Cat urine odor receptor inhibitor, deodorant against cat urine odor, method for eliminating cat urine odor, and method for searching for cat urine odor inhibitor

Abstract

One embodiment of the present invention provides a cat urine odor receptor inhibitor that is suitable for eliminating cat urine odor by suppressing the response of a cat urine odor receptor exhibiting specific responsiveness to cat urine odor.　In a preferred example, the cat urine odor receptor inhibitor includes at least one selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxol-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, and 3-(4-tert-butylphenyl)propanal. The cat urine odor receptor is at least one selected from the group consisting of TAAR5 and polypeptides functionally equivalent to TAAR5.

Description

Inhibitor of cat urine odor receptor, deodorizer for cat urine odor, method for deodorizing cat urine odor, and method for searching for cat urine odor inhibitor

[0001] The present invention relates to an inhibitor of cat urine odor receptors, a deodorizer for cat urine odor, a method for deodorizing cat urine odor, and a method for searching for an inhibitor of cat urine odor.This application claims priority to Japanese Patent Application No. 2024-123631, filed on July 30, 2024, the entire contents of which are incorporated herein by reference.

[0002] The odor of animal excrement, particularly cat urine, is said to be stronger than the odor of human or dog urine, and various deodorizing products have been proposed (for example, Patent Documents 1 and 2). However, conventional methods such as those described in Patent Documents 1 and 2 are not effective in deodorizing open or large spaces.

[0003] In recent years, the use of olfactory receptor response control has been proposed to obtain a more excellent deodorizing effect (for example, Patent Documents 3 and 4).

[0004] Patent Document 3 discloses a cat urine odor suppressant for cats. The cat urine odor suppressant of Patent Document 3 contains at least one active ingredient selected from the group consisting of acetylcedrene and florhydral.

[0005] Patent Document 4 discloses a method for searching for cat urine odor suppressants. The searching method in Patent Document 4 includes adding a test substance and a substance that causes cat urine odor to at least one olfactory receptor polypeptide selected from the group consisting of OR2M3 and polypeptides having equivalent functions thereto, measuring the response of the olfactory receptor polypeptide to the substance that causes cat urine odor, and identifying a test substance that suppresses the response of the olfactory receptor polypeptide based on the measured response.

[0006] JP 2007-229151 A JP 2005-65750 A JP 2022-18279 A JP 2020-112520 A

[0007] In Patent Documents 3 and 4, 3-mercapto-3-methylbutyl formate and 3-mercapto-3-methyl-1-butanol, which are compounds specifically contained in cat urine, are used as the substances that cause cat urine odor. However, because the actual cat urine odor is a complex odor caused by multiple types of compounds, a search method using specific compounds as in Patent Documents 3 and 4 will overlook compounds that originally function as cat urine odor suppressants.

[0008] In addition, the number of cat urine odor receptor candidates that specifically respond to cat urine odor is vast, and most of them are unknown. The number of cat urine odor receptor ligand candidates is also vast.

[0009] One aspect of the present invention provides an inhibitor of cat urine odor receptors, which is suitable for deodorizing cat urine odor by suppressing the response of cat urine odor receptors that exhibit specific responsiveness to cat urine odor.

[0010] Another aspect of the present invention provides a method for searching for cat urine odor inhibitors, which allows comprehensive analysis of inhibitors of cat urine odor receptors.

[0011] The present invention has the following aspects: [1] An inhibitor that suppresses the response of at least one cat urine odor receptor selected from the group consisting of TAAR5 and polypeptides having a function equivalent to TAAR5, and is selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litsea Cubeba fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, 4,8-dimethyl-4,9-decadienal, and 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]butanal. a cat urine odor receptor inhibitor comprising at least one selected from the group consisting of 2-ethyl-3-hydroxy-γ-pyrone, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, and 3-methyl-5-phenylpentan-1-ol. [2] trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litsea Cubeba fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentanal The cat urine odor receptor inhibitor according to [1], which contains at least one selected from the group consisting of 4-en-2-ol, 4,8-dimethyl-4,9-decadienal, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citronellyl butyrate, 1,1-dimethyl-2-phenylethyl acetate, p-cymene, γ-dodecanolactone, and 2-ethyl-3-hydroxy-γ-pyrone.[3] The cat urine odor receptor inhibitor according to [1] or [2], which contains at least one selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, and 3-(4-tert-butylphenyl)propanal. [4] trans-2-hexenal, 2-ethyl-3-hydroxy-γ-pyrone, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citral, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, trans-cinnamaldehyde, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 3-(4-tert-butylphenyl)propanal, 3-(3-isopropylphenyl)butanal, α-damascone, ethyl dehydrocyclogeranate, Litsea Cubeba fruit oil, and α-methyl-1,3-benzodioxole-5-propanal. The cat urine odor receptor inhibitor according to [1], comprising at least one selected from the group consisting of: [5] A deodorizer for cat urine odor, comprising the inhibitor of cat urine odor receptors according to any one of [1] to [4]. [6] A method for deodorizing cat urine odor, using the inhibitor of cat urine odor receptors according to any one of [1] to [5].

[0012] [7] A method for searching for cat urine odor suppressants, comprising mixing a test substance with at least one olfactory receptor selected from the group consisting of TAAR5 and polypeptides having a function equivalent to TAAR5. [8] The method of searching described in [7], further comprising contacting the olfactory receptor with cat urine after mixing the olfactory receptor with the test substance. [9] The method of searching described in [7], further comprising contacting the olfactory receptor with cat urine before mixing the olfactory receptor with the test substance.

[0013] According to one aspect of the present invention, there is provided an inhibitor of cat urine odor receptors that is suitable for deodorizing cat urine odor by suppressing the response of cat urine odor receptors that exhibit specific responsiveness to cat urine odor.

[0014] Another aspect of the present invention provides a method for searching for cat urine odor inhibitors, which allows comprehensive analysis of inhibitors of cat urine odor receptors.

[0015] Figure 1 shows the results of an experiment measuring the receptor response intensity (normalized luminescence) to cat urine, and Figure 2 shows the results of an experiment measuring the concentration dependency of the receptor response intensity to cat urine.

