KR102664264B1 - Composition for prevention or treatment of xerostomia comprising ferrostatin-1 - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating dry mouth, especially dry mouth caused by menopause, containing Ferostatin-1 as an active ingredient. According to the present invention, the above-mentioned composition is used in an animal model that induces menopause. When treated with ferrostatin-1, the increased fat accumulation and lipid peroxidation in the salivary glands were reduced, the decreased mitochondrial function and activity of antioxidant enzymes were restored, and along with anti-inflammatory and antifibrotic effects, ultimately the decreased salivary secretion function was achieved. As this recovery has been confirmed, it can be usefully used to prevent, improve, or treat dry mouth of various causes, including dry mouth caused by menopause.

Description

Composition for preventing or treating xerostomia comprising ferrostatin-1 as an active ingredient {Composition for prevention or treatment of xerostomia comprising ferrostatin-1}

The present invention provides a composition for preventing, improving or treating dry mouth, especially dry mouth caused by menopause (dry mouth caused by menopausal hormonal fluctuations), containing Ferrostatin-1 as an active ingredient, or salivary secretion. It relates to a composition for augmentation.

Dry mouth (xerostomia) is a condition in which the mouth becomes dry because saliva is not secreted continuously for some reason. It occurs when the saliva secretion volume falls below 0.1 ml per minute. Saliva is secreted by three major salivary glands - the parotid, submandibular, and sublingual glands - and small minor salivary glands distributed evenly throughout the oral cavity. When the function of these tissues decreases, saliva secretion decreases and symptoms of dry mouth appear. When dry mouth occurs, the protective effect in the oral cavity by salivary solutions, proteins, and mucins is significantly reduced, leading to destruction of salivary gland tissue. When salivary secretion is reduced and chronic dry mouth develops, it changes pronunciation, makes chewing and swallowing food difficult, causes a burning sensation in the mouth, and increases the risk of cavities and oral diseases.
Meanwhile, postmenopausal is when a woman's menstruation completely stops. During menopause, many physical and psychological changes occur due to decreased female hormones. Among 93% of middle-aged women in Korea, the incidence of menopausal symptoms is reported to be as high as over 50% and they are experiencing considerable discomfort (Ae-ri Song, 2005, Study on health factors related to menopause management in menopausal women, Women's Health) Journal of Nursing, 11(1):12-19).
When menopause occurs, various diseases occur due to hormonal changes, such as a decrease in estrogen production and an increase in follicle-stimulating hormone and progesterone. Estrogen prevents the accumulation of visceral fat, lowers cholesterol levels, and protects blood vessels. Therefore, in postmenopausal women, visceral fat accumulates, cholesterol easily accumulates in blood vessels, and as estrogen secretion decreases, the concentration of total cholesterol and low-density lipoprotein increases, increasing the risk of arteriosclerosis and cardiovascular and liver-related diseases. do. Additionally, symptoms of menopause may include facial redness and weakened bones.
In addition, examples of symptoms that may occur due to menopause include dry mouth, dry eyes, vaginal dryness, decreased elasticity and dryness of the skin. These symptoms greatly reduce the quality of life of postmenopausal women.
One of the most common methods used to prevent or treat menopausal symptoms is hormone replacement therapy. The hormone replacement therapy treatment is a method of improving menopausal symptoms by periodically supplementing female hormones. However, long-term use of hormone replacement therapy is known to have side effects such as breast cancer or stroke. Physical symptoms include nausea, headache, breast pain, abdominal distension, edema, and uterine bleeding, and mental symptoms include anxiety, nervousness, and depression. It may appear. In addition, this hormone replacement therapy has the limitation that it cannot completely eliminate various symptoms caused by decreased estrogen secretion, and there is the inconvenience and resistance to frequent tests. Accordingly, there is a need for the development of treatments that are effective in preventing or treating menopausal symptoms and have no side effects.
In addition, many studies are currently being conducted to treat dry mouth, but since a fundamental treatment for dry mouth has not been developed, popular treatments are mainly used. Increasing water intake to keep the mouth moist, chewing sugar-free gum, drinking lemon-flavored drinks, using artificial saliva, and gargling solutions with added citric acid are used. In addition, newly developed saliva preparations for sprays and electrical stimulation methods are being used experimentally. However, there is a problem that the effect is temporary and not limited. Currently, chemical drugs used as salivary secretion stimulants include anethole trithion, known as a muscarinic receptor agonist, and cevimeline hydrochloride, but their effects are unstable and cause side effects in the digestive system such as nausea, vomiting, loss of appetite, and abdominal discomfort. Potential disadvantages are being raised.
