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]butanal, 3-(3-isopropylphenyl)butanal, and ethylene brassylate.

Description

Inhibitor for ammonia-responsive receptors, deodorant for ammonia odor, method for deodorizing ammonia odor

[0001] The present invention relates to an inhibitor for ammonia-responsive receptors, a deodorant for ammonia odor, and a method for deodorizing ammonia odor. The present application claims priority based on Japanese Patent Application No. 2025-49320 filed with the Japan Patent Office on March 25, 2025, the contents of which are incorporated herein by reference.

[0002] Odors in living spaces include human or animal excrement odor, sweat odor, age-related body odor, garbage odor, tobacco odor, and the like, and there are also complex odors formed by a mixture of these odors. Regarding deodorization of these odors, physical deodorization using adsorption treatment and chemical deodorization using neutralization reaction have been proposed. For example, Patent Document 1 discloses a chemical deodorant fragrance composition for ammonia odor, which contains an aromatic component such as an aliphatic aldehyde having 6 to 12 carbon atoms as an active ingredient.

[0003] However, in chemical deodorization as disclosed in Patent Document 1, odor is eliminated by converting ammonia, which causes the odor, into another compound using a deodorant or an adsorbent. Therefore, molecular contact between malodor molecules and the deodorant or the like is required. In consideration of this point, for example, the deodorizing effect on ammonia odor can be exerted over time in an enclosed space, but it is difficult to obtain the deodorizing effect in open spaces or large spaces.

[0004] Response control of olfactory receptors can be effective for obtaining a deodorizing effect. When humans perceive ammonia odor, it is considered that TAAR5, one type of trace amine-associated receptors (TAAR) present in olfactory nerve cells, responds. Patent Document 2 discloses α-damascone and β-damascone as fragrance components that inhibit the activation of TAAR5. Patent Document 3 discloses a method for searching for an ammonia odor inhibitor. It discloses contacting a trace amine-associated receptor with ammonia after mixing a test substance with a trace amine-associated receptor such as 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.

[0005] Japanese Patent Publication No. 2001-303090, Japanese Patent Publication No. 2023-174084, Japanese Patent Publication No. 2023-174085

[0006] PLOS ONE, 2015, DOI: 10.1371 / journal. bone. 0144704.

[0007] However, the vast majority of candidate ligands for trace amine-related receptors such as TAAR5 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 ammonia-responsive receptors suitable for deodorizing ammonia odor by suppressing the response of trace amine-related receptors such as TAAR5.

[0009] The present invention has the following aspects: [1] An inhibitor for suppressing the response of ammonia-responsive receptors, comprising at least one selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5, and comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, ethyl dehydrocyclogeranate, 2-ethyl-3-hydroxy-γ-pyrone, citral, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, 3-(3-isopropylphenyl)butanal, and ethylene glycol brasilate. [2] An ammonia-responsive receptor inhibitor according to [1], comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, and ethyl dehydrocyclogeranate. [3] An ammonia-responsive receptor inhibitor according to [1] or [2], comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, and Litsea cubeba fruit oil. [4] An ammonia-responsive receptor inhibitor according to any one of [1] to [3], comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, and 2-ethyl-3-hydroxy-γ-pyrone. [5] An ammonia odor deodorizer comprising an ammonia-responsive receptor inhibitor according to any one of [1] to [4]. [6] A method for deodorizing ammonia odor using an ammonia-responsive receptor inhibitor according to any one of [1] to [4].

[0010] The present invention provides an inhibitor of ammonia-responsive receptors that is suitable for deodorizing ammonia odor by suppressing the response of trace amine-related receptors such as TAAR5.

[0011] Figure 1 shows the experimental results of measuring the response intensity of ammonia-responsive receptors (NORMALIZED LUMINESCENCE) in an experimental example.

[0012] [Explanation of Terms] "Polypeptide having equivalent function to TAAR5" refers to a polypeptide that can be expressed on the cell membrane and which, upon ligand binding, induces intracellular cAMP production, or promotes the influx of calcium ions from outside the cell into the cell upon ligand binding. "Agonist" is a compound that activates the response of a receptor by binding to that receptor. "Antagonist" is a compound that inhibits the activation of a receptor by an agonist by binding to that receptor. The "~" indicating a numerical range means that the values ​​written 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, the number of amino acid residues of matching amino acids is calculated in the reference amino acid sequence and the target amino acid sequence, 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 ... Equation (1)

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

[0016] [Inhibitors of ammonia-responsive receptors] Inhibitors of ammonia-responsive receptors suppress the response of at least one ammonia-responsive receptor selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5.