[0016] [Explanation of Terms] A "polypeptide having a function equivalent to TAAR5" refers to a polypeptide that can be expressed on a cell membrane and that, upon binding to one or more compounds contained in cat urine, induces the production of cAMP within the cell, or promotes the influx of calcium ions from the outside into the cell. An "agonist" is a compound that activates the response of a receptor by binding to that receptor. An "antagonist" is a compound that inhibits the activation of that receptor by an agonist by binding to a receptor. The symbol "to" indicating a numerical range means that the numbers before and after it are included as the lower and upper limits.

[0017] The sequence identity (homology) of an amino acid sequence can be determined as the sequence identity of a subject amino acid sequence to a reference amino acid sequence as follows: First, the reference amino acid sequence and the subject 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 the matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (1):

[0018] Sequence identity (%) = (number of matching amino acid residues / total number of amino acid residues in the target amino acid sequence) × 100 Formula (1)

[0019] Hereinafter, the embodiments of the present invention will be described in detail. However, the following description relates to some examples of embodiments of the present invention and is not limited to these examples.

[0020] [Cat urine odor receptor inhibitor] In one embodiment, the cat urine odor receptor inhibitor inhibits the response of at least one cat urine odor receptor selected from the group consisting of TAAR5 and polypeptides having a function equivalent to TAAR5.

[0021] Hereinafter, in this specification, an olfactory receptor for cat urine odor will be abbreviated as a "cat urine odor receptor." Additionally, an olfactory receptor for at least one cat urine odor selected from the group consisting of TAAR5 and polypeptides having a function equivalent to TAAR5 will be referred to as a "specific cat urine odor receptor."

[0022] The present inventors investigated various olfactory receptors and found that specific cat urine odor receptors, such as TAAR5, exhibit specific response strength to cat urine. TAAR5 has been confirmed to be expressed in human olfactory receptor neurons. Human TAAR5 is registered in GenBank (NCBI) under Gene ID: 9038. Human TAAR5 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0023] The cat urine odor receptor may be mouse TAAR5. Mouse TAAR5 is registered in GenBank (NCBI) under Gene ID: 215854. Mouse TAAR5 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0024] Specific cat urine odor receptors such as TAAR5 show specific response to cat urine odor.TAAR5 can be replaced with the polypeptide with the same function as TAAR5.The amino acid sequence of the polypeptide with the same function as TAAR5 preferably shows 80% or more homology with the amino acid sequence of TAAR5, more preferably shows 85% or more homology, more preferably shows 90% or more homology, even more preferably shows 95% or more homology, particularly preferably shows 98% or more homology, and most preferably shows 99% or more homology.

[0025] In addition to TAAR5, any polypeptide with the same function can be used as cat urine odor receptor.For example, include homologous cat urine odor receptors from animals other than humans and mice.For example, include animals other than humans and mice, such as rats and other experimental model organisms.

[0026] The present inventors have tested and investigated various substances and found that trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litopentas crescens fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, 4,8-dimethyl-4,9-decadienal, 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]propanal, methyl methyl cyclopenta-3-en-1-yl]pent-4-en-2-ol ... We have found that each of the following substances suppresses the response of specific cat urine odor receptors, such as TAAR5, to the odor of cat urine: 1,1-dimethyl-2-phenylethyl acetate, p-cymene, γ-dodecanolactone, 2-ethyl-3-hydroxy-γ-pyrone, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, and 3-methyl-5-phenylpentan-1-ol.

[0027] Among these, trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litsea Cubeba fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopentasiloxane), 3-(4-tert-butyl ... At least one selected from the group consisting of (octahydro-4,7-methano-5H-inden)-5-ylidene]butanal, 4,8-dimethyl-4,9-decadienal, 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]butanal, citronellyl butyrate, 1,1-dimethyl-2-phenylethyl acetate, p-cymene, γ-dodecanolactone, and 2-ethyl-3-hydroxy-γ-pyrone is preferred as the inhibitor of cat urine odor receptors.

[0028] Additionally, at least one selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, and 3-(4-tert-butylphenyl)propanal is more preferred as an inhibitor of the cat urine odor receptor, as it provides a more significant improvement in the inhibitory performance of the cat urine odor receptor.

[0029] As the inhibitor of the cat urine odor receptor, trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, and citral are more preferred, trans-2-hexenal and trans-cinnamaldehyde are particularly preferred, and trans-2-hexenal is most preferred.

[0030] The inhibitor of cat urine odor receptors may be used alone or in combination of two or more.

[0031] The cat urine odor receptor inhibitor of one embodiment is believed to function as an antagonist of a specific cat urine odor receptor because it satisfies the following two requirements: - After mixing with the inhibitor, the cat urine odor receptor is less likely to respond to the cat urine odor when it comes into contact with the cat urine odor compared to when the inhibitor is not mixed in. - After mixing with the inhibitor, the cat urine odor receptor does not respond to the inhibitor.

[0032] When verifying the inhibitory effect of an inhibitor, a cat urine odor receptor can be used experimentally. The cat urine odor receptor can be used in any manner as long as it does not lose its responsiveness to cat urine. For example, the cat urine odor receptor can be used in the following manner: cells or tissues naturally expressing the cat urine odor receptor and their cultures; membranes of olfactory receptor cells carrying the cat urine odor receptor; genetically modified cells expressing the cat urine odor receptor and their cultures; membranes of genetically modified cells expressing the cat urine odor receptor; lipid bilayer membranes expressing the cat urine odor receptor; tissues containing olfactory mucus (such as the olfactory epithelium or olfactory mucosa) can also be used as the cat urine odor receptor.

[0033] In one embodiment, the cat urine odor receptor is preferably a cell that naturally expresses the cat urine odor receptor, a genetically modified cell that expresses the cat urine odor receptor, or a culture of these. It is particularly preferred to use a human-derived genetically modified cell that expresses the cat urine odor receptor. Human-derived genetically modified cells can be prepared, for example, by transforming cultured human cells with a vector incorporating a gene encoding the cat urine odor receptor.