Therefore, there is a need to develop an effective treatment for dry mouth, especially dry mouth caused by menopause.

Non-patent Document 1: Authored by Song Ae-ri, “Study on health factors related to menopause management in menopausal women”, Journal of Women’s Health Nursing, published in March 2005, pp. 12~19 (Non-patent document 0002) Non-patent document 2: “Diagnosis and treatment of dry mouth,” co-authored by Jeong-seok Choi and Jae-yeol Lim, Korean J Otorhinolaryngol-Head Neck Surg, published in 2016, pp. 424~429 (Non-patent document 0003) Non-patent document 3: “Dry Mouth” by Hyeongeun Kwon, Jinchun Lee, and Byeongju Lee, J Clin Otolaryngol, published in 2020, pp. 11~17

While the present inventors were researching effective treatments for dry mouth, especially dry mouth caused by menopause, when treating an animal model with menopause with Ferrostatin-1, the symptoms caused by menopause were alleviated. The present invention was completed by confirming that the decreased salivary secretion function was restored.
Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating dry mouth.
Another object of the present invention is to provide a quasi-drug for preventing or improving dry mouth.
Another object of the present invention is to provide a food composition for preventing or improving dry mouth.
Another object of the present invention is to provide a quasi-drug for enhancing saliva secretion.
Another object of the present invention is to provide a food composition for enhancing saliva secretion.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating dry mouth containing ferrostatin-1 as an active ingredient.
In addition, in order to achieve the above other objects, the present invention provides a quasi-drug for preventing or improving dry mouth containing Ferrostatin-1 as an active ingredient.
In addition, in order to achieve the above other object, the present invention provides a food composition for preventing or improving dry mouth containing ferrostatin-1 as an active ingredient.
In addition, in order to achieve the above other object, the present invention provides a quasi-drug for enhancing salivary secretion containing Ferrostatin-1 as an active ingredient.
In addition, in order to achieve the above other object, the present invention provides a food composition for enhancing salivary secretion containing Ferrostatin-1 as an active ingredient.

The present invention confirmed the improvement or treatment effect of Ferostatin-1, a ferroptosis inhibitor, on dry mouth, especially dry mouth caused by menopause, in an animal model induced by menopause. When treated with 1, increased fat accumulation and lipid peroxidation in the salivary glands were reduced, the decreased mitochondrial function and activity of antioxidant enzymes were restored, and along with anti-inflammatory and antifibrotic effects, the ultimately decreased salivary secretion function was confirmed to be restored. Therefore, it can be usefully used to prevent, improve, or treat dry mouth of various causes, including dry mouth caused by menopause.

Figure 1 shows the results of confirming the effect of ferrostatin-1 on fat accumulation (Figure 1A and Figure 1B) and lipid peroxidation (Figure 1C and Figure 1D) due to menopause in the salivary glands of an animal model that induced menopause.
Figure 2 shows the results of confirming the effect of ferrostatin-1 on mitochondrial function (Figure 2A) and antioxidant enzyme activity (Figure 2B) decreased by menopause in the salivary glands of an animal model that induced menopause.
Figure 3 shows the results of confirming the effect of ferrostatin-1 on inflammation caused by menopause in the salivary glands of an animal model that induced menopause.
Figure 4 shows the effect of ferrostatin-1 on fibrosis caused by menopause in the salivary glands of an animal model inducing menopause, showing collagen I, an indicator of fibrosis (Figure 4A), and Col1a1 and Col3a, mRNA subtypes of collagen (Figure 4B). , a result confirmed through analysis of TGFβI (Figure 4C), TGFβI, and TGFβII (Figure 4D) expression.
Figure 5 shows the effect of ferrostatin-1 on the decline in salivary secretion function due to menopause in the salivary glands of an animal model inducing menopause, showing the mRNA of AQP5 (Figure 5A), AQP5 and AQP3, which are indicators of salivary secretion function (Figure 5B). This result was confirmed through expression analysis.

Hereinafter, the present invention will be described in detail.