[0017] Hereinafter, for the sake of simplicity, in this specification, at least one ammonia-responsive receptor selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5 will be referred to as a "specific ammonia-responsive receptor."

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

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

[0020] Certain ammonia-responsive receptors, such as TAAR5, exhibit a specific response intensity to ammonia. 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 an ammonia-responsive receptor. Examples include homologous ammonia-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 at least one substance selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, ethyl dehydrocyclogeranate, 2-ethyl-3-hydroxy-γ-pyrone, citral, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, 3-(3-isopropylphenyl)butanal, and ethylene glycol brasilate suppresses the response of specific ammonia-responsive receptors such as TAAR5 to ammonia.

[0023] In particular, at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, and ethyl dehydrocyclogeranate is preferred as an inhibitor of ammonia-responsive receptors, as it further improves the inhibitory performance of ammonia-responsive receptors.

[0024] In addition, at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, and Litsea cubeba fruit oil is more preferred as an inhibitor of ammonia-responsive receptors because it significantly improves the inhibitory performance of ammonia-responsive receptors.

[0025] As an inhibitor of ammonia-responsive receptors, 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, and α-methyl-1,3-benzodioxol-5-propanal are more preferred, trans-cinnamaldehyde and α-methyl-1,3-benzodioxol-5-propanal are particularly preferred, and trans-cinnamaldehyde is the most preferred.

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

[0027] Ammonia-responsive receptor inhibitors are thought to function as antagonists of specific ammonia-responsive receptors because they satisfy the following two conditions: • When ammonia-responsive receptors are mixed with the inhibitor, they become less responsive to ammonia when in contact with ammonia compared to when the inhibitor is not mixed. • Ammonia-responsive receptors do not respond to the inhibitor after being mixed with it.

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

[0029] In one embodiment, the ammonia-responsive receptor can be a cell that naturally expresses the ammonia-responsive receptor, a genetically modified cell expressing the ammonia-responsive receptor, or a culture thereof. In particular, the use of human-derived genetically modified cells expressing the ammonia-responsive receptor is preferred. Human-derived genetically modified cells can be prepared, for example, by transforming human cultured cells using a vector incorporating the gene encoding the ammonia-responsive receptor.

[0030] When using ammonia-responsive receptors experimentally, metal ions may be used. The manner in which metal ions are used and their presence are not particularly limited. For example, the following methods can be used: Mixing the ammonia-responsive receptor and the test substance in a metal ion-containing solution. Mixing a membrane carrying the ammonia-responsive receptor or cells or tissues expressing the ammonia-responsive receptor with the test substance while immersed in a metal ion-containing solution. Adding metal ions to a culture medium for cells or tissues expressing the ammonia-responsive receptor, and then adding the test substance. Mixing the metal ion-containing solution together with the test substance in a culture medium for cells or tissues expressing the ammonia-responsive receptor. Examples of metal ions include copper ions and silver ions.

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

[0032] Reference Data 1: Data measuring the response of ammonia-responsive receptors before contact with ammonia in the presence of the test substance. Test Data 1: Data measuring the response of ammonia-responsive receptors after contact with ammonia 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 ammonia-responsive receptors in the presence of the test substance before and after contact with ammonia. For example, if the response of ammonia-responsive receptors is suppressed before and after contact with ammonia, the test substance may be useful as an ammonia odor suppressant.

[0034] Alternatively, to verify the inhibitory performance, the olfactory receptor may be brought into contact with ammonia before mixing the ammonia-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 ammonia-responsive receptors after contact with ammonia but before the addition of the test substance. Test Data 2: Data measuring the response of ammonia-responsive receptors after the addition of the test substance following contact with ammonia.

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

[0037] (Mechanism of Action) Ammonia-responsive receptor inhibitors function as antagonists of specific ammonia-responsive receptors that respond specifically to ammonia. Therefore, an ammonia-responsive receptor inhibitor according to one embodiment can suppress the response of these specific ammonia-responsive receptors.

[0038] Ammonia-responsive receptor inhibitors can suppress the response of specific ammonia-responsive receptors to ammonia. 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 ammonia-responsive receptor inhibitor in the target space at a higher concentration than ammonia. For these reasons, ammonia-responsive receptor inhibitors are useful as active ingredients in ammonia odor deodorizers.

