Response sensitivity enhancer for olfactory receptor

Abstract

To provide a substance that enhances the response sensitivity of olfactory receptors to odorants.SOLUTION: To solve the foregoing problem, the present inventors searched for substances that enhance the response sensitivity of olfactory receptors regarding combinations of some olfactory receptors and odorants obtained from human olfactory mucus. As a result, fibronectin was identified as such a substance. The present invention provides, as a solution: a response sensitivity enhancer for olfactory receptors comprising fibronectin; a sensitivity enhancer for odor sensors comprising fibronectin; and a pharmaceutical composition comprising fibronectin for improving olfactory dysfunction.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a substance having an effect of enhancing the response of olfactory receptors to odor substances, and an agent or composition containing the substance.

Background Art

[0002] In terrestrial vertebrates including humans, the process of perceiving odor starts when air containing an odor substance flows into the nasal cavity. The air containing the odor substance reaches the olfactory epithelium present at the back of the nasal cavity, where the gaseous odor substance dissolves in the olfactory epithelial mucosa (OEM), diffuses, and reaches olfactory sensory neurons (OSNs). The cilia of olfactory sensory neurons express olfactory receptors (ORs). When an odor substance binds to an olfactory receptor, signal transduction is induced that activates the olfactory sensory neuron and induces information transmission to the brain.

[0003] Olfactory receptors are G-protein coupled receptors (GPCRs), seven-transmembrane receptors, and belong to class A group. Class A GPCRs have a ligand-binding pocket formed by transmembrane domains. An odor substance binds to the ligand-binding site of the olfactory receptor, activates the olfactory receptor, and transmits a signal through the G αolf pathway. G αolf is a G αs type of G protein, which is specifically expressed in olfactory sensory neurons and cells expressing other olfactory receptors. G αolf activates adenylate cyclase, causes activation of cyclic nucleotide-sensitive channels through an increase in intracellular cAMP concentration, induces influx of calcium ions into the cell, depolarization of the nerve cell, and action potential. The number of olfactory receptors encoded in the genome varies among animal species, with mice having over 1200, while humans have approximately 400. In mammals, birds, and lizards, olfactory receptors are classified into two classes based on phylogenetic relationships, and other classes of receptors also exist in fish and amphibians. In mammals, Class II olfactory receptor genes account for approximately 90% of all olfactory receptors. Class I olfactory receptors are thought to recognize hydrophilic odorants, and Class II olfactory receptors are thought to recognize hydrophobic odorants.

[0004] Although the olfactory system is known to be highly sensitive, many aspects of the mechanism enabling this sensitivity remain unclear. Patients with a disorder in the olfactory mucus covering the cilia expressing olfactory receptors show very low olfactory sensitivity compared to normal individuals, and there are reports that olfactory sensitivity increases when the secretion of olfactory mucus is improved by medical treatment (see Non-Patent Document 1, etc.). Also, it has been reported that in mice with a disorder in the secretion of olfactory mucus, olfactory sensitivity is significantly reduced compared to wild-type mice. Imaging assays of the olfactory bulbs of these mice with secretion disorders have revealed that the response threshold to the odorant eugenol was 0.1% eugenol in wild-type mice, whereas it increased to 10% eugenol in secretion-disordered mice (Non-Patent Document 2). Furthermore, electrophysiological experiments ex vivo using turtles and bullfrogs have reported that when a part of the olfactory mucus is removed, the response of olfactory nerve cells in the removed part decreases or disappears (Non-Patent Document 3). From the above reports, it is suggested that some factor present in the olfactory mucus is important for olfactory sensitivity.

[0005] By the way, in recent years, odor sensors that mimic the olfactory organs of living organisms for detecting odorants have been reported (Patent Document 1, Non-Patent Documents 4 and 5, etc.). However, there is room for improvement in their sensitivity.

[0006] As described above, factors that enhance olfactory sensitivity are expected to exhibit high utility in the medical and industrial fields, such as in improving olfactory decline and increasing the sensitivity of odor sensors. However, at present, no such factors have been found. The complexity of the composition of olfactory mucus is considered to be one of the reasons why it is difficult to identify factors that enhance olfactory sensitivity. In addition, for example, the fact that an assay system (an assay system close to in vivo conditions) for detecting the response of olfactory receptors to odor substances in the gas phase has not been established is also considered to be one of the reasons why the identification of such factors has not progressed.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0009] In view of the above circumstances, an object of the present invention is to provide a substance that enhances the response sensitivity of olfactory receptors to odor substances.

Means for Solving the Problems

[0010] In order to solve the above problems, the present inventors searched for a substance that enhances the response sensitivity of olfactory receptors to odor substances from human olfactory mucus, and as a result, identified fibronectin as such a substance. In identifying fibronectin, the inventors developed an assay system closer to the olfactory system of a living body, that is, a system capable of detecting the response of olfactory receptors to odor substances in the gas phase. In this assay system, it was confirmed that fibronectin enhances the response sensitivity of olfactory receptors. Furthermore, it was also confirmed that fibronectin enhances the response sensitivity of olfactory receptors in an ex vivo system using mouse olfactory epithelium. From the above, it was suggested that fibronectin may function as a factor that enhances olfactory sensitivity in the olfactory system of a living body.

[0011] That is, the present invention is as follows: (1) to (6). (1) A response sensitivity enhancer for olfactory receptors containing fibronectin. (2) The response sensitivity enhancer according to (1) above, wherein the olfactory receptor is derived from a mammal. (3) A sensitivity enhancer for odor sensors containing fibronectin. (4) The sensitivity enhancer for odor sensors according to (3) above, wherein the odor sensor comprises an olfactory receptor as a constituent element. (5) A pharmaceutical composition for improving olfactory disorders containing fibronectin. (6) The pharmaceutical composition according to (5) above, wherein the olfactory disorder is olfactory hypofunction or anosmia. In this specification, the symbol "~" indicates a numerical range including the values on both its left and right.

Effects of the Invention

[0012] The olfactory receptor response sensitivity enhancer (agent containing fibronectin) according to the present invention exhibits an effect of improving olfactory decline and an effect of enhancing the sensitivity of an odor sensor using an olfactory receptor.

Brief Description of Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] Hereinafter, embodiments of the present invention will be described. When referred to as "this embodiment", unless otherwise specified, it refers to all embodiments described in this specification or any one of the embodiments. The first embodiment is a sensitivity enhancer for olfactory receptors containing fibronectin. The present inventors have first found that fibronectin enhances the response sensitivity of olfactory receptors to odorant substances. Fibronectin is a well-known protein with many research reports so far and is a glycoprotein modified with sugar chains. Fibronectin exists as a dimer or multimer in plasma, extracellular matrix, etc., and the molecular weight of the monomer is approximately 260 kDa. Although there is one fibronectin gene, in the case of humans, at least 20 isoforms exist due to the selective splicing of the mRNA precursor. In the case of humans, the nucleic acid sequence and amino acid sequence of fibronectin are reported as NM_002026 and NP_002017, NM_001306129 and NP_001293058, NM_001306130 and NP_001293059, NM_001306131 and NP_001293060 (Genbank Accesion numbers above), etc.

