Composition for preventing, ameliorating or treating olfactory disorder comprising nicotinamide adenine dinucleotide as active ingredient

Abstract

The present invention relates to a use of nicotinamide adenine dinucleotide (NAD) for preventing or treating olfactory disorders. The NAD increases the thickness of olfactory epithelial tissue and promotes the proliferation of olfactory stem cells and the differentiation thereof into olfactory neurons, and thus can effectively restore lost olfactory sense. Therefore, the NAD can be effectively used for preventing, ameliorating or treating olfactory disorders.

Description

A composition for the prevention, improvement, or treatment of olfactory disorders comprising nicotinamide adenine dinucleotide as an active ingredient

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0156437 dated November 06, 2024, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0003] [Technology Field]

[0004] The present invention relates to a composition for the prevention, improvement, or treatment of olfactory disorders comprising nicotinamide adenine dinucleotide as an active ingredient.

[0005] The sense of smell is an important sense that enables us to perceive changes or dangers in the surrounding environment and plays a significant role in the quality of life. Therefore, when an olfactory disorder occurs, not only does it lower an individual's quality of life, such as the loss of the pleasure of eating due to the inability to smell food, but it can also lead to serious problems that threaten life, as the sense of smell acts as a defense mechanism against harmful gases or spoiled food, making it difficult to respond appropriately to the surrounding environment.

[0006] However, due to the impact of rapid industrialization and modernization, contact with pollutants and chemicals that can act as olfactory toxins in the living environment has increased, leading to a growing prevalence of olfactory disorders. Upper respiratory tract infections, aging, diabetes, and smoking have been reported as causes of olfactory disorders.

[0007] Various treatments for such olfactory disorders have been proposed to date, including the use of steroids, vitamins, strychnine, zinc, and aminophylline. However, it has been reported that no drug or treatment method, including steroids, has yet shown a high cure rate in patients with olfactory disorders, and most patients are currently being neglected.

[0008] Against this technical background, the applicant has confirmed that nicotinamide adenine dinucleotide (NAD) can effectively restore lost olfaction by increasing the thickness of olfactory epithelial tissue and differentiating olfactory stem cells into olfactory neurons; thus, the present invention is expected to be usefully utilized for the prevention, improvement, or treatment of olfactory disorders.

[0009] The present invention aims to provide a composition for the prevention, improvement, or treatment of olfactory disorders.

[0010] To achieve the above objective, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of olfactory disorders comprising nicotinamide adenine dinucleotide (NAD).

[0011] In addition, to achieve the above objective, another aspect of the present invention provides a quasi-drug for the prevention or improvement of olfactory disorders comprising nicotinamide adenine dinucleotide (NAD).

[0012] The present invention relates to the use of nicotinamide adenine dinucleotide (NAD) for the prevention or treatment of olfactory disorders. Since the NAD can effectively restore lost olfactory senses by increasing the thickness of olfactory epithelial tissue and promoting the proliferation of olfactory stem cells and differentiation into olfactory neurons, it can be usefully utilized for the prevention, improvement, or treatment of olfactory disorders.

[0013] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0014] Figure 1 shows the experimental process of performing behavioral experiments after treating a mouse model of olfactory loss prepared by treating with ZnSO4 with NAD (nicotinamide adenine dinucleotide) or dexamethasone.

[0015] Figure 2 shows a schematic diagram of behavioral experiments (buried food test and T-maze test) in which mice are made to find hidden food to test olfactory function in an olfactory loss mouse model.

[0016] Figure 3 shows the results of measuring the time it takes for mice to find food by performing behavioral experiments after administering NAD intranasally or intraperitoneally, or dexamethasone intraperitoneally, to a mouse model of olfactory loss.

[0017] Figure 4a shows H&E stained images of olfactory epithelial tissue collected after administering NAD intranasally or intraperitoneally, or dexamethasone intraperitoneally, to a mouse model of olfactory loss.

[0018] Figure 4b compares the thickness of olfactory epithelial tissue after administering NAD intranasally or intraperitoneally, or dexamethasone intraperitoneally, to a mouse model of olfactory loss.

[0019] Figure 5 shows images of olfactory epithelial tissue collected and subjected to immunofluorescence staining after administering NAD intranasally or dexamethasone intraperitoneally to a mouse model of olfactory loss, and the results of measuring the number of cells expressing the olfactory neuron marker OMP (olfactory marker protein) and the neuron marker Tuj1 (beta-III tubulin).

[0020] Figure 6 shows the results of measuring the expression levels of SOX2 and Nestin, which are proliferation-related markers of olfactory stem cells, and the expression levels of OMP and Tuj1, which are olfactory neuron markers, after treating olfactory stem cells with NAD.

[0021] The present invention will be described in detail below.