The present invention provides a pharmaceutical composition for preventing or treating dry mouth containing Ferrostatin-1 as an active ingredient.
Iron induces lipid peroxidation of saturated fatty acids in the body and generates reactive oxygen species along with ATP synthesis in mitochondria. As a result, a large amount of reactive oxygen species and adducts accumulate within the cells, cell dysfunction occurs due to lipid peroxidation products, and this causes cell death, which is called ferroptosis. The characteristics of the ferroptosis mechanism are 1) generation of reactive oxygen species, especially lipid reactive oxygen species, 2) reduction of the antioxidant enzyme GPX4 (Glutathione peroxidase 4), 3) accumulation of lipid hydroperoxides, 4) This can be explained simply by an increase in iron. It is reported that this ferroptosis mechanism is related to the development mechanism of various cancers and diseases such as Parkinson's disease, sepsis, and liver disease.
Ferroptosis inhibitors that inhibit ferroptosis are classified into type 1 (class I) and type 2 (class II). Type 1 inhibits iron accumulation within cells, and type 2 inhibits lipid oxidation. Deferoxamine, included in type 1, is an iron and aluminum ion chelator that has been used to treat aluminum toxicity in hemochromatosis and dialysis patients due to iron overdose, blood transfusion, or genetic factors. Ferrostatin-1, which belongs to type 2, is a substance with the structure of the following formula (1) and is known to inhibit ferroptosis by inhibiting lipid oxidation similar to GPX4.
[Formula 1]

However, as in the present invention, the therapeutic effect of ferroptosis inhibitors in dry mouth, especially dry mouth caused by menopause, has not been reported.
According to one embodiment of the present invention, when ferrostatin-1 was administered to an animal model that induced menopause, the decreased mitochondrial function and the activity of antioxidant enzymes, which are indicators of ferroptosis induction, were restored in the salivary glands. Therefore, it was confirmed that ferrostatin-1 inhibits salivary gland ferroptosis.
In addition, according to another embodiment of the present invention, when ferrostatin-1 is administered to an animal model that induces menopause, symptoms such as menopause-related fat accumulation, lipid peroxidation, inflammation, or fibrosis in the salivary glands are alleviated, especially salivary glands. The expression of AQP5 (Aquaporin-5), an indicator of secretion function, was recovered.
The AQP5 is a major transport protein expressed in the terminal membrane of acinar cells that secrete saliva. AQP5 plays a role in specifically transporting water and acts as an essential element in secreting water, which accounts for 99% of saliva. It has been reported that the expression of AQP5 is reduced in non-obese diabetic mice and autoimmune diseases such as Sjögren's syndrome, which is considered a major cause of dry mouth.
Therefore, according to the present invention, ferrostatin-1 increases the expression of AQP5, and ferrostatin-1 can be used as a composition for preventing, improving, or treating dry mouth of various causes, including dry mouth caused by menopause. You can.
The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. . Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include ferrostatin-1 of the present invention and at least one or more excipients, such as starch, calcium carbonate, and sucrose. Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.
The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 1000 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.
The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection.
Forms for parenteral administration of the pharmaceutical composition of the present invention include toothpaste, mouthwash, topical administration agents (cream, ointment, dressing solution, spray, other coating agents, etc.). As an example of the formulation of the topical administration agent, the pharmaceutical composition for preventing or treating dry mouth according to the present invention may be fixed to a film or patch containing a water-soluble polymer and formulated so that it can be attached to teeth and surrounding areas. .
In the present invention, the pharmaceutical composition for preventing or treating dry mouth includes, in addition to the active ingredient, any compound or natural extract that has proven safety and has the activity of treating dry mouth or enhancing or promoting salivary secretion in order to increase or reinforce the effect of treating dry mouth. may additionally be included.
In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemical therapy, and biological response regulators to prevent or treat dry mouth.
As used herein, the term “administration” means providing a given composition of the invention to an individual by any suitable method.
In the present invention, the term “prevention” may refer to any act of suppressing or delaying the onset of dry mouth by administering the pharmaceutical composition for preventing or treating dry mouth according to the present invention to an individual.
In the present invention, the term "treatment" may mean any action that improves or benefits the symptoms of xerostomia or related diseases by administering the composition according to the present invention to a subject suspected of having xerostomia.
In the present invention, the term "improvement" may mean any action that reduces at least the degree of a parameter, for example, a symptom, related to the condition being treated.
