JP7488709B2 - Cat defecation behavior inhibitor - Google Patents

Description

The present invention relates to an agent for suppressing inhibition of excretory behavior in cats.

Cats are generally clean animals and tend to dislike litter boxes that are soiled with excrement. If the litter box environment is unpleasant for a cat, it may make an accident in a place other than the litter box, or it may hold it in and develop cystitis. For example, when observing cats' excretory behavior in two different environments, it has been reported that in a small litter box in a small room (bad environment), cats urinate less frequently and take longer each time than in a large litter box in a large room (good environment) (Non-Patent Document 1). In order for cats to excrete in peace without feeling uncomfortable, it is desirable to eliminate elements that cats dislike, such as the smell of cat urine, from the litter box environment.

On the other hand, in a processing tool for processing excrement of pets, etc., a deodorizer is provided in the processing tool in order to prevent the owner from perceiving the odor generated by the excrement. For example, Patent Document 1 discloses a pet deodorant containing an aluminosilicate represented by a specific chemical formula. Patent Document 2 discloses a deodorant-containing product containing a deodorant containing a phenolic compound and/or a mediator and an enzyme capable of oxidizing them. Patent Documents 1 and 2 disclose the use of a fragrance such as acetyl cedrene in a pet deodorant.
However, there have been no reports examining the effects of acetylcedrene and florhydral on the excretory behavior of cats.

JP 2007-229151 A JP 2005-65750 A

Anim. Behav. Sci., 2017, 194,: 67-78

The present invention relates to providing an inhibitor of cat excretory behavior inhibition that can suppress inhibition of cat excretory behavior.

The inventors have discovered that acetylcedrene and florhydral can suppress inhibition of excretory behavior in cats.

That is, the present invention relates to the following 1) and 2).
1) An agent for suppressing inhibition of excretory behavior in cats, comprising at least one active ingredient selected from the group consisting of acetylcedrene and florhydral.
2) A method for suppressing inhibition of excretory behavior in a cat, comprising applying at least one member selected from the group consisting of acetylcedrene and florhydral to an object in the environment in which the cat excretes.

The present invention can suppress inhibitions to cats' excretory behavior and provide a comfortable litter box environment where cats can excrete without having to hold it in.

1 is a thickness-wise cross-sectional view of the sheet 1 used in Test Example 1, cut widthwise in the central region along the longitudinal direction. FIG. 2 is an exploded perspective view showing a cat litter box; FIG. 3 is a perspective view showing the cat litter box shown in FIG. 2 in use; FIG. 4 is a schematic cross-sectional view taken along line VV in FIG. 3 . A graph showing the results of urination frequency and time spent in the cat litter box. (A) is a diagram showing the number of sniffing behaviors and the total time for the sheet 1. (B) is a diagram showing the number of approaches to the sheet 1 and the duration of stay. Responses of olfactory receptor polypeptides to various concentrations of substances that cause cat urine odor. A: FcOR2M3, B: FcOR2M3-like, C: HsOR2M3. In all cases, the left side shows the response to MMF, and the right side shows the response to MMB. Data are the mean ± SE (n = 3 for each). Inhibition of the response of olfactory receptor polypeptides to substances that cause cat urine odor by acetylcedrene. A: FcOR2M3, B: FcOR2M3-like, C: HsOR2M3. In all cases, the left side shows the response to MMF, and the right side shows the response to MMB. The horizontal axis shows the acetylcedrene concentration, and the vertical axis shows the response intensity. Data are the mean ± SE (n = 3). Inhibition of the response of olfactory receptor polypeptides to substances that cause cat urine odor by florhydral. A: FcOR2M3, B: FcOR2M3-like, C: HsOR2M3. In all cases, the left side shows the response to MMF and the right side shows the response to MMB. The horizontal axis shows the concentration of florhydral, and the vertical axis shows the response intensity. Data are the mean ± SE (n = 3).