[0034] Metal ions may be used when using cat urine odor receptors experimentally. There are no particular limitations on the manner in which the metal ions are used or present. For example, the following methods are envisioned: - A method in which the cat urine odor receptor is mixed with a test substance in a metal ion-containing solution. - A method in which a membrane carrying the cat urine odor receptor or cells or tissues expressing the cat urine odor receptor is immersed in a metal ion-containing solution and mixed with the test substance. - A method in which metal ions are added to a culture medium in which cells or tissues expressing the cat urine odor receptor are cultured, and then the test substance is added. - A method in which a metal ion-containing solution is mixed with a test substance into a culture medium in which cells or tissues expressing the cat urine odor receptor are cultured. Examples of metal ions include copper ions and silver ions.

[0035] To observe the responsiveness of the cat urine odor receptor, the volume of the buffer solution may be reduced. For example, when using a 96-well plate, the cat urine odor receptor and the cat urine odor can be contacted under conditions where 25 μL or less of the buffer solution is used. If the volume of the buffer solution is relatively small, the cat urine odor is less likely to be converted by the action of the buffer solution, making it easier to observe the responsiveness.

[0036] When considering application to cat urine odor deodorizers and deodorizing methods, it is more preferable to use inhibitors whose deodorizing effects have been confirmed in sensory tests.

[0037] (Action and Effect) The cat urine odor receptor inhibitor functions as an antagonist of specific cat urine odor receptors that specifically respond to the odor of cat urine, and therefore can suppress the response of these specific cat urine odor receptors.

[0038] Cat urine odor receptor inhibitors can suppress the response of specific cat urine odor receptors to cat urine. Therefore, the concentration required to achieve the same level of deodorizing effect in the target space can likely be set lower than in the case of conventional physical or chemical deodorization. Therefore, there is no need to present the cat urine odor receptor inhibitor in the target space at a higher concentration than cat urine. For these reasons, cat urine odor receptor inhibitors are useful as active ingredients in cat urine odor deodorizers.

[0039] [Application Example of Cat Urine Odor Receptor Inhibitor] According to one embodiment, a cat urine odor deodorizer is provided that contains the cat urine odor receptor inhibitor described above. According to one embodiment of the deodorizer, the cat urine odor receptor inhibitor binds to the cat urine odor receptor, thereby inhibiting the response of the cat urine odor receptor to cat urine. As a result of inhibiting the perception of cat urine odor, a deodorizing effect is exerted.

[0040] Since cat urine odor receptor inhibitors are antagonists, they compete with cat urine to inhibit the response of cat urine odor receptors and the perception of cat urine odor. Therefore, there is less need to maintain a diffused state beforehand, as is the case with conventional physical and chemical deodorizers. Therefore, cat urine odor receptor inhibitors are also suitable for instantaneous deodorizing applications such as sprays.

[0041] The deodorizer may be in the form of a composition or may be comprised of an inhibitor of cat urine odor receptors. In the case of a composition, the inhibitor of cat urine odor receptors is contained in the composition as an active ingredient for suppressing cat urine odor.

[0042] The composition may further contain other ingredients besides the cat urine odor receptor inhibitor, such as fragrances and additives, as long as the cat urine odor suppressing effect of the cat urine odor receptor inhibitor is not impaired.

[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. Conventionally known oil-based fragrances can be used without any restrictions as the fragrance.

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

[0045] The animal-derived fragrances are not particularly limited, but examples thereof include musk, spirit cat fragrance, and dragon jasmine fragrance.

[0046] The vegetable flavorings are not particularly limited, but examples thereof include abies oil, accion oil, almond oil, angelica root oil, peper oil, bergamot oil, perch oil, bois basil oil, kajabuchi 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, dill 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. The animal flavorings and vegetable flavorings may be used alone or in combination of two or more.

[0047] Artificial fragrances such as synthetic fragrances and extracted fragrances are not particularly limited, but examples thereof 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 3-(4-tert-butylphenyl)propanal, n-butyraldehyde, isobutyraldehyde, hexylaldehyde, citral, citronellal, benzaldehyde, cinnamic aldehyde, and cumin aldehyde; Examples of suitable artificial fragrances include ketone fragrances such as 1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethan-1-one, carvone, menthone, camphor, acetophenone, and ionone; lactone fragrances such as γ-butyrolactone, coumarin, and cineole; ester fragrances such as methyl dihydrojasmonate, 1,1-dimethyl-2-phenylethyl acetate, 2,2,2-trichloro-1-phenylethyl acetate, hexyl acetate, octyl acetate, benzyl acetate, styrallyl acetate, cinnamyl acetate, linalyl acetate, butyl propionate, and methyl benzoate; and benzopyran fragrances such as galaxolide. These artificial fragrances may be used singly or in combination. The additives may be used alone or in combination of two or more.

[0048] The content of the cat urine odor receptor inhibitor in the blended fragrance is preferably at least 1% by mass, more preferably 5% by mass or more, and even more preferably 10% by mass or more. If the content of the cat urine odor receptor inhibitor is equal to or greater than the lower limit, it is considered that when used as a deodorant, it is sufficient to deodorize the cat urine odor by suppressing the response of the cat urine odor receptor.

[0049] The deodorizer may be carried to the object to be suppressed from cat urine odor, may be placed in a space where cat urine odor may occur, or may be mixed with a substance that may cause cat urine odor.

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

[0051] Another embodiment provides a method for suppressing cat urine odor using a cat urine odor receptor inhibitor. In the method for suppressing cat urine odor, the cat urine odor receptor inhibitor is applied to a subject (individual) whose perception of cat urine odor is to be suppressed. The cat urine odor receptor inhibitor may be applied before the subject is exposed to cat urine, after the subject is exposed to cat urine, or simultaneously with the subject being exposed to cat urine.

[0052] In one embodiment, a cat urine odor receptor inhibitor is applied to a subject whose cat urine odor is to be inhibited before the subject is exposed to cat urine. The applied cat urine odor receptor inhibitor inhibits the response of the cat urine odor receptor of the subject. As a result, even if the subject is exposed to cat urine, the response of the cat urine odor receptor to cat urine is inhibited, and the perception of the cat urine odor is inhibited.

[0053] Examples of applications include animal toilets or excrement treatment, animal medical facilities, excrement treatment at commercial facilities, waste treatment, odor treatment at animal medical facilities and commercial facilities, odor treatment at trash cans, fabric products, cosmetics, detergents, external preparations such as deodorants, etc. However, the applications of the inhibitor of cat urine odor receptors are not limited to these examples.