In addition, the present invention provides a quasi-drug for preventing or improving dry mouth containing Ferrostatin-1 as an active ingredient.
In the present invention, “saliva” is also called saliva and refers to the colorless, viscous digestive juice secreted from the salivary glands in the mouth. In general, the amount of saliva secreted in a normal person for 24 hours is about 1000 to 1500 ml. Normally, when not stimulated by the autonomic nerves, 0.001 to 0.2 ml is produced per minute, and when stimulated, the outflow increases to 0.18 to 1.7 ml per minute, with an average secretion amount of about 1 ml per minute. It is about 99% water and contains various minerals.
According to the present invention, ferrostatin-1 can restore the decreased salivary secretion function, and the present invention provides a quasi-drug for enhancing salivary secretion containing ferrostatin-1 as an active ingredient. .
The quasi-drug may be one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, oral spray, oral gel, oral ointment, mask, poultice, patch, and transdermal absorbent, but is not limited thereto. Additionally, the quasi-drugs can be applied to oral hygiene products such as toothbrushes, dental floss, interdental brushes, tongue cleaners, and oral tissues.
In the present invention, the term 'quasi-drug' refers to products that have a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, quasi-drugs under the Pharmaceutical Affairs Act exclude products used for pharmaceutical purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.
When using the composition according to the present invention as a quasi-drug additive, the composition can be added as is or used together with other quasi-drugs or quasi-drug components, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. The above-mentioned quasi-drug can be used as an auxiliary treatment for dry mouth, and can be administered simultaneously or sequentially with a dry mouth treatment.
When the quasi-drug of the present invention is toothpaste, in addition to containing ferrostatin-1 as an active ingredient, it may further contain an abrasive, binder, moisturizer, foaming agent, sweetener, whitening agent, or flavoring agent.
The abrasives include aluminum hydroxide, anhydrous silicic acid, aluminum silicate, dicalcium phosphate dihydroxide and anhydrous, tricalcium phosphate, calcium carbonate, calcium pyrophosphate, insoluble sodium metaphosphate, tribasic magnesium phosphate, magnesium carbonate, calcium sulfate, Polymethyl methacrylate, etc. can be used singly or in combination. The content of the abrasive may generally be 20% to 90% by weight based on the total composition, but is not limited to this content. When the quasi-drug is a paste-like composition, the binder may be garagenine, various cellulose derivatives for thickening, xanthan gum, gums such as tragacanth gum, polyvinyl alcohol, sodium polyacrylate, polyacrylic acid/maleic acid, etc. Organic binders such as polymers and synthetic polymer derivatives such as carboxyvinyl polymer and inorganic binders such as silica and laponite can be used alone or in combination. The content of the binder may generally be 0.3% to 5% by weight based on the total composition, but is not limited by the content.
In addition, the quasi-drug may additionally contain sorbitol, glycerin, ethylene glycol, propylene glycol, polyether glycol, polypropylene glycol, etc. as moisturizers during its manufacture.
In addition, the quasi-drugs may additionally contain flavorings or sweeteners. The sweetener may be sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, glycerin, sodium saccharin, stevioside, aspartame, etc., and the flavoring agent may be peppermint, menthol, anethole, eugenol, It may be limonene, citronol, alpha-terpineol, salicylmethyl, cineol, linalol, ethyllinalol, vanillin, thymol, spearmint oil, sage oil, rosemary oil, cinnamon oil, etc., and the sweetener or flavoring agent may be used alone. It can be used alone or in combination.
In addition, the quasi-drug of the present invention may contain surfactants used as foaming ingredients or additional effective ingredients singly or in combination, and the surfactants used as foaming ingredients include anionic surfactants, nonionic surfactants, and cationic surfactants. It may be any one selected from the group consisting of, and more specifically, an anionic surfactant, sodium lauryl sulfate, a nonionic surfactant, polyoxyethylene copolymer of polyoxypropylene (poloxamer), polyoxyethylene hydrogenated castor oil, poly Oxyethylene sorbitan fatty acid ester, etc. can be used without limitation.