The acetyl cedrene used in the present invention is a compound also known as methyl cedryl ketone, which is 1-[(1S,2R,5R,7S)-2,6,6,8-tetramethyltriclo[5.3.1.0 ^1,5 ]undec-8-en-9-yl]ethane, CAS registration number: 32388-55-9.
Florhydral is β-methyl-3-(1-methylethyl)benzenepropanal, CAS registration number: 125109-85-5.
Both acetyl cedrene and florhydral can be purchased commercially. For example, these compounds can be obtained from International Flavors & Fragrances, Inc., Givaudan SA, etc. Alternatively, the same substances as the commercially available products or compositions containing them can be produced by chemical synthesis.

In the present invention, either or both of acetyl cedrene and florhydral are used. Preferably, both acetyl cedrene and florhydral are used. In this case, the ratio of acetyl cedrene to florhydral is preferably 1:0.001 to 1:1 by mass, more preferably 1:0.005 to 1:0.5, and most preferably 1:0.01 to 1:0.2.

Acetyl cedrene and florhydral are antagonistic fragrances that function as substances that inhibit the activation of cat olfactory receptors FcOR2M3 and FcOR2M3-like. FcOR2M3 is an olfactory receptor expressed in cat olfactory cells and is registered in GenBank under Accession number: XP_011281372. FcOR2M3-like is a putative cat olfactory receptor registered in GenBank under Accession number: XP_003980593.
Research by the present inventors has revealed that FcOR2M3 and FcOR2M3-like are cat olfactory receptors that respond to thiols, 3-mercapto-3-methylbutylformate (MMF) and 3-mercapto-3-methyl-1-butanol (MMB), which are components characteristic of cat urine odor, and that acetyl cedrene and florhydral function as substances that inhibit the activation of said FcOR2M3 and FcOR2M3-like (see Reference Examples below). Acetyl cedrene and florhydral have been known as fragrance materials, but it was not known that they have antagonistic effects on FcOR2M3 and FcOR2M3-like, or that they have a function of suppressing a cat's sense of smell to the cat urine odor caused by thiols.

In this specification, an antagonist refers to a substance that binds to a receptor but does not activate the receptor or suppresses the response of the receptor to an agonist (or inhibits the receptor activity), and an antagonist fragrance refers to a molecule that is used to inhibit the activation of the olfactory receptor to the target odor molecule by applying the target odor molecule together with another molecule when the binding site is an olfactory receptor and the receptor is activated to transmit odor information, thereby suppressing the odor recognized by an individual. In other words, the use of an antagonist fragrance can achieve odor suppression by olfactory receptor antagonism. Odor suppression by olfactory receptor antagonism is a means of applying the target odor molecule together with another odor molecule to inhibit the receptor response to the target odor molecule by the other odor molecule, thereby suppressing the odor (including bad odor) recognized by an individual. Odor suppression by olfactory receptor antagonism is distinct from methods that use other odor molecules as well, such as fragrances, to counteract a target odor with a strong odor (so-called odor masking).
In this way, acetylcedrene and florhydral can suppress the odor of thiols through odor suppression by olfactory receptor antagonism, thereby suppressing the recognition of the cat urine odor in individual cats.

Cats have a tendency to try to hide the smell of their own excrement after urination. For example, cats dig in the sand after urination to hide their presence from enemies and predators by burying their urine in sand (Spooner, S. K., 1990, Determination of the Normal Ethogram of Feline Urinary Behavior. University of Montreal Masters Thesis.). If the smell of a cat's own excrement does not disappear easily, the time spent digging in the sand after excretion will be extended and the number of times it will urinate will be reduced. In other words, for cats, the smell of excrement such as urine is a factor that inhibits their own excretory behavior, and on the other hand, if a cat does not recognize the smell of excrement after excretion, the time spent in the toilet after excretion will be shorter and the number of times it will urinate will be higher, which can be said to be a comfortable toilet environment for cats (Non-Patent Document 1; Anim. Behav. Sci., 2017, 194,: 67-78).