[0054] [Method for searching for cat urine odor inhibitors] The method for searching for cat urine odor inhibitors involves mixing a cat urine odor receptor with a test substance. If the test substance inhibits the response of the cat urine odor receptor when added to the cat urine odor receptor, the test substance is likely to exhibit a sufficient deodorizing effect against cat urine odor. A test substance that inhibits the response of the cat urine odor receptor to cat urine is considered to be useful as a cat urine odor inhibitor. Unlike Patent Documents 3 and 4, this embodiment uses actual cat urine, allowing for comprehensive analysis of inhibitors of the cat urine odor receptor. As a result, compounds that function as cat urine odor inhibitors are less likely to be overlooked.

[0055] (Mixing of cat urine odor receptor with test substance) When mixing the cat urine odor receptor with the test substance, the cat urine odor receptor may be the cat urine odor receptor after contact with cat urine, or the cat urine odor receptor before contact with cat urine.

[0056] By mixing the cat urine odor receptor with a test substance, data can be obtained to determine whether the test substance functions as an antagonist of the cat urine odor receptor. If the test substance functions as an antagonist, it can be expected to be useful as a cat urine odor suppressant.

[0057] If the cat urine odor receptor after mixing with the test substance satisfies the following two conditions, the test substance can be considered to function as an antagonist: - The cat urine odor receptor after mixing with the test substance is less likely to respond to cat urine than when the test substance is not mixed. - The cat urine odor receptor after mixing with the test substance does not respond to the test substance.

[0058] The cat urine odor receptor can be used in any manner as long as it does not lose its responsiveness to cat urine.For example, the cat urine odor receptor can be used in the following manner: cells or tissues naturally expressing the cat urine odor receptor and their cultures; membranes of olfactory receptor cells carrying the cat urine odor receptor; genetically modified cells expressing the cat urine odor receptor and their cultures; membranes of genetically modified cells expressing the cat urine odor receptor; lipid bilayer membranes expressing the cat urine odor receptor.Tissues having olfactory mucus (olfactory epithelium, olfactory mucosa, etc.) can also be used as the cat urine odor receptor.

[0059] In one embodiment, the cat urine odor receptor is preferably a cell that naturally expresses the cat urine odor receptor, a genetically modified cell that expresses the cat urine odor receptor, or a culture of these. It is particularly preferred to use a human-derived genetically modified cell that expresses the cat urine odor receptor. Human-derived genetically modified cells can be prepared, for example, by transforming cultured human cells with a vector incorporating a gene encoding the cat urine odor receptor.

[0060] The specific method for measuring the response of the cat urine odor receptor is not particularly limited. For example, the amount of intracellular cAMP can be measured. The response of the cat urine odor receptor can be evaluated by using the amount of intracellular cAMP as an indicator of the response of the cat urine odor receptor. Examples of methods for measuring the amount of intracellular cAMP include ELISA and reporter gene assay.

[0061] Other examples include calcium imaging and electrophysiological measurements. In electrophysiological measurements, for example, test cells (e.g., Xenopus oocytes) co-expressing the cat urine odor receptor with other ion channels may be prepared, and the action potential of the ion channel on the test cell may be measured by patch clamping, two-electrode voltage clamping, or the like.

[0062] The test substance is a substance to be tested to determine its suitability as an inhibitor of cat urine odor receptors. Therefore, the test substance is not particularly limited. The test substance is typically a substance desired to be used as an inhibitor of cat urine odor. The test substance may be a naturally occurring substance or a synthetic substance.

[0063] Considering that the cat urine odor suppressant will be commercialized as a deodorizer, the test substance is preferably a substance that gives off a different odor from the cat urine odor, and more preferably a volatile substance. It is also preferable to use an odorless test substance or a test substance with a low odor intensity.

[0064] The test substance may be a single substance or a mixture containing two or more substances. For example, a single fragrance substance or a blend of fragrance substances may be used.

[0065] The method for adding the test substance is not particularly limited. The test substance may be mixed into the culture medium of the cells expressing the cat urine odor receptor. The test substance may be dropped, sprinkled, or sprayed onto the cells, tissues, etc. expressing the cat urine odor receptor.

[0066] When adding a test substance to a cat urine odor receptor, it may be mixed in the presence of a metal ion. The manner in which the metal ion is used and the manner in which it is present are not particularly limited. For example, the following methods can be used.

[0067] - A method of mixing a cat urine odor receptor with a test substance in a metal ion-containing solution. - A method of mixing a membrane carrying a cat urine odor receptor or cells or tissues expressing the cat urine odor receptor with a test substance while immersed in a metal ion-containing solution. - A method of adding metal ions to a culture medium for culturing cells or tissues expressing the cat urine odor receptor, and then adding the test substance. - A method of mixing a metal ion-containing solution together with a test substance into a culture medium for culturing cells or tissues expressing the cat urine odor receptor.

[0068] To observe the responsiveness of the cat urine odor receptor, the volume of the buffer solution may be reduced. For example, when using a 96-well plate, the cat urine odor receptor and the cat urine odor can be contacted under conditions where the buffer solution is 25 μL or less. If the volume of the buffer solution is relatively small, the conversion of the cat urine odor due to the action of the buffer solution is less likely to occur, making it easier to observe the responsiveness. For example, when culturing the cat urine odor receptor in a well plate, the well plate is turned upside down with the lid removed. At this time, some of the liquid in the well remains on the bottom of each well, and the volume of liquid in the well can be reduced by turning the well plate upside down. After reducing the volume of liquid in the well, mixing the cat urine odor receptor with the test substance may make it easier to observe the responsiveness of the cat urine odor receptor to the test substance.

[0069] (Contacting the Cat Urine Odor Receptor with Cat Urine) The method for searching for cat urine odor suppressants may further include mixing the cat urine odor receptor with the test substance, and then contacting the cat urine odor receptor with cat urine. In this case, the following Reference Data 1 and Test Data 1 can be obtained.

[0070] Reference data 1: Data measuring the response of the cat urine odor receptor before contact with cat urine in the presence of a test substance. Test data 1: Data measuring the response of the cat urine odor receptor after contact with cat urine in the presence of a test substance.