In addition, if the quasi-drug of the present invention is a toothpaste, it can be manufactured using a conventional toothpaste manufacturing method except that it contains ferrostatin-1 as an active ingredient, and if the quasi-drug of the present invention is a mouthwash (cleanser), it can be manufactured using a conventional toothpaste manufacturing method. It can be prepared by mixing ferrostatin-1 with a phosphorus solution and formulating it into a mouthwash, and dry mouth can be prevented or improved by washing the mouth 2 to 10 times a day.
In addition, when the quasi-drug of the present invention is a mouthwash (cleanser), it may additionally contain a toothpaste carrier, more specifically, non-toxic alcohol. In the present invention, the ferrostatin-1 may be included in an amount of 0.00001 to 10% by weight relative to the total weight of the composition, preferably in an amount of 0.0005 to 5% by weight relative to the total weight of the composition, and more preferably, in an amount of 0.0005 to 5% by weight relative to the total weight of the composition. It may be included at 0.005 to 1% by weight, but is not limited thereto.
In addition, the present invention provides a food composition for preventing or improving dry mouth containing ferrostatin-1 as an active ingredient.
Additionally, the present invention provides a food composition for enhancing salivary secretion containing Ferrostatin-1 as an active ingredient.
The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food, health functional food, and food additives. .
In the present invention, the term "health functional food" refers to a food manufactured or processed using specific ingredients as raw materials or by extracting, concentrating, refining, or mixing specific ingredients contained in food raw materials for the purpose of health supplementation, It refers to a food designed and processed so that the above ingredients can fully exert bioregulatory functions on the living body, such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc., and performs functions related to prevention of disease or recovery of health. Say what you can do.
The food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art.
For example, as a health food, ferrostatin-1 of the present invention itself can be granulated, encapsulated, and powdered and consumed, or it can be prepared and consumed in the form of tea, juice, and drinks. Additionally, ferrostatin-1 of the present invention can be prepared in the form of a composition by mixing it with known substances or active ingredients known to have an effect in treating dry mouth or enhancing saliva secretion.
In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, marmalade, etc.), fish, meat and their processed foods (e.g. ham, sausage corn beef, etc.) , bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort. It can be manufactured by adding ferrostatin-1 of the present invention to food, frozen food, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.).
The preferred content of ferrostatin-1 of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the finally manufactured food, preferably in the finally manufactured food. It may be 0.01 to 50% by weight.
The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrate may be generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g, per 100 ml of the composition of the present invention.
In addition to the above, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonating agents used in carbonated drinks, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.
The contents of the present invention described above are applied equally to each other unless they contradict each other, and implementation by a person skilled in the art with appropriate changes is also included in the scope of the present invention.
Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited to the following examples.
Example 1. Preparation of menopause-induced xerostomia animal model
An animal model of xerostomia was established by removing both ovaries from female rats under inhalation anesthesia. At this time, 9-week-old female rats, at which the sexual maturity of rodents was completed, were used. They were divided into a control group without ovaries (Sham), an experimental group inducing xerostomia due to menopause (OVX), a ferrostatin-1 administration group (FER), or a similar group. Patients were classified into the group receiving deferoxamine, a roptosis inhibitor (DFO). In the ferrostatin-1 administration group (FER) and deferoxamine administration group (DFO), ferrostatin-1 at a concentration of 2.5 μM/kg or ferrostatin-1 at a concentration of 100 mg/kg was administered by intraperitoneal injection three times a week for 6 weeks. The induction of menopause was confirmed by a decrease in blood estrogen concentration using the Rat Estradiol (E2) ELISA Kit (MyBioSource).
Example 2. Confirmation of effects on fat accumulation and lipid peroxidation in salivary glands
As in Example 1, ferrostatin-1 or deferoxamine was administered to a menopause-induced animal model, and fat accumulation and lipid peroxidation in the salivary glands of each experimental group were measured using a known method using Hematoxylin & Eosin staining. Confirmed.
As a result, as shown in Figure 1, it was confirmed that fat accumulation increased in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM), but significantly decreased in the ferrostatin-1 administration group (FER) (Figure 1A and Figure 1B). In addition, in the xerostomia animal model group (OVX), lipid peroxidation indicators MDA (Malondialdehyde) and HAE (4-hydroxy-2-alkenals) were measured compared to the control group (SHAM) using the BIOXYTECH®MDA-586™ (OxisResearch™) kit. As a result, it was confirmed that MDA and HAE increased due to increased fat, but significantly decreased in the ferrostatin-1 administration group (FER). In particular, it was significantly reduced compared to the deferoxamine administered group (DFO) (Figure 1C and Figure 1D).