As shown in the Examples below, acetyl cedrene and florhydral shorten the time a cat spends in the bladder after urination and increase the frequency of urination. In other words, acetyl cedrene and florhydral can suppress the recognition of cat urine odor by cats through olfactory receptor antagonism, and have the effect of suppressing the inhibition of excretory behavior in cats.
Therefore, at least one selected from the group consisting of acetyl cedrene and florhydral (hereinafter, in this specification, "at least one selected from the group consisting of acetyl cedrene and florhydral" may be referred to as "acetyl cedrene, etc.") can be an excretion behavior inhibition inhibitor that suppresses inhibition of excretion behavior in cats, and can be used to manufacture an excretion behavior inhibition inhibitor in cats. In addition, acetyl cedrene, etc. can be used to suppress inhibition of excretion behavior in cats. By suppressing inhibition of excretion behavior, cats can excrete in peace, creating a comfortable litter box environment for cats.
In this specification, the feline excretory behavior is preferably the feline urination.

The feline excretory behavior inhibition inhibitor of the present invention contains at least one compound selected from the group consisting of acetyl cedrene and florhydral as an active ingredient for inhibiting feline excretory behavior inhibition, and may essentially consist of said compound, or may be a composition further containing other ingredients.
Examples of other components include antibacterial agents, surfactants, deodorants, chelating agents, water-soluble solvents, oils, pH adjusters, moisturizers, thickeners, stabilizers, preservatives, natural extracts, colorants, fragrances other than acetyl cedrene and florhydral, organic solvents, and the like. Among these, from the standpoint of antibacterial properties and uniform blending, it is preferable to include antibacterial agents, surfactants, and deodorants.

The antibacterial agent is preferably one that suppresses the growth of bacteria having urease activity. In this specification, the bacteria having urease activity means bacteria that produce an enzyme (urease) that hydrolyzes urea contained in urine into carbon dioxide and ammonia, and grow in urine to generate ammonia, and examples of such bacteria include Brevundimonas intermedia, Sporosarcina ureae, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella genus, Morganella morganii, Corynebacterium, etc. By containing an antibacterial agent that suppresses the growth of bacteria having urease activity, the growth of bacteria having urease activity can be suppressed, and the generation of ammonia with a very strong irritating odor due to the action of the bacteria can be suppressed, so that the cat urine odor can be more effectively suppressed.
Examples of antibacterial agents include piroctone olamine, didecyldimethylammonium chloride, benzalkonium chloride, benzethonium chloride, paraoxybenzoic acid esters, alkyltrimethylammonium chloride, isopropylmethylphenol, etc. Although florhydral is also an antibacterial agent, it is not included in the antibacterial agents in this specification.
The antibacterial agent may be used alone or in combination of two or more. Among them, piroctone olamine, didecyldimethylammonium chloride, and benzalkonium chloride are preferred from the viewpoint of being effective at a low concentration.

Examples of the surfactant include ionic surfactants (anionic surfactants, cationic surfactants, amphoteric surfactants), nonionic surfactants, and polymeric surfactants. The surfactants may be used alone or in combination of two or more. Among them, anionic surfactants, nonionic surfactants, and amphoteric surfactants are preferred from the viewpoint of uniform blending.
As the anionic surfactant, for example, anionic surfactants having a carboxylic acid group, such as fatty acid or a salt thereof, ether carboxylic acid or a salt thereof, and N-acylamino acid salt, are preferable.

Examples of nonionic surfactants include glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene alkenyl ethers, polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene polyoxypropylene alkenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl polyglucosides, polyglycerin fatty acid esters, etc.

Examples of amphoteric surfactants include alkyl dimethyl amino acetate betaine, alkyl dimethyl amine oxide, alkyl amidopropyl betaine, and alkyl hydroxysulfobetaine.

Cationic surfactants (excluding the antibacterial agents mentioned above) have positively charged hydrophilic groups as hydrophilic sites. Examples of hydrophilic groups include primary, secondary or tertiary amine salts, quaternary ammonium salts, etc.

A deodorant is a substance that can directly act on odors (odor-causing substances) and exert a deodorizing effect by adsorbing, neutralizing, decomposing, etc., the odors (odor-causing substances), and those that are generally used for deodorizing purposes can be used. Examples of deodorants include porous particles and water-soluble pH buffers.
The porous particles are particles having a large number of pores at least on the particle surface, and are capable of collecting, adsorbing and/or encapsulating volatile odorous components in the pores. Examples of the porous particles include inorganic porous substances such as activated carbon, silica, silicon dioxide (silica gel), calcium silicate, high silica zeolite (hydrophobic zeolite), sepiolite, cancrinite, zeolite, and hydrated zirconium oxide.
Water-soluble pH buffers are agents that neutralize acidic or alkaline odor components generated by cat urination, etc., and reduce changes in pH. Examples include weak acids such as citric acid and their conjugate bases, weak bases such as polyhydroxyamine compounds such as tris(hydroxymethyl)aminomethane and their conjugate acids, mixtures of these, and salts of these.