[0015] The monomer of fibronectin consists of a plurality of functional domains, that is, it is composed of three repeating structures called type I, type II, and type III domains. In the isoforms of fibronectin, the number of type I domains and type II domains is common, but the number of type III domains varies depending on the isoform depending on the position of selective splicing.

[0016] The species from which fibronectin in this embodiment is derived (for example, mammals, birds, amphibians, fish, insects, etc.) is not particularly limited and may be any species, but is preferably derived from mammals. Also, it may be something collected from a living body or a recombinant expressed in various cells. The form of fibronectin in this embodiment may be any of a monomer, dimer, or multimer, but is preferably a dimer. In addition, in this embodiment, the protein used as fibronectin is a protein having an amino acid sequence of fibronectin derived from various species and having a sequence identity of, for example, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and having a known activity of fibronectin (e.g., cell adhesion activity), or a protein having a similar primary structure (the above-described domain and module structure). In fact, the amino acid sequence identity between bovine plasma fibronectin and human plasma fibronectin is about 90%, and it has been confirmed that both enhance the sensitivity of olfactory receptors (see Examples).

[0017] In addition, the species from which the olfactory receptor in this embodiment is derived (e.g., mammals, birds, amphibians, fish, worms, insects, etc.) is not particularly limited, and any species may be used. In addition, olfactory receptors are classified into Class I type and Class II type, and the olfactory receptor in this embodiment may be either Class I type or Class II type.

[0018] Those skilled in the art can easily measure the response sensitivity of olfactory receptors by appropriately selecting methods known in the art. As described above, when an odorant binds to an olfactory receptor expressed on the cell membrane, a signal is transmitted into the cell via a conjugated G protein, activating intracellular adenylate cyclase, increasing the intracellular cAMP concentration, and then inducing the activation of cyclic nucleotide-sensitive channels, causing calcium ions to flow into the cell. Therefore, the response sensitivity of olfactory receptors can be evaluated using as an index an increase in the concentration of cAMP or an increase in calcium concentration in cells expressing olfactory receptors. In addition, whether the sensitivity of olfactory receptors to odorants is enhanced can be determined using as an index a decrease in the EC 50 value of the odorant concentration to which the olfactory receptor that is evaluated using an increase in the cAMP concentration or an increase in calcium in olfactory receptor-expressing cells responds, etc.

[0019] The "agent" of the "enhancer" according to this embodiment includes all preparations containing fibronectin as an active ingredient, and its purposes of use are wide-ranging, such as for pharmaceuticals, experimental reagents, and auxiliary agents for detecting odor substances, and are not particularly limited. Further, the "agent" according to this embodiment may contain substances that are effective in detecting odor substances in addition to fibronectin as the active ingredient, and its dosage form is also not particularly limited, and may be tablets, granules, powders, liquids, etc.

[0020] The enhancer for enhancing the response sensitivity of olfactory receptors according to this embodiment can also be used for detecting or screening novel or known odor substances using olfactory receptors.

[0021] The second embodiment is a sensitivity enhancer for an odor sensor containing fibronectin. In recent years, devices for detecting odor substances (hereinafter also referred to as "odor sensors") that utilize constituent molecules of the olfactory organ of a living body (such as olfactory receptors, peptides that are part of olfactory receptors and contain regions that bind to odor substances) as their detection means have been reported (Patent Document 1, Non-Patent Documents 4 and 5, etc.). For example, odor sensors that utilize peptides that bind to odor substances are already commercially available (Ayballe). Some of these odor sensors use olfactory receptors or the odor substance binding regions of olfactory receptors as their components. In the case of an odor sensor that uses an olfactory receptor or the like as a detection means for odor substances, it is possible to enhance the detection sensitivity of odor substances with fibronectin. That is, an agent containing fibronectin as an active ingredient can be used as a sensitivity enhancer for an odor sensor that has an olfactory receptor or the like as a main component.

[0022] The third embodiment is a pharmaceutical composition for improving olfactory disorders containing fibronectin (hereinafter also referred to as "the pharmaceutical composition according to this embodiment"). Here, "improvement" means making the olfactory disorder at a certain point in time into a more favorable state. Olfactory disorders are generally classified into quantitative disorders and qualitative disorders. Quantitative disorders are divided into hyposmia, in which the sense of smell is reduced, and anosmia, in which no smell is felt at all. Qualitative disorders are divided into irritant parosmia (a symptom in which the way of feeling the smell of a substance changes in a situation where a smelly substance is present in the surroundings), and spontaneous parosmia (a symptom in which a smell is felt in a situation where no smelly substance is present in the surroundings). The pharmaceutical composition according to the present embodiment is expected to have an effect of improving quantitative disorders in particular. However, the application of the pharmaceutical composition according to the present embodiment to qualitative disorders is not excluded.

[0023] In addition to fibronectin as an active ingredient, the pharmaceutical composition according to the present embodiment may contain other substances effective for improving olfactory disorders, and may also contain pharmacologically acceptable additives. The pharmaceutical composition according to the present embodiment is not particularly limited, but is expected to exhibit its medicinal effect by nasal administration. Therefore, the dosage form of the pharmaceutical composition is preferably a form suitable for nasal administration, and examples thereof include liquid preparations (such as nasal drops and sprays), ointments, creams, and the like.

[0024] The types of formulation additives used in the manufacture of the pharmaceutical composition according to this embodiment, the ratio of the formulation additives to the active ingredient, the manufacturing method, etc. can be appropriately selected by those skilled in the art. As the formulation additives, inorganic or organic substances, or solid or liquid substances can be used, and generally, they can be formulated in an amount between 1% by weight and 90% by weight based on the weight of the active ingredient. Specifically, examples of formulation additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethyl cellulose, hydroxypropyl starch, calcium carboxymethyl cellulose, ion exchange resin, methyl cellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerol ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water, and the like.

[0025] To produce liquid formulations (such as nasal drops and sprays), the active ingredient is dissolved in distilled water for injection together with pH adjusters such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, and sodium dihydrogen phosphate, and isotonic agents such as sodium chloride and glucose as needed, and then aseptically filtered and filled into ampoules, or alternatively, mannitol, dextrin, cyclodextrin, gelatin, etc. are added and vacuum freeze-dried to obtain a ready-to-dissolve type injection. Also, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. can be added to the active ingredient and emulsified in water to obtain a liquid formulation.