[0039] [Examples of Application of Ammonia-Responsive Receptor Inhibitors] According to one embodiment, an ammonia odor deodorant containing the above-mentioned ammonia-responsive receptor inhibitor is provided. According to the deodorant according to one embodiment, the ammonia-responsive receptor inhibitor binds to the ammonia-responsive receptor, thereby suppressing the ammonia-responsive receptor's response to ammonia. As a result of inhibiting the perception of ammonia odor, a deodorizing effect is exerted.

[0040] Since the inhibitor of ammonia-responsive receptors is an antagonist, it antagonizes ammonia and inhibits the response of ammonia-responsive receptors and the perception of ammonia odor. Therefore, there is less need to maintain a pre-diffusion state unlike conventional physical deodorization and chemical deodorization. Thus, the inhibitor of ammonia-responsive receptors is also suitable for instantaneous deodorization applications such as sprays.

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

[0042] The composition may further contain other components besides the inhibitor of ammonia-responsive receptors, as long as the ammonia odor suppressing effect of the inhibitor of ammonia-responsive receptors is not impaired. Examples of other components include fragrances and additives.

[0043] The fragrance may be a natural fragrance, a single fragrance separated from natural fragrances, a synthesized single fragrance, or a blended fragrance of these. Conventionally known oily fragrances can be used without limitation as the fragrance.

[0044] The fragrance is not particularly limited, and examples include animal fragrances, plant fragrances, synthetic fragrances, and extracted fragrances. One fragrance may be used alone, or two or more fragrances may be used in combination.

[0045] Animal fragrances are not particularly limited, and examples include musk, civet, and dragon's blood incense. Vegetable fragrances are not particularly limited, and examples include abies oil, ajowan oil, almond oil, angelica root oil, parsley oil, bergamot oil, birch tar oil, bois de rose oil, cajeput oil, cananga oil, capsicum oil, caraway oil, cardamom oil, cassia oil, celery oil, cinnamon oil, citronella oil, cognac oil, coriander oil, cumin oil, camphor oil, clove oil, estragole 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. One of animal fragrances and vegetable fragrances may be used alone, or two or more of them may be used in combination.

[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; and aldehyde fragrances such as 3-(4-tert-butylphenyl)propanal, n-butyraldehyde, isobutyraldehyde, hexylaldehyde, citral, citronellal, benzaldehyde, cinnamic aldehyde, and cumin aldehyde. Examples of artificial fragrances include ketone-based fragrances such as 1-(2,3,8,8-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. Additives may be used individually or in combination of two or more.

[0048] In the case of a composition, the content of the ammonia-responsive receptor inhibitor is preferably at least 0.03% by mass, more preferably 0.1% by mass, and even more preferably 1.0% by mass. If the content of the ammonia-responsive receptor inhibitor is above the lower limit, it is considered sufficient to deodorize ammonia odor by suppressing the response of ammonia-responsive receptors when used as a deodorant.

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

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

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

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

[0053] Examples of applicable applications include, for example, toilets or waste disposal for humans and animals, waste disposal in medical and nursing care facilities, waste disposal, disposable diapers, sanitary products, fishing facilities, seafood processing facilities, odor treatment in medical and nursing care facilities, garbage cans, kitchen spaces, bathrooms, odor treatment in nursing care spaces, odor treatment specifically for food waste, clothing such as underwear, masks, face shields, and linens, textiles, fabrics, laundry detergents, fabric softeners, permanent wave treatments, cosmetics, cleaning agents, deodorants and other topical agents, pharmaceuticals, food products, and manufacturing facilities for products that generate ammonia odor. However, the application of ammonia-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 Ammonia-Responsive Receptor-Expressing Cells] Based on sequence information registered in GenBank, genes encoding olfactory receptors listed in Table 1 were cloned. Each gene was cloned by PCR using 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. Furthermore, the receptor gene was incorporated downstream of the Rho tag sequence using the MuI 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 MuI restriction enzyme site and NotI restriction enzyme site of the pCI vector. Hereafter, cells that do not incorporate the gene encoding the olfactory receptor will be referred to as "Rho".

[0057]

[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, the cells were incubated at 37°C and 5% CO2. ２ Hana3A cells expressing each of the 18 human and mouse receptors shown in Table 1 were prepared by culturing them for 24 hours in an incubator maintained at a controlled atmosphere. The amount of TAAR gene added was varied from 1 to 50 μg depending on the ease of expression of each gene.