The cat excretory behavior inhibition inhibitor of the present invention can be applied to objects in the environment where cats live, such as indoors or facilities where cats are kept, particularly in the environment where cats excrete.
Objects in the environment in which a cat excretes include, but are not limited to, interior items such as floors, walls, furniture, curtains, and carpets that are present in the environment in which a cat excretes, toilets, toilet products (toilet sand and sheets), cages, houses, beds, etc.
Preferably, the cat excretion behavior inhibition inhibitor of the present invention is incorporated into products or preparations used in cat litter boxes.
The form of the product or preparation can be appropriately selected depending on the application, and can be, for example, liquid, gel, paste, or solid.
In addition, acetyl cedrene and florhydral may be applied to or impregnated into woven or nonwoven fabrics of paper, natural fibers, regenerated fibers (rayon, etc.) and/or synthetic fibers (polyethylene, polypropylene, polyester, etc.).
Specific examples of the product or formulation form include cleaning agents, deodorants, odor control agents, fragrances, and wet wipes.

The content of acetyl cedrene, etc. in the feline excretion behavior inhibition inhibitor of the present invention varies depending on the form, but is preferably 0.01 to 10% by mass in total, more preferably 0.05 to 5% by mass, and most preferably 0.1 to 3% by mass.

In a further aspect, the present invention provides a method for suppressing cat elimination inhibition, comprising applying acetyl cedrene or the like to an object in the cat's environment where the cat excretes. The object in the cat's environment where the cat excretes is preferably a cat litter box.
Since thiols are naturally contained in cat urine, the odor of thiols is generated immediately after urination and continues to be generated even after time has passed. Therefore, it is preferable to apply acetyl cedrene or the like before or after the cat excretes.
In order to suppress the inhibition of cats' excretory behavior, acetyl cedrene or the like may be brought into contact with objects in the environment in which cats excrete. The method of contact is not particularly limited, and examples include coating, spraying, immersion, addition, and evaporation.
The amount of acetyl cedrene, etc. used in the present invention may be any amount that can suppress the recognition of cat urine odor in individual cats and suppress inhibition of excretory behavior in cats, and can be appropriately determined depending on the form of use.

Details of the antagonist fragrances and cat urine odor causative substances used in the tests below are shown below.
(Antagonist Fragrances)
Acetyl cedrene (methyl cedryl ketone; [1-[(1S,2R,5R,7S)-2,6,6,8-tetramethyltricyclo[5.3.1.0 ^1,5 ]undec-8-en-9-yl]ethane); (CAS Registry Number: 32388-55-9; Trade Name: Vertofix; International Flavors & Fragrances, Inc.)
Florhydral (β-Methyl-3-(1-methylethyl)benzenepropanal); (Registered Trademark (International Registration 0534879)) (CAS Registration Number: 125109-85-5) (Givaudan SA)
Acetyl cedrene and florhydral were mixed in a mass ratio of 98:2 and used as the antagonist flavoring.
(Substances that cause cat urine odor)
3-Mercapto-3-methylbutyl Format (MMF) (Sigma-Aldrich Co.)
3-Mercapto-3-methy-1-butanol (MMB) (Tokyo Chemical Industry Co., Ltd.)

Test Example 1
A liquid preparation was prepared according to the recipe in Table 1. This liquid preparation was sprayed onto the sheet 1 shown in Fig. 1 in an amount of 10 g/sheet, and dried at 50°C for 2 hours.