[0022]

[0023] 1. Pharmaceutical composition for the prevention or treatment of olfactory disorders

[0024] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of olfactory disorders comprising nicotinamide adenine dinucleotide (NAD).

[0025] The above NAD is a coenzyme composed of two nucleotides linked by a phosphate group; it plays an essential role in energy production processes such as cellular respiration by transferring electrons in redox reactions during metabolism, and has the structure of Chemical Formula 1 below:

[0026] [Chemical Formula 1]

[0027]

[0028] The above NAD can effectively restore lost olfaction by increasing the thickness of olfactory epithelial tissue and promoting the proliferation of olfactory stem cells and differentiation into olfactory neurons, and can be usefully utilized for the prevention, improvement, or treatment of olfactory disorders.

[0029] In the present invention, "prevention" refers to any act of suppressing a specific disease or disorder or delaying its onset by administering a pharmaceutical composition according to the present invention, and "treatment" refers to any act of improving or beneficially altering the symptoms of a specific disease or disorder by administering a pharmaceutical composition according to the present invention.

[0030] The above "olfactory disorder" refers to a condition in which the sense of smell becomes dulled or absent, and includes damage to the olfactory center or its neural pathways, as well as disorders within the nasal cavity. The causes of olfactory disorder may include sinus diseases such as sinusitis, the common cold, allergic rhinitis, loss of olfactory sensory cells due to head injury, exposure to toxic substances such as drugs or industrial hazardous materials, diabetes, hormonal imbalances, Alzheimer's disease, Parkinson's disease, brain tumors, or complications after sinus surgery.

[0031] In the present invention, the olfactory disorder may be at least one disease selected from the group consisting of anosmia, hyposmia, hyperosmia, dysosmia, anosmia blindness, and phantomosmia. Specifically, the olfactory disorder may be anosmia, but is not limited thereto. Anosmia refers to a disease in which one cannot smell at all; hyposmia refers to a disease in which one can smell strong odors but cannot smell weak odors; hyperosmia refers to a disease in which one smells more sensitively; dysosmia refers to a disease in which one misperceives a certain odor as another; anosmia refers to a disease in which one cannot smell only a specific odor; and phantomosmia refers to a disease in which one complains of smelling even when there is no odor-producing substance present.

[0032] More specifically, the aforementioned anosmia (loss of smell) refers to a pathological condition in which one cannot smell at all, that is, the sense of smell is completely lost. This anosmia may occur when the nasal cavity is obstructed due to acute or chronic rhinitis, nasal septal abnormalities, or neoplasms such as nasal polyps, preventing air from reaching the olfactory nerve; when abnormalities occur in the olfactory region due to sinusitis, tuberculosis, or tumors; or when there are abnormalities or dysfunctions in the central olfactory function, such as brain tumors or hysteria; or it may result from the death of olfactory nerve cells. The aforementioned hyposmia (hyposmia) refers to a disease in which the degree to which stimuli are perceived in the olfactory region—that is, the olfactory area—is reduced, allowing one to smell strong odors but not weak odors. This condition may occur due to a decline in the function of olfactory nerve cells and can be clinically clearly distinguished from the aforementioned anosmia in that it is not a state of complete loss of the sense of smell.

[0033] The pharmaceutical composition of the present invention containing NAD has excellent olfactory recovery activity and can have a therapeutic effect on all olfactory disorders, and since the NAD can promote the differentiation of olfactory stem cells into olfactory neurons, the therapeutic activity for olfactory loss is particularly superior.

[0034] The pharmaceutical composition of the present invention may be for nasal administration. Depending on the purpose, the pharmaceutical composition of the present invention may be administered by an appropriate method such as intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration, or oral administration; however, since the therapeutic activity for olfactory disorders is particularly superior when administered by the nasal method, nasal administration is most preferable.

[0035] In a specific embodiment of the present invention, NAD was administered intranasally or intraperitoneally to a mouse model of olfactory loss, and behavioral experiments were performed to test olfactory function to compare the degree of olfactory recovery. Additionally, olfactory epithelial tissue was collected and its thickness was compared. As a result, it was found that when NAD was administered intranasally, the time taken to find food decreased significantly and the thickness of the olfactory epithelial tissue increased significantly; furthermore, it was confirmed that this olfactory recovery activity was significantly superior compared to when NAD was administered intraperitoneally.

[0036] In addition, the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. For example, the pharmaceutical composition may be administered at a dose of 0.0001 to 1,000 mg / kg per day, specifically 0.01 to 200 mg / kg, and the administration may be performed once a day or divided into several doses.