[0059]

[0060] [Glo-Sensor Assay] The response of the ammonia-responsive receptor was measured by the Glo-Sensor assay. Ammonia was used as the malodorous molecule. The ammonia-responsive receptor expressed in Hana3A cells is coupled with intrinsic Gαs and Gαolf and activates adenylyl cyclase, thereby increasing the amount of intracellular cAMP. The increase in intracellular cAMP was measured as a luminescence value derived from the firefly luciferase gene, and the response intensity of the ammonia-responsive receptor was determined.

[0061] Luciferase activity was measured according to the product protocol for Glo Sensor cAMP Reagent (Promega). For various stimuli, the luminescence value derived from luciferase after odor stimulation was divided by the luminescence value derived from luciferase before odor stimulation. That is, (luminescence value after stimulation) / (luminescence value before stimulation) was calculated. For ammonia stimulation, (luminescence value after stimulation) / (luminescence value before stimulation) was used as the measured response intensity.

[0062] [Searching for ammonia-responsive receptors] TAAR5 is known, as described in Patent Documents 2 and 3. 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 to 3 hours to introduce cAMP Reagent into the cells.

[0063] Next, a 96-well plate and an air circulation fan were placed in a 5L Flex Sampler bag and filled with pure air. Finally, ammonia was injected using a syringe to achieve a final gaseous concentration of 20 ppm as an odor molecule. Olfactory receptor-expressing cells were exposed to the ammonia for 10 minutes. Subsequently, the response intensity of ammonia-responsive receptors (NORMALIZED LUMINESCENCE) was measured by a Glo Sensor assay. The results are shown in Figure 1.

[0064] The vertical axis in Figure 1 shows the relative response intensity to ammonia in cells expressing each receptor (Figure 1). The relative response intensity was calculated by setting the sum of response in 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. As a result, TAAR5 was identified as the ammonia-responsive receptor showing the highest responsiveness to ammonia. Furthermore, it was found that mouse TAAR5, a mouse homolog of human TAAR5, also responds strongly to ammonia (Figure 1).

[0065] [Test Substances] The following compounds were diluted with dimethyl sulfide to prepare a final concentration of 100 mM as test substances (compounds): • trans-cinnamaldehyde • α-methyl-1,3-benzodioxol-5-propanal • 3-(4-tert-butylphenyl)propanal • Litsea cubeba fruit oil • 1,1-dimethyl-2-phenylethyl acetate • trans-2-hexenal • ethyl dehydrocyclogeranate • 2-ethyl-3-hydroxy-γ-pyrone • citral • 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal • 3-(3-isopropylphenyl)butanal • ethylene glycol brasilate • 2,2,6-trimethyl-α-propylcyclohexane-1-propanol

[0066] [Searching for TAAR5 Antagonists] The culture medium was removed from the culture of mouse TAAR5-expressing cells. 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 cAMP Reagent into the cells. Subsequently, the luminescence value of mouse TAAR5 before the addition of the test substance was measured by a GloSensor assay.

[0067] 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. After 5 minutes, a GloSensor assay was performed to measure the response intensity (fold increase) of mouse TAAR5 to the test substance, and baseline data was obtained.

[0068] To make it easier to observe the responsiveness, it is useful to reduce the amount of liquid in the wells. In this experiment, after measuring the reference data, the lid of the 96-well plate was removed and the plate was inverted to discard the liquid in each well. At this time, some of the liquid in each well remained attached to the bottom, and the amount of liquid in the wells was reduced by returning the 96-well plate to its original orientation.

[0069] Next, a 96-well plate with reduced well volume and an air circulation fan were placed in a 5L Flex Sampler bag and filled with pure air. Finally, ammonia was injected using a syringe to achieve a final gaseous concentration of 20 ppm as an odor molecule. Mouse TAAR5 was exposed to the ammonia gas for 10 minutes, and then a GloSensor assay was performed to measure the response intensity (fold increase) of mouse TAAR5 to the ammonia gas.

[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 ammonia and the test substance were mixed was calculated as (luminescence value 10 minutes after ammonia stimulation) / (luminescence value 5 minutes after adding the test substance). Next, the response intensity of Rho without the test substance was set to 0, and the obtained response intensity was divided by the response intensity when only ammonia was added. That is, ((response intensity when ammonia and test substance are mixed) - (response intensity of Rho)) / ((response intensity of ammonia alone) - (response intensity of Rho)) was calculated to obtain the test data. The measurement results of the response intensity are shown in Table 3.