The sheet 1 had a length of 45 cm and a width of 35 cm, and the absorbent body 4 had a size of 40 cm and 30 cm.
Surface sheet 2: Polyolefin nonwoven fabric, 20 gsm
Back sheet 3: Polyethylene sheet, 20 gsm
・Core wrap sheet 44a on the surface side: thin paper, 16 gsm
Back side core wrap sheet 44b: thin paper, 16 gsm
Liquid-absorbent material 46 upper layer: pulp, 100 gsm
Superabsorbent Polymer 47: Crosslinked water-absorbent resin of sodium polyacrylate, 117 gsm
Liquid-absorbent material 46 lower layer: pulp, 100 gsm
The liquid agent sprayed over the entire surface of the sheet 1 from the surface material side is mostly absorbed by the surface side of the absorbent body 4, i.e., the thin paper 44a.

The dried sheet 1 was stored in the sheet storage tray 16 of the system toilet 10 as shown in Figs. 2 to 4, and wooden chips 32 were placed on the grate 12.
The wooden chips 32 are made by solidifying crushed plant-derived material with a binder, and crushed coniferous trees are used as the crushed plant-derived material. Synthetic resin is used as the binder to increase the water repellency of the chips, so that urine is not absorbed by the chips but is absorbed by the sheet placed underneath.
The following experiment was carried out using two system toilet sets in which sheets sprayed with the liquid of Example 1 or Comparative Example 1 were placed in the sheet storage section 16.

Things to check: Number of times the child urinates in the toilet and the time spent in the toilet (before, after, and after urination)
Location: Cat petting facility (indoors)
Area: ^50m2
Number of cats: 15 Litter box placement: Litter boxes are placed on the left and right with 30cm intervals between each litter box, with the left and right litter boxes swapped every 30 minutes Implementation time: 9AM - 4PM (7 hours)

The test results are shown in FIG.
As shown in Figure 5, in the litter box of the Example sprayed with the liquid agent containing acetyl cedrene and florhydral, the frequency of urination was increased and the time spent in the litter box after urination was shortened compared to the litter box of the Comparative Example. This result shows that the litter box environment of the Example is more favorable for cats (Non-Patent Document 1).

Test Example 2
(1) Behavioral test of cats with respect to urine-added sheets 5 mL of a sterilized and filtered mixture of urine from male cats (ages 1-10) was dropped onto a central area of 5 cm diameter of the Example 1 liquid-added sheets and Comparative Example 1 liquid-added sheets produced in Example 1. The Example 1 liquid-added sheets and Comparative Example 1 liquid-added sheets onto which the cat urine was dropped were placed in an environment in which 24 cats (ages 3-13, 12 males and 12 females) were kept in a free-moving environment. When a cat recognizes the cat urine odor, it shows a sniffing behavior with curiosity. If the recognition of the cat urine odor by a cat is suppressed by the Example 1 liquid, the sniffing behavior of the cat urine odor should decrease. In this test, cats that reached the urine-added area at the center of either sheet and showed a sniffing behavior within the 60-minute test time were analyzed.

Seven cats sniffed the center of one of the sheets. The number of times and total time that the cats sniffed the sheets are shown in Figure 6A. The cats' sniffing of the urine-added area was mainly directed at the urine odor on the Comparative Example 1 liquid-added sheet, and was hardly observed on the Example 1 liquid-added sheet (Figure 6A, left). There was a statistically significant difference between the number of times that the cats sniffed the Example 1 liquid-added sheet and the Comparative Example 1 liquid-added sheet (p=0.0082, paired t-test). The total time that the cats sniffed also tended to be shorter on the Example 1 liquid-added sheet (Figure 6A, right). This demonstrates that the antagonist fragrance suppresses the cat's sense of smell for the cat urine odor, and shows that the antagonist fragrance provides a comfortable litter box environment for cats.

(2) Cat Preference for Example Liquid-Added Sheets A test was conducted to confirm that the reduction in cat urine odor sniffing behavior when using the Example 1 liquid-added sheet in (1) above was due to the cat urine odor suppressing effect of the antagonist fragrance. That is, it was considered that the odor of the antagonist fragrance emitted from the sheet was unpleasant to cats, so that the cats refrained from sniffing the sheet even when they recognized the urine odor. In this test, such a possibility was verified. The Example 1 liquid-added sheet and the Comparative Example 1 liquid-added sheet were placed in an environment where 24 cats, the same as in Test Example 2, were kept in a free-moving condition. Cats that reached and stayed on either sheet within the 30-minute test time were analyzed.