[0071] The response of the cat urine odor receptor in the presence of the test substance can be evaluated both before and after contact with cat urine by comparing Reference Data 1 and Test Data 1. For example, if the response of the cat urine odor receptor is suppressed both before and after contact with cat urine, the test substance may be useful as a cat urine odor suppressant.

[0072] The method for screening cat urine odor suppressants may further include contacting the olfactory receptor with cat urine before mixing the cat urine odor receptor with the test substance. In this case, the following Reference Data 2 and Comparative Data 2 can be obtained.

[0073] Reference data 2: Data measuring the response of the cat urine odor receptor after contact with cat urine but before the addition of the test substance. Test data 2: Data measuring the response of the cat urine odor receptor when the test substance is added after contact with cat urine.

[0074] The response of cat urine odor receptors already activated by cat urine can be evaluated before and after the addition of the test substance by comparing Reference Data 2 and Test Data 2. For example, if the response index of the cat urine odor receptor in Test Data 2 is statistically significantly reduced compared to Reference Data 2, the test substance may be useful as a cat urine odor suppressant.

[0075] The type of buffer solution is not particularly limited, but examples thereof include PBS (phosphate buffered saline), DPBS (Dulbecco's phosphate buffered saline), HBSS (Hank's balanced salt solution), and EBSS (Earle's balanced salt solution). HBSS is preferably used.

[0076] The buffer solution may contain various aminoethanesulfonic acids or aminopropanesulfonic acid derivatives having a Zwitterion structure, such as EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-morpholinoethanesulfonic acid), MOPS (3-morpholinopropanesulfonic acid), and PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)). The concentration thereof is not particularly limited, and may be, for example, 0.1 to 100 mmol / L, 1 to 125 mol / L, or 5 to 10 mmol / L.

[0077] The manner in which the cat urine odor receptor is brought into contact with cat urine is not particularly limited. For example, the following methods can be mentioned.

[0078] A method in which a membrane carrying a cat urine odor receptor or a cell or tissue expressing a cat urine odor receptor is placed in a sealed container containing cat urine. A method in which a membrane carrying a cat urine odor receptor or a cell or tissue expressing a cat urine odor receptor is placed in a sealed container, and then cat urine is supplied into the sealed container.

[0079] When cat urine is brought into contact with the cat urine odor receptor, the cat urine may be in a gaseous, liquid, or solid state. Cat urine consisting of a single component may be used, but it may also be a complex odor combined with malodorous substances other than cat urine, or an odor collected from any space containing cat urine.

[0080] In terms of reproducing actual olfactory behavior, it is preferable to use gaseous cat urine odor. By using cat urine odor, the ligand search conditions can be made closer to the reaction conditions of the cat urine odor receptor in the actual nose. Then, it is possible to search for inhibitors or antagonists of the cat urine odor receptor that respond to cat urine odor. It is believed that cat urine odor inhibitors selected in this way are likely to exhibit deodorizing effects when actually used in products such as deodorants.

[0081] However, in other embodiments, liquid cat urine may be vaporized in a sealed container, etc. A culture plate, a petri dish, or a circulator may be used to bring the cat urine into contact with the cat urine odor receptor in the sealed container.

[0082] In one embodiment, a cell culture plate having multiple wells may be used. While the number of wells is not particularly limited, a 96-well plate is preferably used. The shape of the well bottom can be any of flat, round, V-bottom, U-bottom, easy-wash bottom, and half-area solid, with flat bottoms being preferred. The opening diameter is also not particularly limited, but is 3 to 8 mm, preferably 5 to 7 mm, the bottom diameter is 2 to 7 mm, preferably 3 to 6.5 mm, and the maximum volume is 30 to 450 μL, preferably 200 to 400 μL. For example, cells or tissues expressing a cat urine odor receptor and a test substance are dispensed and mixed into each of multiple wells of a cell culture plate, and cat urine is supplied around the periphery of the cell culture plate or into the gaps in the well plate to bring the cat urine into contact with the cat urine odor receptor.

[0083] By premixing the test substance with the cat urine odor receptor in this way, the cat urine odor receptors in each well can be brought into contact with the cat urine so that the response time to the malodor of the cat urine odor receptors in each well is synchronized. Therefore, there is less of a time lag between the response time of each well, which occurs when cat urine is added to the cat urine odor receptors in each well one by one in advance and then the response of the cat urine odor receptor is measured.

[0084] In this case, it is possible to obtain cat urine odor receptor response data (the above-mentioned Reference Data 1 and Test Data 1) with less measurement error. When using a cell culture plate with multiple wells, the cat urine odor receptors in each well may be identical or different.

[0085] To observe the responsiveness of the cat urine odor receptor, the volume of the buffer solution may be reduced. For example, when using a 96-well plate, the cat urine odor receptor and the cat urine odor can be contacted under conditions where the buffer solution is 25 μL or less. If the volume of the buffer solution is relatively small, the conversion of the cat urine odor due to the action of the buffer solution is less likely to occur, making it easier to observe the responsiveness. For example, when culturing the cat urine odor receptor in a well plate, the well plate is turned upside down with the lid removed. At this time, some of the liquid in the well remains on the bottom of each well, and the volume of liquid in the well can be reduced by turning the well plate upside down. After reducing the volume of liquid in the well, mixing the cat urine odor receptor and cat urine may make it easier to observe the responsiveness of the cat urine odor receptor.

[0086] (Sensory Test) In one embodiment, a sensory test may be further performed. For example, a test substance that is expected to be useful as a cat urine odor suppressant may be used as a candidate substance for a cat urine odor deodorizer, and the cat urine odor suppression effect of the candidate substance may be evaluated by a sensory test. A candidate substance that is confirmed to have a cat urine odor suppression effect in the sensory test may be selected as a cat urine odor deodorizer.

[0087] The sensory test can be performed according to a normal evaluation procedure for deodorants. For example, the evaluator may smell the cat urine odor simultaneously with the odor of the candidate substance and evaluate the intensity of the cat urine odor, or may smell the cat urine odor separately from the odor of the candidate substance and evaluate the intensity of the cat urine odor. The evaluation results obtained from the sensory test are compared with the intensity of the cat urine odor alone. A candidate substance that is evaluated as having reduced the intensity of the cat urine odor as a result of the sensory test can be expected to be useful as a cat urine odor suppressant.