[0072]

[0073] In the test data shown 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 ammonia-responsive receptors is considered to be.

[0074] From the results of the response intensity measurements shown in Table 3, one or more substances selected from 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, ethyl dehydrocyclogeranate, 2-ethyl-3-hydroxy-γ-pyrone, citral, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, 3-(3-isopropylphenyl)butanal, or ethylene glycol brasilate suppressed the response of TAAR5 to ammonia, suggesting that they function as antagonists of TAAR5. These are considered to be inhibitors that readily exhibit sufficient deodorizing effects against ammonia odor.

[0075] In contrast, no inhibitory effect of TAAR5 on the ammonia response was observed with 2,2,6-trimethyl-α-propylcyclohexane-1-propanol.

[0076] [Test substances used in sensory evaluation 1] The following test substances were used to test the deodorizing effect on ammonia odor: 3-(4-tert-butylphenyl)propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, 2-ethyl-3-hydroxy-γ-pyrone, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, γ-dodecanolactone, 3,3-dimethyl-5-(2,2,3-trimethylcyclopenta-3-en-1-yl)penta-4-en-2-ol

[0077] [Contents of Sensory Test 1] The sensory test was conducted by 10 evaluators. The 10 evaluators consisted of men and women in their 20s or 30s who had passed a separate olfactory test.

[0078] The scented air samples were prepared as follows: A 3cm strip of test paper was prepared and soaked with one drop (approximately 0.02g) of each test substance. The test paper strips were then sealed in a 3L bag and filled with odorless air. The bag was then left to stand overnight at room temperature to obtain the scented air samples for each sample.

[0079] The evaluation sample was prepared as follows: Ammonia (2.06% / N) in a 3L bag. ２ 10 mL of fragrant air and odorless air were injected to adjust the ammonia concentration in the bag. To equalize the intensity of the fragrant air for each sample, the amounts of fragrant air listed in Table 4 were injected into the bag to obtain evaluation samples. Furthermore, a 3 L bag filled with odorless air was placed in another bag containing ammonia (2.06% / N). ２ ) 10 mL was added (a sample containing only ammonia).

[0080] The evaluation test was conducted as follows. Each sample was presented to the evaluators. Each evaluator scored the "odor intensity" according to the following criteria. Table 4 shows the average score of 11 evaluators regarding the difference in odor intensity between a sample containing only ammonia and a sample in which scented air was injected into ammonia.

[0081] [Criteria for Odor Intensity] The following evaluation criteria were used to evaluate odors on a 6-point scale from +5 to 0 in 1-point increments: +5: Intense +4: Strong +3: Easily detectable +2: Weak +1: Barely detectable 0: Odorless

[0082]

[0083] 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.

[0084] In this test, the sensory deodorization test standards set by the Japan Fragrance and Deodorizing Agents Association were adopted. The Japan Fragrance and Deodorizing Agents Association defines "deodorizing effect" as a reduction of one level or more in malodor intensity. From the results of the sensory test, the sensory deodorizing effect on ammonia odor was confirmed for 3-(4-tert-butylphenyl)propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, 2-ethyl-3-hydroxy-γ-pyrone, γ-dodecanolactone, and 3,3-dimethyl-5-(2,2,3-trimethylcyclopenta-3-en-1-yl)penta-4-en-2-ol. Among these, 3-(4-tert-butylphenyl)propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, and 2-ethyl-3-hydroxy-γ-pyrone are also antagonists of ammonia-responsive receptors, and are therefore considered promising candidate compounds for deodorizing ammonia odor by suppressing the response of trace amine-related receptors such as TAAR5.

[0085] [Confirmation of the inhibitory effect of blended fragrances on mouse TAAR5] As shown in Table 5 below, fruity-like blended fragrances were prepared by combining each of the test substances. The blended fragrances contain the compounds indicated by "+" in Table 5. The fruity-like blended fragrances were provided by Takasago International Corporation.