Twelve cats reached either sheet. The number of times the cats reached the sheets and the duration of their stay are shown in Figure 6B. The cats reached the liquid-added sheet of Example 1 more frequently and stayed longer there than the liquid-added sheet of Comparative Example 1. These results indicate that the odor of the antagonist fragrance is not an unpleasant odor that would induce aversion in cats. Therefore, it was confirmed that the decrease in cat urine odor-sniffing behavior observed in (1) above was due to the suppression of cat urine odor by the antagonist fragrance.

Test Example 3
The solutions were formulated according to the recipe in Table 2.
In this test, 5 mL of the same urine mixture as in Test Example 2 was added to the center of a piece of carpet (30 cm x 30 cm). The same area was then sprayed with 10 mL of the solution. The subsequent treatments are listed in Table 2 as A, B, or C according to the type of solution below.
The evaluation results immediately after the post-treatment and after leaving the sample for one week are shown in Table 2. The evaluation method and evaluation criteria are as follows.
(Post-processing method)
A: None B: Wipe off C: Rinse off with water (Evaluation method)
The carpets that had been post-treated in each case were lined up in a cat breeding facility in the same manner as in Test Example 2, and the behavior of the cats was observed. The evaluation criteria were as follows: if the number of times that the cats went to sniff the carpet was one-third or less of that of the comparative example, it was given an ⊚; if it was one-half or less, it was given an 0; and if it was more than one-half, it was given an X.

As shown in Table 2, the number of times that cats sniffed the carpet sprayed with the liquid formulation of the embodiment was low, demonstrating that the antagonist fragrance suppresses the cat's sense of smell for the odor of cat urine.

Reference Example 1 Identification of olfactory receptor polypeptides that respond to cat urine odor causative substances 1) Synthesis of olfactory receptor polypeptide genes Based on the sequence information registered in GenBank, genes encoding the cat olfactory receptor polypeptides FcOR2M3 and FcOR2M3-like (SEQ ID NOs: 1 and 2, respectively) were synthesized. For gene synthesis, a DNA synthesis service provided by Thermo Fisher Scientific was used. In addition, based on the sequence information registered in GenBank, a gene encoding the human olfactory receptor HsOR2M3 (GenBank Accession number: KP290147, see Patent Application No. 2019-005550) that responds to cat urine odor causative substances was cloned from human genomic DNA female (G1521: Promega). Each gene was integrated downstream of the Flag-Rho tag sequence on the pME18S vector.

2) Construction of pME18S-human RTP1S Vector The gene encoding human RTP1S (SEQ ID NO:3) was inserted into the EcoRI and XhoI sites of the pME18S vector.

3) Preparation of Olfactory Receptor-Expressing Cells HEK293 cells were prepared by expressing each olfactory receptor polypeptide. A reaction solution having the composition shown in Table 3 was prepared and left to stand in a clean bench for 15 minutes. 10 μL of the reaction solution was added to each well of a multi-well plate (96-well plate, BioCoat), and then 90 μL of HEK293 cells (2×10 ⁵ cells/cm ² ) were added to seed the cells. The cells were cultured for 24 hours in an incubator maintained at 37° C. and 5% CO ₂ . As a control, cells that do not express olfactory receptors (mock) were prepared.