[0026] Regarding ointments and creams, they can be manufactured by kneading and mixing the active ingredient, base, and additives. For example, an oily ointment can be manufactured by heating and melting an oily base such as fats and oils, waxes, and hydrocarbons such as paraffin, adding the active ingredient, mixing and dissolving or dispersing it, and kneading and mixing until the whole becomes homogeneous. A water-soluble ointment can be manufactured, for example, by heating and melting a water-soluble base such as macrogol, adding the active ingredient, and kneading and mixing until the whole becomes homogeneous. For example, creams can be manufactured by adding the active ingredient to either the oil phase containing petrolatum, higher alcohols, etc. as they are or with additives such as emulsifiers, or to the liquid phase containing purified water as it is or with additives such as emulsifiers, heating each, combining the oil phase and the liquid phase, and stirring and emulsifying until the whole becomes homogeneous.

[0027] A fourth embodiment is a method for preventing and / or treating olfactory disorders, including administering to a patient a pharmaceutical composition for improving olfactory disorders (the pharmaceutical composition according to this embodiment) containing fibronectin. Here, "treatment" means preventing or alleviating the progression and worsening of a pathological condition caused in a mammal to be treated. Also, "prevention" means preventing in advance the onset of a disease in a mammal that is at risk of developing the disease.

[0028] The pharmaceutical composition according to this embodiment may be administered via the nasal mucosa. Examples of the method include administering into the nasal cavity by spraying, applying, dropping, etc. to the nasal mucosa. The dosage of the pharmaceutical composition according to this embodiment can be appropriately determined according to the age, weight, etc. of the administration subject, but contains a pharmaceutically effective amount of the vaccine antigen. A pharmaceutically effective amount refers to the amount of antigen necessary to induce an immune response against the vaccine antigen. For example, it can be administered once a day to several times at a dose of several μg to several tens of mg of the vaccine antigen per administration, and a total of several times, for example, about 1 to 5 times, at intervals of 1 to several weeks.

[0029] The subject to which the pharmaceutical composition according to this embodiment is administered may be any animal classified as a mammal, and is not particularly limited. For example, in addition to humans, animals belonging to primates, pet animals such as dogs, cats, rabbits, ferrets, and livestock animals such as cows, pigs, sheep, and horses. Particularly preferred subjects for administration are humans.

[0030] The disclosure content of all documents cited in this specification is incorporated herein by reference in its entirety. Also, throughout this specification, when the words in the singular form "a", "an", and "the" are included, unless the context clearly indicates otherwise, it shall include not only the singular but also the plural. Further, in this specification, "about" and "degree" mean a numerical range of ±10%. Examples will be shown below to further explain the present invention, but the examples are merely illustrative of the embodiments of the present invention and do not limit the scope of the present invention.

Examples

[0031] 1. Materials and methods 1-1. Odorous substances and fibronectin Those shown in Table 1 were used.

Table 1

[0032] 1-2. Buffer solutions, etc. Ringer's solution; 140 mM NaCl, 5.6 mM KCl, 5 mM HEPES, 2.0 mM sodium pyruvate, 1.25 mM KH2PO4, 2 mM CaCl2, 2 mM MgCl2, 9.4 mM D-glucose, pH 7.4 Note that the Ringer's solution without glucose was the one without adding glucose from the above composition.

[0033] 1-3. In vitro imaging assay 1-3-1. Imaging Assay (Method Common to All Imaging Assays) (1) Data Acquisition Fluorescence was measured using an Olympus IX3 microscope (Olympus) equipped with an ORCA R2 camera (Hamamatsu Photonics, C10600) and a CoolLED pE-340 fura (CoolLED). Data acquisition was performed using HCImage (Hamamatsu Photonics) at a sampling rate of 1 frame per second. Data analysis was carried out using Igor Pro 9.0 (WaveMetrics) with custom scripts. (2) Selection of Responding Cells for Analysis Cells that responded when each odorant added was at a concentration equal to or higher than the EC 50 concentration were defined as "responding cells". Specifically, cells that did not respond when stimulated at a concentration below the EC 50 concentration and responded when stimulated once with an odorant at the EC 50 concentration were used in the experiment.

[0034] 1-3-2. cAMP Imaging As the cells, HKE293T cells ("cAMPr cells") that constitutively express the cAMP sensor cAMPr (Hackley et al., Science Signaling 11:eaah3738 2018) were used. The cells were seeded in 35 mm glass dishes and then transfected with 2 μg of pME18S-Rho-tag OR and 0.5 μg of pME18S-RTP1S using the XtremeGene HP DNA Transfection Reagent (XTGHP-RO, Roche Life Sciences). "Rho-tag" is the 20 amino acids on the N-terminal side of bovine rhodamine. 46 - 52 hours after transfection, the medium was replaced with Ringer's solution, and the cells were incubated at 37 °C for 20 - 30 minutes and then left to stand at room temperature for 10 - 20 minutes. (1) Stimulation with Odorants in the Liquid Phase The odorant substances were added to the cells using the perfusion system constructed by the inventors. Pentyl acetate, eugenol, cyclotene, isovaleric acid, methyl anthranilate, and 2,3-dimethylpyrazine were each directly dissolved in the Ringer's solution. Musk ketone and muscone were prepared as a 100 mM (in DMSO) stock and then added to the Ringer's solution for use. The stimulation with the odorant substances was performed for 10 seconds. (2) Substances, samples, etc. used other than odorant substances The olfactory mucus sample, fibronectin solution, and control solution were added to the cells at a flow rate of 200 μL / min using a syringe pump (KD Scientific, KDS-100, 78-0100J) equipped with a 1 mL syringe (Terumo). To add the test substance, perfusion was temporarily stopped, and 200 μL of the test substance was added to the cells over 1 minute. After adding the test substance, the following (i) or (ii) was performed; (i) Immediately resume perfusion and perform stimulation with the odorant substance. (ii) Incubate the test substance and the cells for 5 minutes before resuming perfusion. In the experiment of olfactory mucus, the above (i) was adopted.

[0035] (3) Ultrafiltration When performing in vitro assays on the filtered sample and the flow-through after ultrafiltration, it was considered possible that the yield decreased as the active substance was not completely dissolved and / or the active substance adsorbed to the filtration membrane. Therefore, the filtered sample was incubated in a 37°C water bath for 10 - 15 minutes, and after confirming that the particulate matter present in the sample had disappeared, it was added to the cells. After incubating for 5 minutes from the addition, perfusion was resumed (the above (ii)).

[0036] 1-3-3. Assay in the gas phase (1) Method common to all gas phase assays A nasal mucus sample prepared 30 minutes before the experiment or 30 μg / mL of fibronectin was added to cells washed three times with Ringer's solution, incubated for 1 minute, and then the sample was removed with a pipette. Next, the cells were set in the experimental apparatus and data acquisition was started. Control cells were treated in the same manner except that Ringer's solution was used instead of the nasal mucus sample and fibronectin. Immediately before imaging, the liquid around the cells was removed, and the stimulation device fabricated by the inventors was set on the cells. Inside this device, the cells are exposed to air in a small chamber, and a metal mesh plate (hole diameter: 55 μm) is placed at the top of the chamber. The metal mesh plate constitutes the bottom surface of the well through which the odorant solution flows by perfusion. After installing this device and starting perfusion, the upper part of the well was capped to prevent the odorant from volatilizing upward. Data acquisition was started within 4 minutes after removing the liquid from the cells (see Figure 3A). Using this device, it is possible to prevent the perfusion fluid from directly contacting the cells. Furthermore, the odorant volatilized from the perfusion fluid reaches the cells through the gas phase, binds to the olfactory receptors expressed on the cell surface, and elicits a response.