[0037] The above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other olfactory disorder therapeutic agents, and may be administered sequentially or simultaneously with conventional olfactory disorder therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0038] Meanwhile, the pharmaceutical composition of the present invention may be provided as a pharmaceutical composition comprising an active ingredient alone or comprising one or more pharmaceutically acceptable carriers, excipients, or diluents. When the pharmaceutical composition of the present invention is formulated, it may be prepared using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0039] In particular, when the pharmaceutical composition of the present invention is used by a nasal administration method, it may be formulated as aerosols, drops, or insufflation, and may also be formulated as a powder or microsphere. When the pharmaceutical composition of the present invention is administered in a drop formulation, the pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one that is commonly used in formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0040]

[0041] 2. Quasi-drugs for the prevention or improvement of olfactory disorders

[0042] In addition, another aspect of the present invention provides a quasi-drug for the prevention or improvement of olfactory disorders comprising nicotinamide adenine dinucleotide (NAD).

[0043] The above description regarding NAD and olfactory disorders is the same as that described above in '1. Pharmaceutical composition for the prevention or treatment of olfactory disorders,' and specific descriptions are to be taken from therein. Below, only the compositions specific to quasi-drugs will be described.

[0044] In the present invention, the term "improvement" refers to any act that causes the symptoms of a disease or disability to improve or become beneficial.

[0045] The quasi-drug of the present invention may be for nasal administration. Depending on the purpose, the quasi-drug of the present invention may be administered by an appropriate method such as nasal administration, oral administration, transdermal administration, or oral administration; however, since the activity of improving olfactory disorders is particularly superior when administered by the nasal method, nasal administration is most preferable.

[0046] The above "quasi-drugs" refer to items used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, and among these, items with milder effects than pharmaceuticals. According to the Pharmaceutical Affairs Act, quasi-drugs are defined as items excluding those used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans or animals, or products that have mild effects on the human body or do not act directly on it.

[0047] The quasi-drug of the present invention is used for the purpose of preventing or improving olfactory disorders, and there are no particular limitations on its formulation. In each formulation, other ingredients may be arbitrarily selected and combined according to the formulation or purpose of use of other quasi-drugs. The amount of the active ingredient can be appropriately determined according to the purpose of use (inhibition or alleviation). For example, it may include conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0048]

[0049] Another aspect of the present invention provides a method for preventing, improving, or treating olfactory disorders, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition or quasi-drug to a subject who requires prevention, improvement, or treatment of olfactory disorders.

[0050]

[0051] The present invention will be explained in detail below through examples.

[0052] However, the following examples are intended to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0053]

[0054] [Example 1]

[0055] Confirmation of olfactory recovery activity by intranasal administration of NAD in anosmia mouse model

[0056] 1-1. Measurement of Olfactory Recovery Activity through Behavioral Experiments

[0057] To confirm the activity of NAD (nicotinamide adenine dinucleotide) in restoring olfactory loss, an olfactory loss mouse model was prepared, NAD was administered, and behavioral experiments were performed to determine whether the mice's sense of smell was restored.

[0058] First, an olfactory loss model was prepared by administering 20 μL of ZnSO4 (170 μM) into the left and right nostrils of mice three times a week for 5 weeks. Normal control mice were administered 20 μL of PBS (phosphate-buffered saline) into the left and right nostrils (Fig. 1).

[0059] Subsequently, NAD was administered intranasally (in) or intraperitoneally (ip) to mice with anosmia at a concentration of 100 mg / kg five times a week for 4 weeks, and behavioral tests (buried food test and T-maze test) were performed to test olfactory function by having the mice find hidden food (Fig. 2). As a positive control (treatment control), mice with anosmia that were administered dexamethasone intraperitoneally at a concentration of 1 mg / kg once a week were used.

[0060] As a result of behavioral experiments, it was found that the time taken to find food decreased starting from day 14 in mice administered nasally with NAD, and on day 28, the time taken to find food was most similar to that of the normal control group, confirming that the sense of smell was significantly restored in mice with olfactory loss due to the nasal administration of NAD (Fig. 3). This olfactory recovery activity was found to be significantly higher than that of the positive control group (treatment control group), the dexamethasone treatment group.

[0061] In contrast, in mice administered NAD intraperitoneally, the time to find food was temporarily reduced, but increased again on day 28 of the experiment, indicating that the sense of smell was not restored in mice with olfactory loss.

[0062] Through the above experiment, it was confirmed that administering NAD nasally can effectively restore the sense of smell in mice with olfactory loss, and that this effect is significantly superior compared to when NAD is administered peritoneally.

[0063]

[0064] 1-2. Measurement of Olfactory Recovery Activity through Analysis of Olfactory Epithelial Tissue

[0065] Mice that underwent the behavioral experiment in 1-1 above were sacrificed to collect olfactory epithelial tissue, and H&E staining and immunofluorescence staining were performed. Subsequently, the thickness of the olfactory epithelial tissue and the number of cells expressing olfactory neuron markers were measured.