4) Luciferase assay of olfactory receptor response The olfactory receptor polypeptide expressed in HEK293 cells increases the amount of intracellular cAMP by coupling with the intrinsic Gαs and activating adenylate cyclase. In this embodiment, a luciferase reporter gene assay was used to measure the response of cells to the substances causing cat urine odor, which monitors the increase in the amount of intracellular cAMP as the luminescence value derived from the firefly luciferase gene (fluc2P-CRE-hygro). In addition, a Renilla luciferase gene fused downstream of the CMV promoter (hRluc-CMV) was simultaneously introduced into the cells and used as an internal standard to correct errors in gene introduction efficiency and cell number. The Dual-Glo ^™ luciferase assay system (Promega) was used to measure luciferase activity. After the operation of 3), the medium was removed from the culture of olfactory receptor expressing cells, and 75 μL of medium containing or not containing the cat urine odor causative substance was added to each well. A DMEM solution containing 300 μM CuCl ₂ was used as the medium. MMF (final concentration 0-0.03 mM) or MMB (final concentration 0-0.1 mM) was used as the cat urine odor causative substance. Four hours after the addition of the cat urine odor causative substance, luminescence was measured according to the product's operation manual, and the value [fLuc/hRluc] was calculated by dividing the luminescence value derived from firefly luciferase by the luminescence value derived from Renilla luciferase. As an index of the responsiveness of the olfactory receptor to the stimulus of the cat urine odor causative substance, the fold increase was calculated according to the following formula.
Fold increase = X/Y
X: fLuc/hRluc induced by stimulation with cat urine odor causative substance
Y: fLuc/hRluc without stimulation of cat urine odor causative substance

The results are shown in Figure 7. No response to MMF or MMB was observed in mock, whereas concentration-dependent responses to both MMF and MMB were observed in cells expressing FcOR2M3 and FcOR2M3-like (Figures 7A and B). These responses were similar to those of human MMF and MMB receptor HsOR2M3 (Patent Application No. 2019-005550 by the present applicant) (Figure 7C).
These results indicate that FcOR2M3 and FcOR2M3-like are feline-derived olfactory receptor polypeptides that recognize MMF and MMB, which are components of cat urine odor.

Reference Example 2 Inhibitory activity of olfactory receptor polypeptide response by acetyl cedrene and florhydral The response of olfactory receptor polypeptide to MMF or MMB in the presence of acetyl cedrene or florhydral was measured, and changes in response intensity were examined. Luciferase assay of olfactory receptor polypeptide-expressing HEK293 cells was performed according to the same procedure as in Reference Example 1. The final concentration of MMF was adjusted to 0.01 mM, and the final concentration of MMB was adjusted to 0.03 mM. The final concentrations of acetyl cedrene and florhydral were adjusted in the range of 0 to 0.3 mM. The fLuc/hRluc value (X) induced by stimulation with MMF or MMB alone, the fLuc/hRluc value (Y) in cells not stimulated with MMF or MMB, and the fLuc/hRluc value (Z) induced by co-stimulation of MMF or MMB with acetyl cedrene or florhydral were obtained. The response strength (Response (%)) of the olfactory receptor polypeptide to MMF or MMB in the presence of acetylcedrene or flurhydral was calculated according to the following formula.
Response (%) = (Z-Y) / (X-Y) x 100
Three independent experiments were performed, and the average response intensity obtained in each experiment was calculated.

The results are shown in Figures 8 and 9. Both acetylcedrene (Figure 8) and florhydral (Figure 9) inhibited the FcOR2M3 and FcOR2M3-like responses to MMF and MMB in a concentration-dependent manner. From the above results, it was confirmed that these two compounds are antagonist fragrances for FcOR2M3 and FcOR2M3-like. Furthermore, these compounds inhibited the HsOR2M3 response to MMF and MMB, similar to previous findings (Patent Application No. 2019-005550).
The above results indicate that acetylcedrene and florhydral, which are antagonists of the olfactory receptor FcOR2M3 or FcOR2M3-like, suppress a cat's sense of smell to the cat urine odor caused by MMF, MMB, etc.

Claims (5)

An agent for inhibiting the excretory behavior of cats, containing at least one active ingredient selected from the group consisting of acetylcedrene and florhydral. The cat excretion behavior inhibition inhibitor according to claim 1, which contains both acetylcedrene and florhydral as active ingredients. The cat excretion behavior inhibition inhibitor according to claim 1 or 2, which is used in combination with at least one antibacterial agent selected from the group consisting of piroctone olamine, didecyldimethylammonium chloride, and benzalkonium chloride. The cat excretion behavior inhibition inhibitor according to any one of claims 1 to 3, which is in the form of a cleaning agent or a deodorant. A method for suppressing inhibition of excretory behavior in cats, comprising applying at least one selected from the group consisting of acetylcedrene and florhydral to an object in the environment in which the cat excretes.