[0037] (2) Calcium imaging HEK293T cells cultured on a 35 mm dish (Iwaki) until 60 - 70% confluent were transfected with 2 μg of pME18S-Rho-tag OR and 1.2 μg of pME18S-G using XtremeGene HP DNA Transfection Reagent (XTGHP-RO, Roche Life Sciences). α15Alternatively, cells were transfected with 0.5 μg of pME18S-RTP1S. Twenty-four to twenty-eight hours after transfection, the cells were loaded with 2.5 μM Fura2 / AM at 37 °C for 20 - 40 minutes. Subsequently, the cells were washed three times with 1 mL of Ringer's solution and then imaged. Excitation was performed at an excitation wavelength of 340 nm or 380 nm, and fluorescence at 510 nm was recorded. Images were recorded at 2 frames per second. Odorant stimuli were applied at 30-second intervals.

[0038] (3) cAMP imaging (stimulation in the gas phase) To use the same combination of olfactory receptor - odorant as in the stimulation in the liquid phase for the stimulation in the gas phase, the following changes were made; First, the readout of the olfactory receptor was changed from calcium imaging to cAMP imaging using the gCarvi cAMP protein (Kawata et al., Proc. Nat. Acad. Sci., 119:e2122618119 2022). gCarvi dissociates from cAMP faster than cAMPr and can shorten the time to return to the baseline. Second, to shorten the time required for the assay and speed up the washing of the odorant, the stimulation interval was shortened from 30 seconds to 5 seconds. HEK293T cells seeded in a 35 mm dish were transfected with 2 μg of pME18S-Rho-tag OR, 0.5 μg of pME18S-RTP1S, or a plasmid carrying 0.5 μg of the gCarvi cAMP sensor protein gene using the XtremeGene HP DNA Transfection Reagent. The imaging assay was performed 22 - 26 hours after transfection.

[0039] 1 - 3 - 4. Imaging assay using methyl anthranilate as an odorant The assay was performed in the same manner as the in vitro assay of odorants in the liquid phase, except for the following points. To prevent fluorescence bleed-through from the GFP-based cAMP sensor, non-transfected HKE293T cells instead of cAMPr cells were used. Also, instead of an excitation wavelength of 480 nm, an excitation wavelength of 340 nm was used to match the excitation wavelength of methyl anthranilate.

[0040] 1-4. For olfactory mucus samples Olfactory mucus samples were collected according to the previous report (Ijichi et al., Scientific Reports 12:9984 2022; Ijichi et al., Chemical Senses 44:465-481 2019). The provider of the olfactory mucus sat in the prone position with the forehead up on the bed and washed the nasal cavity with buffer. Specifically, 1 mL of buffer was injected into the nostril and kept in that state for 1 minute. When the head was kept straight, the liquid flowed out of the nostril and the liquid was collected. Although physiological saline was used as the buffer in the previous report, Ringer's solution was used in this experiment. Olfactory mucus samples were used within 30 minutes after collection or immediately frozen in liquid nitrogen after collection.

[0041] 1-5. FITC (Fluorescein isothiocyanate) labeling of fibronectin 1 mg of bovine plasma fibronectin (ThermoFisher 42805) was dissolved in 300 μL of Milli-Q water. To adjust the pH, 300 μL of 0.1 M NaHCO3 and 300 μL of 0.1 M Na2CO3 were added to the fibronectin solution. To achieve a labeling ratio of 3:1 (approximately 3 molecules of FITC covalently bound to 1 molecule of fibronectin), 2.8 μL of a 10 mg / mL DMSO solution of FITC (ThermoFisher, 46425) was added to the fibronectin solution. The labeling reaction was carried out with the reaction solution shielded from light with aluminum foil and rotated and stirred in the dark for 1 hour. To remove the remaining FITC, the reaction solution was dialyzed overnight with stirring against 1 L of glucose-free Ringer's solution. After dialysis, the reaction solution was further dialyzed with stirring against 1 L of glucose-free Ringer's solution for 8 hours. Then, the dialyzed reaction solution was transferred to an Eppendorf tube, shielded from light with aluminum foil, and stored at 4 °C.

[0042] 1 - 6. Creation of an apparatus for stimulating with odor substances in the gas phase Apparatus used for calcium imaging of mOR (mouse olfactory receptor)-EG (eugenol); Using plastic parts of a frame hanging kit (Graphic Station F673), a chamber was created. A metal mesh (hole diameter: 55 μm) (Ishikawa Kanaami) was adhered with Araldite adhesive and dried overnight before use. Apparatus used for human OR-cAMP imaging; The apparatus with a chamber for cells and wells on its upper part was designed using MoI (Triple Squid Software Design), a computer assisted design (CAD) software. This apparatus was printed using a Value 3D MagiX high-resolution DLP 3D printer system (MUTOH ML-48). The adhesion of the metal mesh (hole diameter: 55 μm) was carried out as described above. For each odor substance, a dedicated apparatus was used.

[0043] 1-7. Animal Experiments 1-7-1. Mice For the experiments, male Slc:ICR mice (SLC Japan) at 6-7 weeks of age were used. The mice were housed in an animal laboratory under a 12-hour light / dark cycle condition with free access to standard food. All animal experiments were conducted in accordance with the animal breeding guidelines approved by the University of Tokyo. 1-7-2. Electrophysiology Experiments Lethal dose of pentobarbital was administered to the mice. Immediately after death, the heads of the mice were collected and the skulls were dissected along the midline. The nasal conchae were exposed and the parietal bone and epithelium were removed. If both sides of the head were intact, one side was used for the experiment with the addition of fibronectin and the other side was used for the experiment with the addition of control serum albumin. The excised heads were placed on 1% agar containing Ringer's solution. The odorant (pentyl acetate) was delivered in a 1.0-second pulse from the headspace of a 60 mL glass vial containing a 100 μM pentyl acetate solution. Since the amplification of the response to pentyl acetate was greatest in endoturbinate II and III, recordings were made at these sites. The change in potential was recorded from the olfactory epithelium using a glass pipette filled with 1% agar containing Ringer's solution, which was connected to an AC / DC differential amplifier (Warner Instrument Corporation, DP-301) with an Ag / AgCl bridge. As a reference electrode, a second Ag / AgCl bridge was connected to another glass pipette inserted into the agar. The electrical signal was detected using PowerLab / 8SP (ADInstruments). Removal of olfactory mucus was performed using a medical sponge (HOGY, Tafpott 3 TTP3-M10) cut into a wedge shape of 2 mm × 0.5 mm × 0.5 mm. The thick part of the wedge-shaped sponge was pinched with forceps and the thin part was gently brought into contact with the recording site of the olfactory epithelium. Specifically, it was gently pressed for 3-8 seconds and then released, and this was repeated about 5-8 times. The sponge containing the olfactory mucus from one individual was collected in an Eppendorf tube and frozen at -80°C. Then, the sponge was used for Western blotting analysis. The test substance (mouse plasma fibronectin (Abcam AB92784) or bovine serum albumin (Wako 014-15151)) was aspirated in an amount of 0.5 μL with a 28-gauge Microfil needle (World Precision Instruments, MF26G67-5) from a sample at a concentration of 300 μg / mL, and the amount of one drop formed at the tip of the needle was extruded and gently added directly near the recording electrode of the nasal concha. Care was taken so that the needle did not touch the electrode or the tissue directly. When a droplet was visible at the position where the sample solution was added, a Kimwipe was gently brought into contact with the meniscus of the droplet near the electrode to absorb the excess solution. After adding the test substance, the response reaction to pentyl acetate volatilized in the headspace of the solution containing 100 μM pentyl acetate was measured periodically for 10 - 12 minutes, and the most amplified response reaction was taken as the "response after (test substance) addition".