[0088] 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 in this specification can be modified in various ways within the scope of the effects of the invention, and can be combined with features described in other embodiments within the scope of feasibility.

[0089] The present invention will be described in more detail below by showing the results of experiments conducted by the inventors, although the present invention is not limited to the results of the experiments.

[0090] [Preparation of Human Olfactory Receptor-Expressing Cells] Based on the sequence information registered in GenBank, genes encoding 415 types of olfactory receptors, as shown in Tables 1 and 2, were cloned. Each gene was cloned by PCR using human genomic DNA Human mixed (G3041: Promega) as a template. Each gene amplified by PCR was incorporated into a pCI vector (Invitrogen) according to the product protocol. Specifically, a Rho-tag sequence was incorporated using the NheI and BamHI restriction enzyme sites present on the pCI vector. The olfactory receptor gene was incorporated downstream of the Rho-tag sequence using the MluI and NotI restriction enzyme sites downstream of the Rho-tag sequence. Next, a gene encoding human RTP1S was incorporated into the MluI and NotI restriction enzyme sites of the pCI vector.

[0091]

[0092]

[0093] Hana3A cells were cultured in a 96-well plate (Corning, BioCoat) to 50% confluence. A reaction solution with the composition shown in Table 3 was prepared. The 96-well plate was left standing in a clean bench for 15 minutes, and then 50 μL of the reaction solution was added to each well. The incubation temperature was 37°C, 5% CO ２ Hana3A cells expressing each of the 415 olfactory receptors shown in Tables 1 and 2 were prepared by culturing the cells for 24 hours in an incubator maintained at ambient atmosphere.

[0094]

[0095] [Glo Sensor Assay] A Glo Sensor assay was performed to measure the response of olfactory receptors. Cat urine was used as the odorant. Because the odor intensity of cat urine varies from day to day, urine was collected from a 6-year-old neutered male cat in separate vials for seven days. Urine collected on five different days was used. Because the odor varied on each collection day, urine was separated into urine with a strong irritating odor and urine with a mild irritating odor. The urine samples with the same level of irritation were then mixed. The mixed strong and mild urine samples were then further mixed at an arbitrary concentration to prepare odorants.

[0096] The olfactory receptors expressed in Hana3A cells couple with endogenous Gαs and Gαlf, activating adenylate cyclase and increasing intracellular cAMP levels. The increase in intracellular cAMP levels was measured as luminescence values ​​derived from the firefly luciferase gene, and the response strength of the olfactory receptors was measured.

[0097] Luciferase activity was measured using Glo Sensor cAMP Reagent (Promega) according to the product protocol. For various stimulation conditions, the luciferase-derived luminescence value after odor stimulation was divided by the luciferase-derived luminescence value before odor stimulation. That is, (luminescence value after stimulation) / (luminescence value before stimulation) was calculated. The value of (luminescence value after stimulation) / (luminescence value before stimulation) was used as a measurement value of the response intensity induced by odorant stimulation.

[0098] [Search for cat urine odor receptors that respond to cat urine odor] (Identification of TAAR5) The medium was removed from the culture of olfactory receptor-expressing cells. Then, 25 μL of Glo Sensor cAMP Reagent diluted with HBSS buffer containing 10 mM HEPES was added to each well of a 96-well plate. The cells were cultured in a light-blocking environment for 2 to 3 hours to introduce the cAMP Reagent into the cells.

[0099] Next, 25 μL of cat urine solution, prepared by mixing cat urine with a strong irritating odor and cat urine with a weak irritating odor, was added between the wells of the 96-well plate as malodorous molecules. The malodorous molecules were allowed to come into contact with human and mouse cat urine odor receptors, including mouse TAAR5, in the gas phase for 15 minutes. The response strength (fold increase) of the cat urine odor receptor to cat urine was measured over time, and the total response strength over 15 minutes was calculated. The results are shown in Figure 1.

[0100] The vertical axis of Figure 1 shows the relative response intensity of each receptor-expressing cell to the cat urine odor (Figure 1). The relative response intensity was calculated assuming the response intensity in the absence of the cat urine odor receptor was 0. The response to the cat urine odor was measured for each of the cells expressing 415 types of olfactory receptors. As a result, mouse TAAR5, which showed the highest response to the cat urine odor, was identified as the cat urine odor receptor (Figure 1).

[0101] (Concentration dependency of TAAR5 response to cat urine odor) 25 μL of cat urine aqueous solution prepared by mixing cat urine with a strong irritating odor and cat urine with a weak irritating odor as malodorous molecules was added between the wells of a 96-well plate, and the response of each receptor-expressing cell to cat urine was measured. The results are shown in Figure 2. As a result, TAAR5 showed a concentration-dependent response to cat urine odor. This confirmed that TAAR5 is an olfactory receptor that specifically responds to cat urine odor.

[0102] [Test Substances] The following compounds were used as test substances (Compounds) and diluted with HBSS buffer containing 10 mM HEPES to a final concentration of 100 μM: trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litopentas umbellata fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, 4,8-dimethyl-4,9-decadienal, and 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]. Butanal, citronellyl butyrate, 1,1-dimethyl-2-phenylethyl acetate, p-cymene, gamma-dodecanolactone, 2-ethyl-3-hydroxy-gamma-pyrone, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, 3-methyl-5-phenylpentan-1-ol, undecanal, nopyracetate, ethylene glycol brassillate

[0103] [Screening for Mouse TAAR5 Antagonists] The medium was removed from cultures of mouse TAAR5-expressing cells. Then, 25 μL of GloSensor cAMP Reagent diluted with HBSS buffer containing 10 mM HEPES was added to each well of a 96-well plate. The cells were cultured in a light-blocking environment for 2-3 hours to allow the cAMP Reagent to be introduced into the cells. The GloSensor assay was then performed to measure the response intensity of mouse TAAR5 before the addition of the test substance.

[0104] Next, the test substance was added to each well of the 96-well plate to give a final concentration shown in Table 4 or Table 5. Five minutes later, the GloSensor assay was performed to measure the response strength (fold increase) of mouse TAAR5 to the test substance.