Example 3. Confirmation of effects on salivary gland mitochondrial function and antioxidant enzymes
As in Example 1, ferrostatin-1 or deferoxamine was administered to a menopause-induced animal model, and changes in salivary gland mitochondrial function and antioxidant enzyme GPX4 activity in each experimental group were compared and analyzed, as shown in Figure 2.
As a result of electron microscopy, death of mitochondria and deterioration of cell function were confirmed in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM), while mitochondria that were deteriorated due to menopause in the ferrostatin-1 administration group (FER) It was confirmed that cell function was restored (Figure 2A).
In addition, for GPX4 activity analyzed using the Glutathione Peroxidase Assay Kit (Colorimetric) (abcam), a decrease in activity was observed in the xerostomia-induced animal model (OVX), but in the ferrostatin-1 administered group (FER) ), it was confirmed that the decreased GPX4 activity due to menopause was significantly recovered. In particular, as in Example 2, it was confirmed that the degree of recovery of GPX4 activity was greater compared to the deferoxamine administered group (DFO) (Figure 2B).
Example 4. Confirmation of effect on inflammation of salivary glands
As in Example 1, ferrostatin-1 or deferoxamine was administered to the menopause-induced animal model, and the effect on salivary gland inflammation in each experimental group was confirmed by real-time PCR and is shown in Figure 3.
For this purpose, primers for IL-6 (Interleukin-6) [Forward: 5'-ATCTGCCCTTCAGGAACAGC-3' and Reverse: 5'-GAAGTAGGGAAGGCAGTGGC-3' were used, and tumor necrosis factor-α (tumor necrosis factor-α) was used. Primers for necrosis factor-α, TNFα) [forward: 5'-GGTCAACCTGCCCAAGTACT-3' and reverse: 5'-CTCCAAAGTAGACCTGCCCG-3'] were used.
As a result, the mRNA of inflammatory cytokines IL-6 and TNF increased in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM), but the mRNA of IL-6 and TNFα was significantly lower in the ferrostatin-1 treated group (FER). A decrease was confirmed. In particular, similar to the previous results, the degree of decrease was greater compared to the deferoxamine administration group (DFO), and in the case of TNFα, it was confirmed that the decrease was similar to that of the control group.
Example 5. Confirmation of effect on fibrosis of salivary glands
As in Example 1, ferrostatin-1 or deferoxamine was administered to a menopause-induced animal model, and the effect on salivary gland fibrosis in each experimental group was confirmed, as shown in Figure 4.
In addition, as a result of performing Masson's trichrome staining using a known method, collagen I, an indicator of salivary gland fibrosis, increased in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM). , a significant decrease was confirmed in the ferrostatin-1 administration group (FER) (Figure 4A)
In addition, the expression of collagen mRNA subtypes Col1a1 and Col3a was confirmed using real-time PCR. For this purpose, primers for Col1a1 [Forward: 5'-CATGTTCAGCTTTGTGGACCT-3' and Reverse: 5'-GCAGCTGACTTCAGGGATGT-3'] were used, and primers for Col3a [Forward: 5'-TCCCCTGGAATCTGTGAATC-3] ' and reverse: 5'-TGAGTCGAATTGGGGAGAAT-3'] were used.
As a result, it was confirmed that the expression of Col1a1 and Col3a was increased in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM), while it was significantly decreased in the ferrostatin-1 administered group (FER) (Figure 4B).
In addition, as a result of immunostaining using a TGFβI (tumor growth factor βI) antibody (abcam), it was found that intracellular TGFβI expression was increased in the xerostomia-induced animal model (OVX) and significantly decreased in the ferrostatin-1 administered group (FER). This was confirmed (Figure 4C).
In addition, primers for TGFβI [Forward: 5'-AAGAAGTCACCCGCGTGCTA-3' and Reverse: 5'-TGTGTGATGTCTTTGGTTTTGTCA-3'] and primers for TGFβII [Forward: 5'-ATCGATGGCACCTCCACATATG-3'] and Reverse: 5'-GCGAAGGCAGCAATTATCCTG-3'] to confirm the expression of TGFβI and TGFβII mRNA.