[0044] 1-8. Biochemical experiments For all Western blotting, CBB staining, and silver staining of acrylamide gels, the samples were loaded onto a 7.5% acrylamide gel for SDS-PAGE and electrophoresed at 30 mA. The samples were treated with 1× sample buffer (0.25 M Tris-HCl pH 6.8, 75 mM sodium dodecyl sulfate, 7% glycerol, 5% β-mercaptoethanol, 2.5% bromophenol blue) before loading onto the gel. The specific operations were carried out according to the conventional method. 1-8-1. Ultrafiltration Ultrafiltration was performed using a Centrisart I polyethersulfone-membrane centrifugal ultrafiltration unit with a 100 kDa MWCO (Sartorius, 13269E). First, the lyophilized olfactory mucus sample was dissolved in Milli-Q water to the volume at the time of collection. Next, the sample was added to the Centrisart I unit and centrifuged twice at 1,500×g for 10 minutes at room temperature. The flow-through (<100 kDa) was transferred to another tube and immediately stored at 4°C. Further, the remaining sample was added to the unit and the same operation was performed. This operation was repeated until the sample reached approximately 0.5 mL. The insoluble components in the ultrafiltered sample (>100 kDa) were removed by centrifuging the sample at 15,000×g for 5 minutes. The olfactory mucus sample was concentrated approximately 10-fold by ultrafiltration compared to the time of collection. The sample (>100 kDa) prepared by ultrafiltration was immediately used for gelatin affinity purification and a part of it was lyophilized. A part of the flow-through (<100 kDa) was also lyophilized. The lyophilized samples were stored at -80°C. When using the sample (>100 kDa) prepared by ultrafiltration for the assay, the lyophilized sample was dissolved in Milli-Q water and adjusted to a 1-fold concentration. On the other hand, since the lyophilized sample of the flow-through (<100 kDa) was not concentrated, it was dissolved in Milli-Q water to the same volume as at the time of collection.

[0045] 1-8-2. Gelatin Affinity Assay A 5 × 50 mm Polypropylene-made Muro-mak (registered trademark) column (Muromachi Chemicals Incorporated) was packed with Gelatin Sepharose 4B (Cytiva, 17095601) to prepare a gelatin affinity column. A Ringer's solution without glucose was used as the running buffer. After equilibrating the column with 10 volumes of the running buffer, an ultrafiltration sample (>100 kDa) was loaded onto the column. Next, the column was washed with the running buffer, then with a running buffer containing 1 M NaCl, and further washed with the running buffer. Fibronectin was eluted with an elution buffer (running buffer containing 1 M arginine), and the eluted fractions were collected. To remove arginine, the eluted fractions were concentrated using a Centrisart I 100 kDa MWCO unit, and the concentrated fractions were washed with 1 mL of Ringer's solution (without glucose).

[0046] 1-9. Statistical Analysis Statistical analysis was performed using Igor Pro 9.0 (WaveMetrics). A significance level of p < 0.05 was set. In in vitro liquid-phase stimulation assays (except for dose-response curves for 7 pairs of olfactory receptors-odorants), the enhancement of responses at each response time of each responding cell was compared by one-way ANOVA. Tukey's test was performed as a post hoc test when ANOVA was significant (p < 0.05). In dose-response experiments in the gas and liquid phases, the responses under control conditions and those under sample addition conditions were compared by t-test. For the electro-olfactogram experiments in mice, after testing the normality of the distribution by the Shapiro-Wilk test, mixed-model ANOVA was performed, followed by Tukey's test.

[0047] 2. Results 2-1. Examination of the presence or absence of olfactory enhancement by olfactory mucus in vitro 2-1-1. Effect of olfactory mucus on the response sensitivity of olfactory receptors to odorants According to a previous report (Ijichi et al., Chemical Senses 44, 465 - 481 2019), human olfactory mucus samples were collected and used in the experiments. Also, as an in vitro assay, an imaging assay system for living cells was used (Figure 1). To test olfactory receptors, cAMP was used as a sensor to detect the responses of olfactory receptors. Specifically, a cell line that constitutively expresses cAMPr (cAMPr cells) was used. This cAMP sensor contains the cAMP - binding portion of protein kinase A fused with circularly permuted GFP (Hackley et al., Science Signalling 11:eaah3738 2018). To investigate whether olfactory mucus contains factors that enhance olfactory sensitivity, the responses of olfactory receptors to odorants were measured before and after adding olfactory mucus samples to olfactory receptor - expressing cells (Figure 2). As olfactory receptors and odorants, human olfactory receptor OR2W1 and pentyl acetate were used in the experiments. As a result, the response of the olfactory receptor to 100 μM pentyl acetate increased by approximately 1.8 - fold immediately after the addition of olfactory mucus compared to before the addition (Figure 2A). The subsequent responses of the olfactory receptor showed no significant difference from the responses before the addition of olfactory mucus.

[0048] To examine the dose - dependence of the effect of olfactory mucus, OR2W1 and 100 μM pentyl acetate were used, and the enhancement rates of response sensitivity were measured with 1 / 10 dilution, 3 / 10 dilution, and 1 / 1 dilution (undiluted) of the olfactory mucus sample. As a result, the enhancement rate decreased as the dilution rate increased (Figure 2B). In subsequent experiments, the olfactory mucus sample was used without dilution.