[0086]

[0087] 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 from which all antagonist-effect test substances were removed. After adding 5 μL of the fragrance formulation at the concentrations listed in Table 6 to each well, the response intensity was measured. The response intensity when ammonia and the test substance were mixed was calculated as (luminescence value 15 minutes after ammonia stimulation) / (luminescence value 5 minutes after test substance addition). Next, the response intensity of Rho without the test substance was set to 0, and the obtained response intensity was divided by the response intensity when only ammonia was added. That is, ((response intensity when ammonia and test substance are mixed) - (response intensity of Rho)) / ((response intensity of ammonia alone) - (response intensity of Rho)) was calculated to obtain the test data.

[0088]

[0089] As shown in Table 6, the results of the response intensity measurement showed that the fruity fragrance formulations containing each test substance suppressed the response of mouse TAAR5 to ammonia, suggesting that they function as antagonists of mouse TAAR5. These are considered to be inhibitors that readily exhibit a sufficient deodorizing effect against ammonia.

[0090] [Sensory Test 2] The deodorizing effect of ammonia was tested using the fragrance formulations shown in Table 5. For comparison, each fragrance formulation was also tested in which all the test substances exhibiting antagonist effects were removed. Sensory Test 2 was conducted by nine evaluators. The evaluators consisted of women in their 20s or 30s who had passed a separate olfactory test.

[0091] The fragrance masterbatch was prepared as follows: The fragrance mixture was dissolved in water using a surfactant, diluted to 1.2%, and added to a container containing the volatile material. This mixture was then sealed in a 10L bag and filled with odorless air. After standing at room temperature for 30 minutes, the masterbatch was obtained.

[0092] The evaluation sample was prepared as follows: 20 ml of the fragrance masterbatch was transferred to a 3 L bag. Then, ammonia (2.06% / N) was added. ２ 30 mL was injected into a 3 L bag and filled with odorless air. In another bag, a 3 L bag filled with odorless air was added to ammonia (2.06% / N). ２ 30 mL was added (a sample containing only ammonia).

[0093] The evaluation test was conducted as follows. Each sample was presented to an evaluator, and each evaluator scored the ammonia intensity according to the following criteria. Table 7 shows the average score of the evaluators' results, which represents the difference between the odor intensity of the sample containing only malodorous odor and the odor intensity of the evaluation sample in which ammonia was injected with scented air.

[0094] [Criteria for Odor Intensity] The following evaluation criteria were used to evaluate odors on a 6-point scale from +5 to 0 in 1-point increments: +5: Intense +4: Strong +3: Easily detectable +2: Weak +1: Barely detectable 0: Odorless

[0095]

[0096] In Table 7, "Difference in Odor Intensity" refers to the difference between the average odor intensity evaluation score for ammonia and the average odor intensity evaluation score when the fragrance masterbatch is added. A value of 1.0 or higher indicates a deodorizing effect.

[0097] In this test, we adopted the sensory deodorization test standards set by the Fragrance and Deodorizing Agents Association. As already mentioned, the Fragrance and Deodorizing Agents Association defines "deodorizing effect" as a reduction of one level or more in malodor intensity. From the results of Sensory Test 2, the fruity-like fragrance blends containing each test substance were confirmed to have a sensory deodorizing effect against ammonia, and are considered to have high value as a deodorizer for ammonia.

[0098] The present invention provides an inhibitor of ammonia-responsive receptors that is suitable for deodorizing ammonia odor by suppressing the response of trace amine-related receptors such as TAAR5.

Claims

1. An inhibitor of the ammonia-responsive receptor, comprising at least one selected from the group consisting of TAAR5 and polypeptides having equivalent function to TAAR5, and including at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, ethyl dehydrocyclogeranate, 2-ethyl-3-hydroxy-γ-pyrone, citral, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, 3-(3-isopropylphenyl)butanal, and ethylene glycol brasilate.

2. An ammonia-responsive receptor inhibitor according to claim 1, comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, trans-2-hexenal, and ethyl dehydrocyclogeranate.

3. An ammonia-responsive receptor inhibitor according to claim 1, comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, and Litsea cubeba fruit oil.

4. An ammonia-responsive receptor inhibitor according to claim 1, comprising at least one selected from the group consisting of 3-(4-tert-butylphenyl)propanal, Litsea cubeba fruit oil, 1,1-dimethyl-2-phenylethyl acetate, and 2-ethyl-3-hydroxy-γ-pyrone.

5. A deodorant for ammonia odor, comprising an ammonia-responsive receptor inhibitor according to any one of claims 1 to 4.

6. A method for deodorizing ammonia odor using an ammonia-responsive receptor inhibitor according to any one of claims 1 to 4.

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