As a result, it was confirmed that the mRNA of TGFβI and TGFβII also increased in the xerostomia-induced animal model (OVX) compared to the control group (SHAM), and was significantly decreased in the ferrostatin-1 administered group (FER) (Figure 4D).
In particular, in Figures 4A to 4D, it was confirmed that the degree of reduction in fibrosis indices in the experimental group administered ferrostatin-1 was more significant than that in the group administered deferoxamine.
Example 6. Confirmation of effect on salivary secretion of salivary glands
As in Example 1, ferrostatin-1 or deferoxamine was administered to the menopause-induced animal model, and the effect on saliva secretion from the salivary glands of each experimental group was confirmed, and is shown in Figure 5.
For this purpose, immunostaining was performed using a known method using an antibody (Santa Cruz) against AQP5 (Aquaporin 5), a function indicator of salivary secretion.
As a result, the intracellular protein of AQP5 was decreased in the salivary glands of the xerostomia-induced animal model (OVX) compared to the control group (SHAM), but the intracellular protein of AQP5 was confirmed to be increased in the ferrostatin-1 administered group (FER) (Figure 5A ).
In addition, the mRNA expression of AQP5 and AQP3, another functional indicator of salivary secretion, was confirmed.
Primers for AQP5 [Forward: 5'-CATGAACCCAGCCCGATCTT-3' and Reverse: 5'-AGAGACCCAGTGAGAGGGG-3'] and primers for AQP3, another functional indicator of salivary secretion [Forward: 5] The expression of AQP5 and AQP3 mRNA was confirmed by real-time PCR using '-AATTGTCTGGAGCCCACTTG-3' and Reverse: 5'-CAGCTTGATCCAGGGCTCTC-3'].
As a result, AQP5 and AQP3 mRNA was also decreased in the xerostomia-induced animal model (OVX), but was confirmed to be significantly increased in the ferrostatin-1 administered group (Figure 5B). Therefore, it was confirmed that administration of ferrostatin-1 restores the salivary secretion function of the salivary glands, which was reduced due to menopause.
Overall, when Ferostatin-1 was treated in an animal model that induced menopause through an example of the present invention, increased fat accumulation and lipid peroxidation in the salivary glands were reduced, and the decreased mitochondrial function and antioxidant function were reduced. It was confirmed that the activity of the enzyme was restored, along with anti-inflammatory and anti-fibrotic effects, and ultimately the decreased salivary secretion function was restored. This can be used to prevent, improve or treat dry mouth caused by various causes, including dry mouth caused by menopause. It can be useful.

Claims (8)

Dry mouth due to menopausal hormonal fluctuations, which includes Ferrostatin-1 as an active ingredient, and the active ingredient increases the expression of AQP (Aquaporin) 5 protein and AQP 5 and AQP 3 mRNA. Pharmaceutical composition for prevention or treatment of. delete According to paragraph 1,
A pharmaceutical composition for preventing or treating dry mouth due to menopausal hormonal fluctuations, wherein the menopausal hormonal fluctuations are at least one selected from the group consisting of a decrease in estrogen production, an increase in follicle-stimulating hormone, and an increase in progesterone. Dry mouth due to menopausal hormonal fluctuations, which includes Ferrostatin-1 as an active ingredient, and the active ingredient increases the expression of AQP (Aquaporin) 5 protein and AQP 5 and AQP 3 mRNA. Quasi-drugs for the prevention or improvement of. According to clause 4,
The quasi-drug is characterized in that it is at least one selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, oral spray, oral gel, oral ointment, mask, poultice, patch, and transdermal absorbent.
Salivary gland function due to menopausal hormonal fluctuations, including Ferrostatin-1 as an active ingredient, wherein the active ingredient increases the expression of AQP (Aquaporin) 5 protein and AQP 5 and AQP 3 mRNA. A quasi-drug to enhance saliva secretion for decreased salivary function. Dry mouth due to menopausal hormonal fluctuations, which includes Ferrostatin-1 as an active ingredient, and the active ingredient increases the expression of AQP (Aquaporin) 5 protein and AQP 5 and AQP 3 mRNA. Food composition for preventing or improving. Salivary gland function due to menopausal hormonal fluctuations, including Ferrostatin-1 as an active ingredient, wherein the active ingredient increases the expression of AQP (Aquaporin) 5 protein and AQP 5 and AQP 3 mRNA. Food composition for enhancing salivation in response to decreased salivary secretion.