[0049] It was examined whether the sensitivity enhancement effect by olfactory mucus occurs only for the combination of OR2W1 and pentyl acetate. Specifically, the effect of olfactory mucus on the response sensitivity in the combination of human olfactory receptor OR5K1 and 2,3-dimethylpyrazine, and the combination of human olfactory receptor OR5AN1 and musk ketone was investigated (Figure 2, E - H). As a result, it was confirmed that the response sensitivity to odorants was enhanced in all combinations. By adding olfactory mucus, the response sensitivities of the combination of OR5K1 and 2,3-dimethylpyrazine, and the combination of OR5AN1 and musk ketone were enhanced 1.4-fold and 2-fold, respectively. Here, it was confirmed that the above-mentioned response sensitivity enhancement effect is not simply due to the addition of protein. Specifically, the effect of BSA on the combination of OR2W1 and pentyl acetate was investigated. First, when the protein concentration in olfactory mucus was examined, it was 630 ± 150 μg / mL (N = 4). Therefore, 500 μg / mL of BSA was added to olfactory receptor-expressing cells, and the effect on the response sensitivity of olfactory receptors was investigated. As a result, the response sensitivity of OR2W1 to pentyl acetate did not change even when BSA was added (data not shown). Therefore, it was confirmed that the effect of olfactory mucus on the sensitivity of olfactory receptors is not due to the simple addition of protein.

[0050] 2-1-2. Fabrication of a device for detecting the response of olfactory receptors to odorants in the gas phase In order to detect the effect of olfactory mucus on the response of olfactory receptors to odorants in a state closer to in vivo, an attempt was made to investigate the effect of olfactory mucus in a gas-phase odorant stimulation assay. The in vitro gas-phase stimulation method for vertebrate olfactory receptors has not yet been fully established. Although there are reports using high-throughput plate assays, there are no reports on single cells in real time. The main problem in establishing the gas-phase stimulation method is that when cells are exposed to air, they rapidly dry out (resulting in cell death). In order for gaseous odorants to reach the cells, it is usually necessary to remove the culture medium or buffer covering the cells and leave only a thin liquid layer, but the moisture in this liquid layer will volatilize immediately unless replenished. Therefore, a device for gas-phase stimulation was devised with the need for humidification in mind (Figure 3). This device has a chamber for covering cells with a thin liquid layer at the bottom and a well for storing liquids such as perfusion fluid at the top. The lower chamber and the upper well are separated by a metal plate with a mesh structure, and only gas can pass through the holes in this mesh (Figure 3A). The operating principle of this device will be explained below. When liquid is perfused through the upper well, water vapor passes through the mesh and humidifies the cells in the lower chamber (Figure 3B(i)). In order to generate volatile odorants for stimulating the cells, a liquid in which the odorant is dissolved (the odorant in the liquid phase) is perfused into the upper well (Figure 3B(ii)). The liquid cannot pass through the mesh, but the odorant can volatilize into a gas and pass through the mesh (Figure 3B(iii)). The odorant in the liquid phase in the upper well is washed away by perfusion (Figure 3B(iv)), and the gaseous odorant in the lower chamber dissolves in the thin liquid layer covering the cells, activating the cells expressing olfactory receptors (Figure 3B(v)). Finally, the odorant in the liquid layer covering the cells also volatilizes, and the response of the olfactory receptors ends (Figure 3B(vi)).

[0051] To confirm whether the gas actually passes through the mesh of the metal plate and hinders the passage of liquid, the effectiveness of the device was examined using receptors that respond to both volatile and non-volatile ligands. For the purpose of examining this effectiveness, rat TRPV1, a sensory receptor that responds to both capsaicin (non-volatile) and menthol (volatile), was used. Experiments were conducted with concentrations well above the threshold for each ligand, namely 100 nM capsaicin and 1 mM menthol. If liquid leaks from the mesh, a response to capsaicin should be observed, and if gas cannot pass through the mesh, the inhibitory effect of menthol should not be observed. As shown in Figure 3C, no response to capsaicin was observed, and a clear response to menthol was observed, confirming that the devised device operates as designed.

[0052] 2-1-3. Examination of the effect of olfactory mucus on the response of olfactory receptors to odorants in the gas phase (in vitro) Using the above-described device, the effect of olfactory mucus on the response of mOR, a mouse olfactory receptor that has been well studied so far, to eugenol was examined. This experiment was conducted by the calcium imaging method. Calcium imaging can complete the measurement before the cells dry because the time until the response is completed is shorter than that of cAMPr imaging. Representative data of cells containing only buffer in the liquid layer and representative data of cells containing olfactory mucus in the liquid layer are shown in Figure 4A. Cells to which olfactory mucus was added (+OEM) had a lower EC 50 value than cells to which only buffer was added (Control) (Figure 4B; EC 50 value of Control = 100 μM, EC 50 value of +OEM = 30 μM). Furthermore, it was found that the response threshold of cells to which olfactory mucus was added was lower than that of cells under control conditions (Figure 4C). In fact, in all cases examined at odorant concentrations below 300 μM, the amplification of the response was significantly higher under the condition of adding olfactory mucus compared to the control condition (p < 0.05, t-test). As described above, since the dose-response curve shifted to higher sensitivity by adding olfactory mucus, it was shown that at least in the combination of mOR-EG-eugenol, even when the olfactory receptor responded to the odorant in the gas phase, the olfactory mucus sample had the effect of enhancing its sensitivity.

[0053] 2-2. Identification of factors enhancing the response of olfactory receptors present in olfactory mucus 2-2-1. Narrowing down using the biochemical characteristics of candidate factors and their abundance in olfactory mucus Since olfactory mucus has the effect of enhancing the response sensitivity of olfactory receptors to odorants, an attempt was made to identify a sensitivity-enhancing factor (a factor enhancing the response of olfactory receptors). Experiments were conducted using the activity of enhancing the response of OR2W1 to pentyl acetate in the liquid phase as an index. Ultrafiltration of olfactory mucus was performed to determine the approximate molecular weight of the enhancing factor. Before performing ultrafiltration, first, the olfactory mucus sample was freeze-dried overnight, and it was examined whether the reconstituted sample of the freeze-dried preparation retained the enhancing activity. As a result, the enhancing activity was also retained in the freeze-dried and reconstituted samples, and the enhancing effect was approximately 1.8 times the same as that of the olfactory mucus sample within 30 minutes after collection (data not shown). Therefore, it was found that freeze-drying is a usable method and the enhancing factor is non-volatile. Next, the stored sample was reconstituted and its ultrafiltration was carried out. When a filter with a cut-off of 100 kDa was used, the filtered solution (100 kDa or more; >100 kDa) enhanced the response of OR2W1 to the odorant (Figure 5A), and the flow-through (100 kDa or less; <100 kDa) did not show an enhancing effect (Figure 5B). From this result, it was suggested that the factor in olfactory mucus enhancing the response of OR2W1 to pentyl acetate (hereinafter also referred to as "enhancing factor") is 100 kDa or more. Since the olfactory mucus contains lipids, small molecules, sugar chains, etc., the enhancer factor might not be a protein. However, since its molecular weight was 100 kDa or more, it was suggested to be a protein. Therefore, the fractions with enhancing activity (>100 kDa) and those without activity (<100 kDa) were subjected to SDS-PAGE. In the fraction with activity, there were two bands at positions of 100 kDa or more, but there were none in the fraction without activity (Figure 5D). These bands were considered to be the first candidates for the enhancer factor.