[0105] Then, 25 μL of cat urine solution, prepared by mixing cat urine with a strong irritating odor and cat urine with a weak irritating odor, was added between the wells of the 96-well plate as malodorous molecules. A GloSensor assay was performed, and mouse TAAR5 was contacted with the malodorous molecules for 15 minutes. The response strength (fold increase) of mouse TAAR5 to the malodorous molecules was measured over time.

[0106] The response intensity to the test substance was calculated by dividing the luciferase-derived luminescence value 5 minutes after the addition of the test substance by the luciferase-derived luminescence value before the addition of the test substance, i.e., (luminescence value 5 minutes after the addition of the test substance) / (luminescence value before the addition of the test substance) was calculated to obtain reference data.

[0107] The response intensity when cat urine and the test substance were mixed was calculated by calculating the response value induced by stimulation with cat urine every minute for 1 minute and summing the values ​​for 15 minutes. The 1-minute response value was calculated as (luminescence value after stimulation with cat urine at each time) / (luminescence value 5 minutes after addition of the test substance). The obtained response value was then divided by the response value when only cat urine was added. In other words, (response value when cat urine and the test substance were mixed) / (response value from cat urine only) was calculated to obtain test data. The measurement results are shown in Tables 4 and 5.

[0108]

[0109]

[0110] As shown in Tables 4 and 5, the test data measurements revealed that the following compounds were detected: trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litopentas crescens fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, 4,8-dimethyl-4,9-decadienal, 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]butanal Since 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, and 3-methyl-5-phenylpentan-1-ol suppressed the response of mouse TAAR5 to cat urine odor, they are considered to function as antagonists of mouse TAAR5. These are considered to be inhibitors that are likely to exert a sufficient deodorizing effect against cat urine odor.

[0111] In contrast, as shown in Table 5, undecanal, nopyracetate and ethylene glycol brassylate were not found to have an inhibitory effect on the response of TAAR5 to the odor of cat urine.

[0112] [Test Substances Used in Sensory Test 1] The following test substances were used to test the deodorizing effect on cat urine odor: trans-2-hexenal, 2-ethyl-3-hydroxy-γ-pyrone, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citral, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, trans-cinnamaldehyde, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 3-(4-tert-butylphenyl)propanal, 3-(3-isopropylphenyl)butanal, α-damascone, ethyl dehydrocyclogeranate, Litopenaeus littoralis fruit oil, α-methyl-1,3-benzodioxole-5-propanal.

[0113] [Contents of Sensory Test 1] Sensory Test 1 was conducted by 10 evaluators. The 10 evaluators were men and women in their 20s or 30s who had passed a separately conducted olfactory test.

[0114] The fragrance master batches were prepared as follows. Scent paper cut to approximately 4 cm was prepared. One drop (approximately 0.02 g) of a solution of each test substance diluted to 1% with DMSO was impregnated onto the scent paper. The scent paper was then sealed in a 3 L bag, and the inside was filled with odorless air. The bag was then left to stand overnight at room temperature to obtain a scent master batch with a concentration of 1% of each test substance.

[0115] Each test sample was diluted to 10% with DMSO, and the resulting solution was further diluted to a concentration of 0.01% with triethyl citrate. Scent paper cut to approximately 4 cm was prepared separately. One drop (approximately 0.02 g) of the diluted solution was impregnated onto the scent paper. The scent paper was then sealed in a 3 L bag, and the bag was filled with odorless air. The test material was then left to stand overnight at room temperature to obtain a scent masterbatch with a concentration of 0.01% for each test substance.

[0116] The cat urine masterbatch was prepared as follows: 4 g of cat urine 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 together with a fan, and the bag was filled with odorless air. The bag was left to stand overnight at room temperature to obtain the cat urine masterbatch.

[0117] Evaluation samples were prepared as follows. The scented air of each fragrance masterbatch was transferred to a 3 L bag. Then, 200 ml to 300 ml of the cat urine masterbatch was poured into the 3 L bag so that the odor intensity, as defined by the Aromatic Deodorizer Council, was 3 to 4. The 3 L bag was then filled with odorless air to obtain each test sample. Another 3 L bag was filled with odorless air, and 200 ml of the cat urine masterbatch was poured into it (cat urine only sample).

[0118] The evaluation test was carried out as follows: Each sample was presented to an evaluator, who scored the cat urine intensity according to the following criteria. The average scores of 9 or 10 evaluators for the difference in malodor intensity between the malodor-only sample and the evaluation sample in which scented air was injected into cat urine are shown in Table 6.

[0119] [Standards for odor intensity] The odors were evaluated on a 6-point scale from +5 to 0 in increments of 1 point using the following evaluation standard: +5: Strong +4: Strong +3: Easily detectable +2: Weak +1: Barely detectable 0: Odorless

[0120]

[0121] This test adopted the sensory deodorizing test standards established by the Japan Deodorizer Association, which considers a product to have a deodorizing effect if it reduces the intensity of the malodor by one level or more. From the results of sensory test 1, it was confirmed that trans-2-hexenal, 2-ethyl-3-hydroxy-γ-pyrone, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citral, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, trans-cinnamaldehyde, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 3-(4-tert-butylphenyl)propanal, 3-(3-isopropylphenyl)butanal, α-damascone, ethyl dehydrocyclogeranate, Litopenaeus littoralis fruit oil, and α-methyl-1,3-benzodioxole-5-propanal have a sensory deodorizing effect against cat urine odor.

[0122] [Confirmation of ability of blended fragrances to inhibit mouse TAAR5] Floral blended fragrance 1, blended fragrance 2, fruity blended fragrance, and citrus blended fragrance were prepared, each containing a test substance, as shown in Table 7 below. Each blended fragrance contains the compound in the column marked with "+" in the table. Floral blended fragrance 1, floral blended fragrance 2, fruity blended fragrance, and citrus blended fragrance were all provided by Takasago International Corporation.