[0066] First, H&E staining was performed to compare the thickness of the olfactory epithelial tissue. The results showed that the thickness of the olfactory epithelial tissue in mice with anosmia was significantly reduced compared to normal control mice, but in mice administered NAD intranasally, the reduced thickness of the olfactory epithelial tissue was found to significantly increase to a level similar to that of normal control mice (Figs. 4a and 4b).

[0067] In contrast, it was found that the thickness of the olfactory epithelial tissue was not significantly restored in mice administered NAD intraperitoneally and mice treated with dexamethasone.

[0068] Next, immunofluorescence staining was performed to compare the number of cells expressing OMP (olfactory marker protein), an olfactory neuron marker, and Tuj1 (beta-III tubulin), a neuron marker, in olfactory epithelial tissue. As a result, the number of cells expressing OMP was significantly reduced in mice with olfactory loss compared to normal control mice, but the number of reduced cells was significantly increased in mice administered intranasally with NAD (Fig. 5). Meanwhile, the number of cells expressing Tuj1 was found to be significantly increased in mice administered intranasally with NAD compared to normal control mice.

[0069] Through the above experiment, it was confirmed that when NAD is administered nasally, the thickness of the reduced olfactory epithelial tissue in mice with olfactory loss significantly increases, and the number of olfactory neuron markers and cells expressing neuron markers also significantly increases, thereby effectively restoring the lost sense of smell.

[0070]

[0071] [Example 2]

[0072] Confirmation of NAD's activity promoting olfactory stem cell differentiation

[0073] To confirm the mechanism by which NAD restores lost olfactory function, olfactory stem cells were treated with NAD, and the level of differentiation of the olfactory stem cells into olfactory neurons was measured.

[0074] Specifically, olfactory epithelial mucosa was collected from the human nasal cavity, washed with DMEM / HAM F12, and cultured at 37°C for 1 hour in a 2.4 IU / mL solution of dispase II (Stem Cell Technologies; CA). Subsequently, only the lamina propria, the proper layer of the olfactory epithelium, was isolated and added to a 10% FCS (fetal calf serum) solution containing DMEM / HAM F12 and antibiotics to culture olfactory stem cells. The cultured olfactory stem cells were differentiated into olfactory neurons by being cultured in an environment containing neurobasal medium, DMEM / F12 medium supplemented with B-27, L-glutamine (Glutamax), glutamate (2 mM), penicillin (50 U / mL), and streptomycin (50 μg / mL). At this time, NAD was added at a concentration of 2.5 or 5.0 mM, and the expression levels of OMP, a marker of olfactory neurons, and Tuj1, a neuron marker, were measured to determine whether NAD could increase the differentiation of olfactory stem cells into olfactory neurons.

[0075] As a result, when NAD was applied, the expression levels of SOX2 and Nestin, markers related to olfactory stem cell proliferation, and the expression levels of OMP and Tuj1, markers related to olfactory neurons, increased in a NAD concentration-dependent manner compared to the untreated control group (Fig. 6).

[0076] Through the above experiment, it was confirmed that NAD can restore lost olfaction by promoting the proliferation of olfactory stem cells and their differentiation into olfactory neurons.

[0077] As described above, NAD can effectively restore lost olfaction by increasing the thickness of olfactory epithelial tissue and differentiating olfactory stem cells into olfactory neurons, and thus can be usefully utilized for the prevention, improvement, or treatment of olfactory disorders.

[0078]

[0079] Although representative embodiments of the present invention have been described above by way of example, the scope of the present invention is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of this application.

Claims

1. A pharmaceutical composition for the prevention or treatment of olfactory disorders comprising nicotinamide adenine dinucleotide (NAD).

2. In Claim 1, A pharmaceutical composition wherein the above-mentioned olfactory disorder is at least one disease selected from the group consisting of anosmia, hyposmia, hyperosmia, dysosmia, anosmia and phantomosmia.

3. In Claim 1, A pharmaceutical composition in which the above-mentioned olfactory disorder is anosmia.

4. In Claim 1, The above pharmaceutical composition is a pharmaceutical composition intended for nasal administration.

5. In Claim 1, The above pharmaceutical composition is a pharmaceutical composition that increases the thickness of the olfactory epithelial tissue.

6. In Claim 1, The above pharmaceutical composition is a pharmaceutical composition that promotes the proliferation of olfactory stem cells.

7. In Claim 1, The above pharmaceutical composition is a pharmaceutical composition that increases the differentiation of olfactory stem cells into olfactory neurons.

8. In Claim 1, The above pharmaceutical composition is a pharmaceutical composition that promotes the regeneration of olfactory epithelial cells.

9. A quasi-drug for the prevention or improvement of olfactory disorders containing nicotinamide adenine dinucleotide (NAD).

10. In Claim 9, The above-mentioned quasi-drug is a quasi-drug intended for nasal administration.

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