[0054] To further narrow down the enhancer factor candidates, the proteomics data of olfactory mucus (provided by Ajinomoto Co., Inc.) was referred to, and the candidate factors were narrowed down based on the following criteria; (i) Narrowing down to proteins >100 kDa (16 proteins out of 359 proteins) (ii) Narrowing down to the top 10% in abundance (6 proteins out of 16 proteins) (iii) Prioritize proteins that are significantly more abundant in olfactory mucus than in respiratory mucus. Based on the above criteria, among all proteins >100 kDa, fibronectin, a 260 kDa glycoprotein, which is the most abundant, the seventh most abundant among all proteins in olfactory mucus, and is significantly more abundant in olfactory mucus than in respiratory mucus, was selected as a candidate for the enhancer factor.

[0055] Next, it was examined whether fibronectin actually existed in the fraction with enhancing activity. The two protein bands of about 250 kDa in SDS-PAGE were similar to the characteristics of the bands of fibronectin electrophoresed under reducing conditions. To confirm whether these two bands were fibronectin, Western blotting was performed, and the two bands were recognized by an anti-fibronectin antibody. Therefore, it was revealed that these two protein bands were fibronectin (Figure 5E).

[0056] 2-2-2. Generation and Activity Measurement of Candidate Factors from Olfactory Mucus To determine whether fibronectin present in olfactory mucus affects the response of olfactory receptors to odorants, fibronectin was purified from olfactory mucus and its effect on the response of OR2W1 to pentyl acetate was examined. After concentrating the ultrafiltration fraction of olfactory mucus samples with a molecular weight cut-off of 100 kDa or more, fibronectin was purified using a gelatin affinity column, a standard method. After removing the arginine used for elution by ultrafiltration, Western blotting confirmed that the concentration of the eluted fibronectin was 1-10 μg / mL (Figure 5F). From the results of silver staining, it was confirmed that other bands present in the sample with a molecular weight of 100 kDa or more were removed from the elution fraction of the affinity column, except for a band of approximately 63 kDa that was thought to be serum albumin, and the purification results were good (Figure 5G). To confirm the response enhancement activity, the purified fibronectin was added to OR2W1-expressing cAMP r cells and the effect on the response to pentyl acetate was measured. As a result, the response was significantly enhanced up to an average of 1.4-fold (Figures 5H and I).

[0057] 2-2-3. Confirmation of Enhancement Activity with Commercially Available Fibronectin To further verify whether the response enhancement effect confirmed in the experiment using olfactory mucus samples was due to fibronectin, commercially available fibronectin was used for investigation. First, bovine plasma fibronectin, which has 93% amino acid sequence identity with human plasma fibronectin, was used. Western blotting was performed on several olfactory mucus samples and commercially available fibronectin with a known concentration as a standard to prepare the concentration of the commercially available fibronectin to be the same as the fibronectin concentration in the olfactory mucus samples. As a result, it was found that the fibronectin concentration in the olfactory mucus samples was approximately 30 μg / mL (Figure 6).

[0058] The effect of 30 μg / mL of commercially available fibronectin (hereinafter simply referred to as "fibronectin") on the responses of the same pairs as the olfactory receptor-odorant combinations used in the olfactory mucus confirmation experiment was examined. As a result, it was found that fibronectin had an effect of enhancing the responses for all three olfactory receptor-odorant combinations (Figs. 7A to F). Furthermore, it was confirmed that the tendency of the response enhancement effect on the three olfactory receptor-odorant pairs (Fig. 7G) was consistent with the results (Fig. 2A) when using olfactory mucus samples. To further examine the effect of fibronectin, the dose-dependence of the effect of fibronectin on the combination of OR2W1 and pentyl acetate was examined. Experiments were conducted in two ways: a method of stimulating with pentyl acetate immediately after adding fibronectin for 1 minute (the method performed when using olfactory mucus), and a method of stimulating with pentyl acetate after adding fibronectin for 1 minute and incubating for 5 minutes (a total incubation of 6 minutes) (the method performed when using a partially purified sample of olfactory mucus) (Fig. 8). As a result, it was found that the dose-response curve in the case of a 6-minute incubation was consistent with the curve when using fibronectin purified from olfactory mucus in terms of the degree of enhancement (1.4-fold at about 3 μg / mL) (see also Fig. 5I). From this result, it was shown that the activity of commercially available bovine plasma fibronectin is equivalent to that of fibronectin purified from olfactory mucus, at least with respect to the combination of OR2W1 / pentyl acetate. Also, it was found that the dose-response curve in the case of a 1-minute incubation was similar to the dose-response curve measured by diluting olfactory mucus, and this result further supported that fibronectin is an active factor in olfactory mucus samples.

[0059] 2-3. Analysis of the effect of fibronectin on the response to odorants in vitro and ex vivo 2-3-1. Examination of the effect of fibronectin on the dose-response curve of human olfactory receptor-odorant in vitro (1) Liquid phase stimulation To confirm that fibronectin is a response enhancer in human olfactory mucus, the following three points were examined: (a) Does fibronectin affect sensitivity (detection threshold), or does it simply enhance the response at concentrations of odorant substances above the threshold? (b) Does fibronectin have an effect on combinations of olfactory receptors from different families and odorant substances with different chemical structures? (c) Regarding the mechanism of action of fibronectin, particularly which of the olfactory receptor and the odorant substance it affects?

[0060] The following combinations were selected as the combinations of olfactory receptors and odorant substances to be examined. Olfactory receptors are classified into Class I and Class II. In this experiment, without bias towards either Class, receptors belonging to Class I and receptors belonging to Class II were both selected. For the odorant substances (see Table 1), substances that are often perceived in daily life (for example, odorant substances such as food, cosmetics, or body odor) were selected. (i) OR5AN1 and musk ketone (ii) OR8D1 and cyclotene (iii) OR2W1 and pentyl acetate (iv) OR5K1 and 2,3-dimethylpyrazine (v) OR5AN1 and muscone (vi) OR5P3 and methyl anthranilate (vii) OR51E1 and isovaleric acid Human plasma fibronectin was used as fibronectin. Fibronectin was found to shift the dose - response curve highly sensitively, that is, shift the threshold (the minimum amount required for the odorant to react with the olfactory receptor), for combinations other than the combination of (iv) above. The dose - response curves of (i) - (vii) above are shown in Fig. 9. For the combination of (iv) above, no shift in the threshold was observed, but an enhancement of the response was recognized when a high concentration of the odorant was present. From Fig. 9, it was suggested that fibronectin enhances the sensitivity regardless of whether the olfactory receptor is of Class I or Class II type. Therefore, it is considered that fibronectin can enhance the sensitivity regardless of the type and species of the olfactory receptor. Among the six combinations in which a shift in the threshold was observed, in five combinations, the threshold shifted by about half a digit, and in the combination of (i) above, a shift of about one digit was observed. Here, in the combination of OR5AN1 and musk ketone of (i) above, in the combination of (v) in which muscone was selected as the odorant, since the shift in the threshold was about half a digit, it was suggested that fibronectin acts on the odorant rather than the olfactory receptor and has an influence (Fig. 9).