[0123]

[0124] The response intensity of mouse TAAR5 was measured for the blended fragrances shown in Table 7, and the results are shown in Table 8. For comparison, the response intensity of mouse TAAR5 was measured for each blended fragrance from which all test substances exhibiting antagonistic effects had been removed, and the results are shown in Table 8. The fruity blended fragrance and the fruity blended fragrance from which all test substances exhibiting antagonistic effects had been removed were measured at a concentration of 10%, while the other fragrance concentrations were measured at 1%. The response intensity when cat urine and the test substance were mixed was calculated as (luminescence value after stimulation with cat urine 20 minutes later) / (luminescence value 5 minutes after addition of the test substance).

[0125]

[0126] As shown in Table 8, the response intensity measurements showed that the floral fragrance 1, floral fragrance 2, fruity blended fragrance, and citrus blended fragrance, each containing the test substance, suppressed the response of mouse TAAR5 to the cat urine odor. This result suggests that they function as antagonists of mouse TAAR5. These are considered to be inhibitors that are likely to fully exert their deodorizing effect against cat urine odor.

[0127] [Sensory Test 2] The compounded fragrances shown in Table 7 were used to test the deodorizing effect on cat urine odor. For comparison, a test was also conducted on each compounded fragrance from which all test substances exhibiting antagonist effects had been removed. Sensory Test 2 was conducted by eight evaluators. The eight evaluators consisted of men and women in their 20s or 30s who had passed a separate olfactory test.

[0128] The fragrance master batches were prepared as follows: Each blended fragrance was diluted to 8% using an isoparaffin-based solvent and a glycol ether-based solvent, and added to a bottle containing the volatiles. The diluted mixture was then sealed in a 10-liter bag, and the inside was filled with odorless air. The bag was left to stand at room temperature for 30 minutes, yielding a master batch.

[0129] The evaluation samples were prepared as follows: The scented air from each scent masterbatch was transferred to a 3-L bag. Then, 50 ml of a cat urine masterbatch made using 2 g of cat urine was poured into the 3-L bag to obtain each test sample. Furthermore, 50 ml of the cat urine masterbatch was poured into another 3-L bag filled with odorless air (cat urine only sample).

[0130] The evaluation test was carried out as follows. Each sample was presented to an evaluator, who scored the cat urine intensity according to the following criteria. The average scores of the eight evaluators for the difference in malodor intensity between the malodor-only sample and the evaluation sample in which scented air was injected into cat urine are shown in Table 9.

[0131] [Standards for odor intensity] The odors were evaluated on a 6-point scale from +5 to 0 in increments of 1 point using the following evaluation standard: +5: Strong +4: Strong +3: Easily detectable +2: Weak +1: Barely detectable 0: Odorless

[0132]

[0133] This test also adopted the sensory deodorizing test standards established by the Japan Association of Deodorizers and Aromatic Fresheners. As previously mentioned, the Association considers a product to have a "deodorizing effect" when the odor intensity is reduced by one level or more. The results of Sensory Test 2 confirmed that the fruity-like compound fragrance and citrus-like compound fragrance containing the test substances each have a sensory deodorizing effect on cat urine odor, and are therefore considered to be highly useful as deodorizers for cat urine odor.

[0134] According to one aspect of the present invention, there is provided an inhibitor of cat urine odor receptors that is suitable for deodorizing cat urine odor by suppressing the response of cat urine odor receptors that exhibit specific responsiveness to cat urine odor.

[0135] Another aspect of the present invention provides a method for searching for cat urine odor inhibitors, which allows comprehensive analysis of inhibitors of cat urine odor receptors.

Claims

1. An inhibitor that suppresses the response of at least one cat urine odor receptor selected from the group consisting of TAAR5 and polypeptides having functions equivalent to TAAR5, and which is selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litopentas crenata fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, 4,8-dimethyl-4,9-decadienal, and 4-[(octahydro-4,7-methano-5H-inden)-5-ylidene]butanal. a cat urine odor receptor inhibitor comprising at least one selected from the group consisting of 2-ethyl-3-hydroxy-γ-pyrone, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopentenyl)-2-buten-1-ol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, and 3-methyl-5-phenylpentan-1-ol.

2. trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, 3-(4-tert-butylphenyl)propanal, Litopentas crenata fruit oil, 3-(3-isopropylphenyl)butanal, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)penta 2. The cat urine odor receptor inhibitor according to claim 1, comprising at least one selected from the group consisting of 1,1-dimethyl-2-phenylethyl acetate, 4-en-2-ol, 4,8-dimethyl-4,9-decadienal, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citronellyl butyrate, 1,1-dimethyl-2-phenylethyl acetate, p-cymene, γ-dodecanolactone, and 2-ethyl-3-hydroxy-γ-pyrone.

3. The cat urine odor receptor inhibitor according to claim 1, which contains at least one selected from the group consisting of trans-2-hexenal, trans-cinnamaldehyde, α-methyl-1,3-benzodioxole-5-propanal, citral, α-damascone, ethyl dehydrocyclogeranate, and 3-(4-tert-butylphenyl)propanal.

4. The cat urine odor receptor inhibitor of claim 1, comprising at least one selected from the group consisting of trans-2-hexenal, 2-ethyl-3-hydroxy-γ-pyrone, 4-[(octahydro-4,7-methano-5H-indene)-5-ylidene]butanal, citral, 3,3-dimethyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-en-2-ol, trans-cinnamaldehyde, 2,2,6-trimethyl-α-propylcyclohexane-1-propanol, 3-(4-tert-butylphenyl)propanal, 3-(3-isopropylphenyl)butanal, α-damascone, ethyl dehydrocyclogeranate, Litsea Cubeba fruit oil, and α-methyl-1,3-benzodioxole-5-propanal.

5. A cat urine odor deodorizer comprising the cat urine odor receptor inhibitor according to any one of claims 1 to 4.

6. A method for deodorizing cat urine odor using the cat urine odor receptor inhibitor described in any one of claims 1 to 4.

7. A method for searching for a cat urine odor suppressant, comprising mixing a test substance with at least one olfactory receptor selected from the group consisting of TAAR5 and polypeptides having functions equivalent to TAAR5.

8. The detection method according to claim 7, further comprising contacting the olfactory receptor with cat urine after mixing the olfactory receptor with the test substance.

9. The detection method according to claim 7, further comprising contacting the olfactory receptor with cat urine before mixing the olfactory receptor with the test substance.

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