[0061] (2) Gas - phase stimulation Next, it was examined whether fibronectin changes the response threshold of the olfactory receptor to the odorant in the gas - phase stimulation assay. To examine the effect of gas - phase stimulation on the dose - response curves of both the olfactory receptor - odorant combinations in which a shift in the dose - response curve was confirmed in the liquid - phase stimulation and the olfactory receptor - odorant combinations in which no shift in the dose - response curve was confirmed, the effect of fibronectin on the combinations of OR2W1 - pentyl acetate and OR5K1 - 2,3 - dimethylpyrazine was examined by the gas - phase stimulation assay (Fig. 10).

[0062] Regarding the combination of OR2W1-pentyl acetate, it was confirmed that the dose-response curve shifted sensitively in the presence of fibronectin (Figure 10A). In the presence of fibronectin (30 μg / mL), when a low concentration of pentyl acetate was present in the gas phase, it was found that both the average response magnification (Figure 10B) and the proportion of responsive cells (Figure 10C) increased. On the other hand, regarding the combination of OR5K1-2,3-dimethylpyrazine, in the presence of fibronectin, the average response magnification increased significantly (Figure 11A). However, in the presence of a low concentration of 2,3-dimethylpyrazine, the difference in the average response magnification between the presence and absence of fibronectin was smaller than the difference in the average response magnification of OR2W1 in the presence of a low concentration of pentyl acetate (Figure 11B). Also, the proportion of responsive cells in the presence of a low concentration of 2,3-dimethylpyrazine also increased significantly in the presence of fibronectin, but was smaller compared to the results of the combination of OR2W1-pentyl acetate (Figure 11C). From the above results, it was shown that fibronectin exerts an effect of reducing the response threshold of olfactory receptors to odorants in in vitro gas-phase stimulation assays.

[0063] 2-3-2. Examination of the effect of fibronectin ex vivo In vitro, it was confirmed that fibronectin enhances the stimulation of olfactory receptors by odorant substances not only in the liquid phase but also in the gas phase, which is closer to the in vivo environment. Next, we decided to examine whether fibronectin also affects olfactory receptor sensitivity in vivo. For this purpose, it would be ideal to knockout mouse fibronectin and compare it with the olfaction of wild-type mice. However, in vivo experiments are difficult due to reasons such as the fact that complete knockout of fibronectin leads to embryonic lethality and the unknown source of fibronectin involved in olfaction. Therefore, we decided to examine the effect of fibronectin in an ex vivo experimental system. In the electroolfactogram (EOG) of mice, the electrical response to gaseous odorant substances can be detected from the olfactory epithelium in the presence of olfactory mucus from euthanized mice. When a part of the olfactory mucus was removed from the olfactory epithelium of turtles and bullfrogs, it was reported that the response to the odorant substance pentyl acetate measured by EOG disappeared or decreased (Shibuya, Science 143:1338 - 1339 1964). Therefore, we decided to examine the effect of fibronectin by confirming whether fibronectin can restore the response to odorant substances after removal of olfactory mucus.

[0064] When the effect of removing olfactory mucus on the response to pentyl acetate was examined by mouse EOG, it was confirmed that the response to gaseous pentyl acetate disappeared or decreased due to partial removal of olfactory mucus (Figure 12). When mouse plasma fibronectin (300 μg / mL) was added to the measurement area of the olfactory epithelium, the response to pentyl acetate was significantly partially restored, whereas addition of control BSA (300 μg / mL) did not result in restoration (Figures 12A, B, and C). From these results, it became clear that fibronectin enhances the response to gaseous odorant substances in the olfactory organs of animals.

[0065] Next, it was examined whether fibronectin was present in the olfactory mucus from which it had been removed. The substances present in the sponge used to remove the olfactory mucus were eluted, and the eluate was subjected to Western blotting with an anti-fibronectin antibody. As a result, it was found that fibronectin was contained in the olfactory mucus, and it is considered that the removal of this fibronectin caused the disappearance of the response (Fig. 12D).

[0066] 3-3. Examination of the mechanism of olfactory sensitivity enhancement by fibronectin In the assay in the liquid phase, it has been suggested that fibronectin may have some effect on odorant substances rather than olfactory receptors (see 2-3-1 above and Fig. 9). Therefore, using methyl anthranilate that emits fluorescence as the odorant substance (excitation wavelength: 340 nm, fluorescence wavelength: 430 nm), it was examined whether the concentration and distribution of the odorant substance were affected in the presence of fibronectin. For comparison with the results of Fig. 9, the addition of fibronectin and the odorant substance was carried out in the same manner as the experimental method of Fig. 9. When the cells were stimulated with methyl anthranilate in the absence of fibronectin, compared with the case where the cells were stimulated with methyl anthranilate after the addition of fibronectin, the fluorescence from methyl anthranilate was stronger at the outer edge of the cells (Fig. 13B). From this result, it is suggested that fibronectin accumulates around the cells and binds temporarily to the odorant substance. Here, if fibronectin binds strongly to the odorant substance, it is considered that there will be a delay in the disappearance of the fluorescence from the odorant substance when the buffer is flowed by perfusion. Therefore, the fluorescence intensity was measured over time from 50 randomly selected cells (Fig. 13C). When the odorant substance was added, the fluorescence intensity around the cells increased, and it decreased as the odorant substance was washed away. The maximum value of the fluorescence intensity around the cells after the addition of fibronectin was larger than the value before the addition of fibronectin. However, the time required for the fluorescence to disappear by washing away the odorant substance was independent of the presence or absence of the addition of fibronectin. From the above results, it was suggested that, at least in the case of methyl anthranilate, the interaction between the odorant and fibronectin is not strong and is transient.

Industrial Applicability

[0067] The present invention is expected to be used in all fields targeting the action of olfactory receptors on odorants.

Claims

1. An enhancer for enhancing the response sensitivity of olfactory receptors, containing fibronectin.

2. The enhancer for enhancing response sensitivity according to Claim 1, wherein the olfactory receptor is derived from a mammal.

3. An enhancer for enhancing the sensitivity of an odor sensor, containing fibronectin.

4. The enhancer for enhancing the sensitivity of an odor sensor according to Claim 3, wherein the odor sensor comprises an olfactory receptor as a component.

5. A pharmaceutical composition for improving olfactory disorders, containing fibronectin.

6. The pharmaceutical composition according to Claim 5, wherein the olfactory disorder is olfactory hypofunction or anosmia.

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